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BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-4-491549622810.1186/1471-2458-4-49Research ArticleTrends in suicide in Scotland 1981 – 1999: age, method and geography Stark Cameron [email protected] Paddy [email protected] Diane [email protected] Tracey [email protected] Alan [email protected] Alistair [email protected] Centre for Rural Health, University of Aberdeen, The Green House, Beechwood Business Park North, Inverness, IV2 3ED, Scotland, UK2 NHS Highland, Inverness, Scotland, UK3 Information and Statistics Division, NHS Scotland, Edinburgh, Scotland, UK4 Health Centre, Durness, Sutherland, Scotland, UK5 New Craigs Hospital, Inverness, Scotland, UK2004 20 10 2004 4 49 49 24 5 2004 20 10 2004 Copyright © 2004 Stark et al; licensee BioMed Central Ltd.2004Stark et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Male suicide rates continued to increase in Scotland when rates in England and Wales declined. Female rates decreased, but at a slower rate than in England and Wales. Previous work has suggested higher than average rates in some rural areas of Scotland. This paper describes trends in suicide and undetermined death in Scotland by age, gender, geographical area and method for 1981 – 1999.
Methods
Deaths from suicide and undetermined cause in Scotland from 1981 – 1999 were identified using the records of the General Registrar Office. The deaths of people not resident in Scotland were excluded from the analysis. Death rates were calculated by area of residence, age group, gender, and method. Standardised Mortality Ratios (SMRs) and 95% confidence intervals were calculated for rates by geographical area.
Results
Male rates of death by suicide and undetermined death increased by 35% between 1981 – 1985 and 1996 – 1999. The largest increases were in the youngest age groups. All age female rates decreased by 7% in the same period, although there were increases in younger female age groups.
The commonest methods of suicide in men were hanging, self-poisoning and car exhaust fumes. Hanging in males increased by 96.8% from 45 per million to 89 per million, compared to a 30.7% increase for self-poisoning deaths. In females, the commonest method of suicide was self-poisoning. Female hanging death rates increased in the time period.
Male SMRs for 1981 – 1999 were significantly elevated in Western Isles (SMR 138, 95% CI 112 – 171), Highland (135, CI 125 – 147), and Greater Glasgow (120, CI 115 – 125). The female SMR was significantly high only in Greater Glasgow (120, CI 112 – 128).
Conclusion
All age suicide rates increased in men and decreased in women in Scotland in 1981 – 1999. Previous findings of higher than expected male rates in some rural areas were supported. Rates were also high in Greater Glasgow, one of the most deprived areas of Scotland. There were changes in the methods used, with an increase in hanging deaths in men, and a smaller increase in hanging in women. Altered choice of method may have contributed to the increased male deaths.
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Background
Compared to the adjacent countries of England and Wales, Scotland had a low suicide rate through most of the twentieth century [1]. This did not appear to be explained by differences in recording of suicide [2]. Suicide rates in Scottish men increased in the 1970s and 1980s [3,4]. Rates in younger men continued to increase in the late 1980s [5] and early 1990s [6], at a time when male rates in England declined [7,8]. By contrast, female rates decreased in Scotland, although not as rapidly as in England and Wales [3].
Several authors have noted the importance of suicide in Scotland as a public health problem [9,10]. There was an increase in hanging and motor vehicle exhaust fumes as methods of male suicide in 1970 – 1989 and Pounder [5] suggested that choice of method might contribute to the increase in Scottish male rates, as some methods are associated with higher case fatality rates. Crombie [11] found that some areas had higher rates of male suicide than the Scottish average, mainly in rural areas. Access to particular methods of suicide may contribute to this [12]. Gender, age, suicide method and geographical area therefore appear to be important considerations in the epidemiology of suicide in Scotland. No recent summary of suicide trends in Scotland has been available, and this paper describes trends in relation to these factors.
Methods
We used anonymised information on deaths by suicide and undetermined deaths provided by the General Register Office for Scotland (GROS). Deaths registered during 1981 – 1999 were included if the cause of death was recorded as suicide or as undetermined cause (ICD-9 E950-E959 and E980-E989 respectively). Undetermined deaths were included as suicide deaths may be misattributed [13,14].
Population figures were taken from the GROS annual reports for the mid-year of each period. For analyses by area, if a death was registered away from the person's home address, the death was allocated to their area of residence, rather than the area in which they died. Deaths of people resident outside Scotland were identified using country codes, and were excluded. As far as possible, therefore, results reflect the rates of suicide and undetermined deaths of people resident in each area of Scotland. Standardised Mortality Ratios were calculated for National Health Service administrative areas, with 95% confidence intervals. In time period descriptions, the periods 1981 – 1985, 1986 – 1990, 1991 – 1995, and 1996 – 1999 were used. Data were analysed using Excel and SPSS.
Results
There were 14502 deaths recorded as suicide or undetermined cause in the time period. Of these deaths, 28.5% occurred in females (n = 4137) and 71.5% in males (n = 10365).
Gender and age group
Table 1 shows changes by gender. The male suicide rate for suicide and undetermined deaths increased from 187 per million in 1981 – 1985 to 252 per million in 1996 – 1999, an increase of 35%. In the same period, the female rate per million decreased from 88 to 82 per million, a 7% decrease. The female decline occurred between 1981 – 1985 and 1986 – 1999. By contrast, male rates increased between all time periods. The female: male rate ratio in 1981 – 1985 was 2.1:1 By 1996 – 1999 this had increased to 3.1:1.
Table 1 Suicide and Undetermined Deaths in Scotland 1981 – 1999 By Gender and Time Period Rate per Million Population
1981 to 1985 1986 to 1990 1991 to 1995 1996 to 1999
Gender No. of deaths Rate/million No. of deaths Rate/million No. of deaths Rate/million No. of deaths Rate/million % change from first to last time period
Males 2324 187 2,587 210 2,948 238 2,506 252 35%
Females 1180 88 1,030 78 1,058 80 869 82 -7%
Total 3,504 136 3,617 142 4,006 156 3,375 165 21%
Examining changes by age group in males (Table 2), there are increases in male rates in the under 15 years, 15– 24 year, 25 – 34 year and 35 – 44 year age groups, of 137%, 97%, 86% and 26% respectively. The increase in the youngest male age group, although based on very small numbers of deaths, occurred between 1986 – 90 and 1991 – 95. In the 15–24 and 25 – 34 year age groups, increases occurred in every time period. There were decreases in the 45 – 54 and 55 – 64 year age groups and increases, of 4% and 10%, in the 65 – 74 and 75 years and over age groups.
Table 2 Suicide and Undetermined Deaths in Males in Scotland 1981 – 1999 By Age Group and Time Period
1981–1985 1986–1990 1991–1995 1996–1999
Age group No. of deaths Rate per million No. of deaths Rate per million No. of deaths Rate per million No. of deaths Rate per million % change from first to last period
<15 years 11 4.1 11 4.5 25 10.1 19 9.7 137%
15–24 317 141.0 440 208.3 467 257.2 369 278.0 97%
25–34 410 226.1 536 276.1 736 355.9 677 421.4 86%
35–44 422 268.5 470 279.2 594 340.8 506 338.0 26%
45–54 437 311.0 420 301.6 473 313.6 377 290.6 -7%
55–64 382 290.7 339 263.3 306 241.3 229 226.4 -22%
65–74 228 244.6 223 238.5 212 216.0 200 254.3 4%
75+ 117 253.6 148 287.8 135 253.6 129 278.8 10%
All Ages 2,324 187.0 2,587 209.7 2,948 237.8 2,506 252.1 35%
In women, there were increases in the three youngest age groups, with a 76% increase in rates in the under 15 year old group, 150% in the 15 – 24 year group and 37% in 25 – 34 year olds (Table 3). There were decreases in every older age group from 35 – 44 years to 75 years and over.
Table 3 Suicide and Undetermined Deaths in Females in Scotland 1981 – 1999 By Age Group and Time Period
1981–1985 1986–1990 1991–1995 1996–1999
Age group No. of deaths Rate per million No. of deaths Rate per million No. of deaths Rate per million No. of deaths Rate per million % change from first to last period
<15 years 7 2.7 7 3.0 7 3.0 9 4.8 76%
15–24 70 32.4 89 44.1 100 57.6 103 81.0 150%
25–34 141 78.9 174 91.4 217 106.4 172 108.3 37%
35–44 179 112.3 158 93.3 192 109.1 157 103.9 -8%
45–54 231 154.9 151 103.3 179 115.0 153 115.1 -26%
55–64 264 176.3 187 129.6 138 98.4 96 86.7 -51%
65–74 174 136.5 153 122.5 107 84.8 101 102.6 -25%
75+ 114 114.3 111 103.0 118 108.3 78 87.0 -24%
All Ages 1,180 88.4 1,030 78.1 1,058 80.1 869 82.4 -7%
Method of suicide
The commonest methods of suicide and undetermined deaths in men were hanging, strangulation and suffocation, poisoning with solid or liquid substances, drowning, use of gases and vapours and jumping from high places (Table 4). Hanging, strangulation and suffocation in males had a similar rate to poisoning with solid or liquid substances in 1981 – 1985, but by 1996 – 1999 it had increased by 96.8% from 45 per million to 89 per million, compared to a 30.7% increase for self-poisoning deaths, from 46 per million to 60 per million. Deaths from jumping and cutting also increased, by 44.2% and 18.8% respectively. 'Other gases and vapours', predominantly car exhaust deaths (data not presented), decreased slightly from the first to last periods, but this concealed a substantial increase between 1981 – 1985 and 1986 – 1990, followed by a decrease in 1996 – 1999. Unspecified means increased by 70.9% from 14 to 23 per million.
Table 4 Methods of Suicide and Undetermined Death in Males in Scotland 1981 – 1999 By Time Period
1981 to 1985 1986 to 1990 1991 to 1995 1996 to 1999
Primary Cause No. of Deaths Rate/million No. of Deaths Rate/million No. of Deaths Rate/million No. of Deaths Rate/million % change from first to last time period
Solid or liquid substances 572 46 594 48 768 62 598 60 30.7
Hanging, strangulation and suffocation 562 45 630 51 774 62 880 89 96.8
Submersion(drowning) 353 28 417 34 358 29 268 27 -5.1
Other gases and vapours 288 23 428 35 448 36 225 23 -2.3
Other, unspecified means 169 14 170 14 226 18 231 23 70.9
Firearms and explosives 166 13 119 10 114 9 66 7 -50.3
Jumping from high place 163 13 176 14 203 16 188 19 44.2
Cutting and piercing instruments 40 3 38 3 38 3 38 4 18.8
Gases in domestic use 8 1 7 1 7 1 8 1 33.4
Late effects of injury 3 . 4 8 1 12 1 4 1 99.9
Total 2,324 187 2,587 210 2,948 238 2,506 252 34.8
In females, the commonest methods of suicide and undetermined death were poisoning with solid or liquid substances, hanging, strangulation and suffocation, and drowning (Table 5). Self-poisoning decreased by 4.8% between first and last periods from 45 to 43 per million, while drowning, and jumping from high places decreased by 54.1% and 30.3% respectively. Gases and vapours showed an increase but, as in males, the rate was higher in the middle two time periods. The rate of hanging, strangulation and suffocation deaths increased by 53.5%. There was an increase in unspecified means of suicide, from 4 to 8 per million.
Table 5 Methods of Suicide and Undetermined Death in Females in Scotland 1981 – 1999 By Time Period
1981 to 1985 1986 to 1990 1991 to 1995 1996 to 1999
Primary Cause No. of Deaths Rate/million No. of Deaths Rate/million No. of Deaths Rate/million No. of Deaths Rate/million % change from first to last time period
Solid or liquid substances 601 45 577 44 570 43 452 43 -4.8
Submersion(drowning) 248 19 159 12 132 10 90 9 -54.1
Hanging, strangulation and suffocation 127 10 95 7 134 10 154 15 53.5
Jumping from high place 89 7 77 6 67 5 49 5 -30.3
Other, unspecified means 60 4 53 4 81 6 83 8 75.1
Other gases and vapours 31 2 51 4 50 4 30 3 22.5
Firearms and explosives 13 1 10 1 6 0.5 3 0.4 -68.9
Cutting and piercing instruments 9 1 8 1 9 1 5 1 -25.0
Late effects of injury 2 0.4 - - 7 1 3 0.4 1.4
Gases in domestic use - - - - 2 0.4 - - -
Total 1,180 88 1,030 78 1,058 80 869 82 -6.8
Geographical areas
In the nineteen-year period as a whole, there was substantial geographical variation (Figures 1 and 2). The highest male rates were in Western Isles, Highland, Orkney, Greater Glasgow and Tayside (Table 6). When considered as Standardised Mortality Ratios, Western Isles, Highland and Greater Glasgow were statistically significantly elevated (Table 6). Six areas, Fife, Ayrshire and Arran, Forth Valley, Lothian, Borders and Lanarkshire had significantly lower SMRs than the Scottish average.
Figure 1 Male Standardised Mortality Ratios for Suicide and Undetermined Deaths in Scotland, 1981 – 1999 by Area
Figure 2 Female Standardised Mortality Ratios for Suicide and Undetermined Deaths in Scotland, 1981 – 1999 by Area
Table 6 Male Standardised Mortality Ratios by Health Service Area Suicide and Death by Undetermined Cause in Scotland 1981 – 1999
Health Board of Residence No. of Deaths Rate/million Standardised Mortality Ratio
Ratio LCI UCI
Argyll & Clyde 909 225 103 97 110
Ayrshire & Arran 676 197 91 84 98
Borders 176 186 84 72 97
Dumfries & Galloway 301 223 101 90 113
Fife 634 197 90 83 97
Forth Valley 496 197 89 82 98
Grampian 1,050 219 99 93 105
Greater Glasgow 2,252 264 120 115 125
Highland 555 294 135 125 147
Lanarkshire 907 174 81 75 86
Lothian 1,399 202 90 85 95
Orkney 50 274 124 92 164
Shetland 47 214 99 72 133
Tayside 828 231 105 98 112
Western Isles 85 300 138 112 171
Scotland 10,365 220 100 - -
In women, the highest rates were in Glasgow, Tayside, Highland and Dumfries and Galloway (Table 7). Only the Greater Glasgow SMR was significantly elevated. One area, Lanarkshire, had a significantly low female SMR.
Table 7 Female Standardised Mortality Ratios by Health Service Area Suicide and Death by Undetermined Cause in Scotland 1981 – 1999
Health Board of Residence No. of Deaths Rate/million Standardised Mortality Ratio
Ratio LCI UCI
Argyll & Clyde 330 77 93 84 104
Ayrshire & Arran 290 78 95 84 106
Borders 83 81 95 77 118
Dumfries & Galloway 122 85 101 84 120
Fife 277 82 100 89 112
Forth Valley 195 73 89 77 102
Grampian 383 77 95 86 105
Greater Glasgow 921 99 120 112 128
Highland 169 86 105 91 123
Lanarkshire 366 66 83 75 92
Lothian 598 81 97 90 105
Orkney 15 80 98 55 162
Shetland 16 74 95 54 154
Tayside 357 92 110 99 122
Western Isles 15 53 65 36 107
Scotland 4,137 82 100 - -
There were changes within areas over the period studied. Male rates increased in all fifteen NHS Board areas (Table 8). The smallest percentage increases were in Orkney, Highland and Greater Glasgow, three of the areas with the highest male rates in the first time period. Shetland and Western Isles had large percentage increases, but this was based on small numbers of suicide and undetermined cause deaths. The largest increases in mainland Scottish Board areas were in Argyll and Clyde (62%), Borders (60%), Forth Valley 59%), Ayrshire and Arran (56%) and Grampian (49%).
Table 8 Male Deaths from Suicide and Undetermined Cause in Scotland 1981 – 1999 Rates By Health Service Area and Time Period
1981 to 1985 1986 to 1990 1991 to 1995 1996 to 1999
Primary Cause No. of Deaths Rate/million No. of Deaths Rate/million No. of Deaths Rate/million No. of Deaths Rate/million % change from first to last time period
Argyll & Clyde 190 175 212 199 272 259 235 283 62
Ayrshire & Arran 143 158 161 178 194 214 178 246 56
Borders 31 128 41 167 62 245 42 205 60
Dumfries & Galloway 71 201 74 209 79 220 77 269 34
Fife 148 177 155 183 169 198 162 239 35
Forth Valley 102 154 127 192 136 205 131 245 59
Grampian 219 181 258 208 291 224 282 270 49
Greater Glasgow 551 234 565 252 666 304 470 270 15
Highland 131 273 155 315 144 284 125 305 12
Lanarkshire 195 140 242 177 255 187 215 197 40
Lothian 307 172 350 195 408 223 334 222 29
Orkney 13 275 11 233 15 308 11 281 2
Shetland 12 202 10 179 7 121 18 388 92
Tayside 193 205 200 214 230 242 205 272 33
Western Isles 18 230 26 343 20 274 21 376 64
Scotland 2,324 187 2,587 210 2,948 238 2,506 252 35
In females, rates changed little or decreased in all mainland Boards other than Argyll and Clyde (16.9% increase) (Table 9). Rates increased in Orkney, Shetland and Western Isles, although these were based on very small numbers of deaths. The greatest declines in mainland Board areas were in Ayrshire and Arran (30% decrease) and Grampian (27.9% decrease).
Table 9 Female Deaths from Suicide and Undetermined Cause in Scotland 1981 – 1999 Rates By Health Service Area and Time Period
1981 to 1985 1986 to 1990 1991 to 1995 1996 to 1999
Primary Cause No. of Deaths Rate/million No. of Deaths Rate/million No. of Deaths Rate/million No. of Deaths Rate/million % change from first to last time period
Argyll & Clyde 96 82 73 64 76 68 85 96 17
Ayrshire & Arran 88 90 73 75 80 82 49 63 -30
Borders 26 98 20 75 18 66 19 86 -12
Dumfries & Galloway 34 91 29 77 32 84 27 89 -2
Fife 79 89 76 85 69 77 53 74 -18
Forth Valley 53 75 44 63 55 78 43 76 0.3
Grampian 133 105 85 66 84 63 81 76 -28
Greater Glasgow 270 104 238 97 235 98 178 94 -10
Highland 41 82 40 79 59 112 29 68 -17
Lanarkshire 98 67 93 64 93 64 82 71 7
Lothian 165 85 156 81 143 73 134 84 -0.3
Orkney 2 41 2 41 3 60 8 202 390
Shetland 1 17 4 72 7 125 4 88 412
Tayside 91 88 93 92 101 99 72 89 0.6
Western Isles 3 38 4 53 3 41 5 88 130
Scotland 1,180 88 1,030 78 1,058 80 869 82 -7
Discussion
The epidemiology of suicide in Scotland has changed greatly between 1981 and 1999. Male suicide rates have increased in all age groups up to and including 35 – 44 years. The highest male suicide and undetermined death rates in 1996 – 1999 were in the 25 – 34 year age group. In women, rates dropped in age groups from 35 – 44 years up to and including 75 years and over. Rates increased in younger women.
There is limited information on the factors underlying individual deaths from suicide in Scotland. Squires and Gorman [15] reviewed the deaths by suicide of a group of young men in Lothian, and reported that a third had experienced recent relationship difficulties with a partner. Half of the group studied had a previous history of attempted suicide. Cavanagh et al [16] reported largely similar findings in a case control study in south-east Scotland. The group who had died by suicide had an odds ratio of 9.0 (95% CI 1.3 – 399) for current family problems, and an odds ratio of 5.0 (95% CI 1.1 – 47) for physical health problems. There was felt to be limited scope to intervene in suicide and deliberate self-harm through family health services because of limited contact, and non-specific presentation of problems [15,17].
Some methods of self-harm have higher case fatality rates [18]. Firearms have the highest case fatality rates, followed by drowning and hanging [19,20]. The most striking changes in male suicide methods in Scotland were the marked increase in hanging deaths, and the increase and subsequent decrease in deaths from 'other gases and vapours', which are mainly car exhausts. It seems likely that the decrease in motor vehicle exhaust fume deaths was related to the introduction of catalytic converters. Not all countries have reported a decrease in suicide from motor vehicle exhausts after catalytic converter introduction [21], but deaths in England decreased [22]. The reduction in deaths from motor vehicle exhaust fumes in England and Wales was associated with an increase in hanging deaths [7]. In Scotland, our data suggest that hanging deaths were increasing in men before deaths from motor vehicle exhaust fumes began to decline. The increase in hanging also appears greater than the decrease in motor vehicle exhaust deaths. The relationship between vehicle exhaust fume and hanging deaths in Scotland does not appear to be identical to that reported in England and Wales, and deserves further investigation.
The difference between areas was also of note. The lower rates of increase in the areas with the highest initial rates may reflect to regression to the mean. Method availability [23] may be important in rural/urban differences. Obafunwa and Busuttil [24] reported that, within the Lothian region of Scotland, hanging was commoner in younger deaths, while use of car exhaust fumes for suicide was particularly important in rural areas [24,25]. In Lothian, an area that includes the capital city of Scotland, suicide by firearms was uncommon. Previous work has suggested higher rates of male suicide in some rural parts of Scotland [11]. Stark et al [12] have suggested that this may be related to the use of methods of self-harm in rural areas, such as firearms, with a high case fatality rate. Gunnell and colleagues [26] have argued, in relation to England and Wales, that changes in method preference, and therefore in case fatality, should be considered before concluding that changes must relate to social trends.
Availability of method would not explain the differences between apparently similar rural areas. Previous work has found that deprived areas of Scotland tend to have higher suicide rates [27,28]. Deprived areas in Scotland were reported to have had the greatest increase in young male suicide between 1981 – 3 and 1991 – 3 [29]. Greater Glasgow, the non-rural area with the highest rate over the time period, is an area with substantial deprivation. Rural deprivation is difficult to measure, and recent work suggests that rural areas of Scotland may suffer greater levels of deprivation than had been realised. It is possible that rural deprivation is underestimated, and deprivation may explain more of the elevation in some rural areas than has been assumed in the past.
Using routine information allowed a large number of suicide and undetermined deaths to be included in this series. There are, however, limitations to the use of anonymised routine data. No qualitative information was available, and our exploration of the data was limited to trends with no examination of possible underlying causes. The increase in the rate of deaths recorded as suicide or undetermined cause of death, but where no detail on method was included, could conceal recent trends. The increase as a percentage of relevant registrations was small, however, increasing from 7.5% in the first period to 9.1% in the last period studied. The classification of deaths as suicide is often difficult, but the inclusion of undetermined deaths as well as deaths recorded as suicide should have helped to minimise bias from under identification [14,30]. Squires et al [31] reported that improved communication between pathologists and the Registrar General for Scotland from 1994 on was associated with a decrease in undetermined deaths and in increase in deaths coded as being caused by dependent or non-dependent use of drugs. It is possible, therefore, that the figures for the final two periods may under-represent deaths that would have been identified as 'unidentified' in the earlier periods. Using information on Scottish residents only allowed identification of the suicide rates of local populations. Deaths of non-residents can account for up to 10% of all suicide and undetermined cause deaths in some rural areas of Scotland [12]. Our findings indicate that, even when these deaths are excluded, rates remain increased in some rural areas.
Conclusions
A divergence between male rates in England and Wales and in Scotland, and in male and female rates within Scotland, had been identified for the first part of the time period described here. This work found that male rates of suicide and undetermined death continued to increase in Scotland, but also identifies increases in younger female age groups. Examination of changes in method by male and female age group will help to establish whether changes in case fatality because of altered method choice [26] may be part of the explanation for these findings. The shift to hanging seems to be a significant trend in men in Scotland. It will be important to understand the reasons for this to allow appropriate intervention strategies to be considered.
Some rural areas of Scotland had significantly elevated male suicide rates. We have suggested that access to lethal means of suicide may be one contributing mechanism for this, and have also noted the higher than expected suicide numbers in some rural occupations [12]. Rural areas are subject to poverty of income and opportunity, so it is also possible that rural deprivation may play an important part. Occupational associations of suicide in Scotland deserve further exploration. Examination of the association between deprivation, rurality and suicide may assist in the identification of possible interventions in rural Scotland.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
CS had the idea for the study, wrote the grant application, contributed to the design and interpretation and drafted the paper. Diane Gibbs and Tracey Rapson analysed the data. Paddy Hopkins contributed to the design, undertook part of the analysis, and commented on the interpretation of the results. Alan Belbin and Alistair Hay contributed to the design and helped interpret the results. All authors read and approved the final draft of the paper.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
This work was undertaken with a grant awarded by the Scottish Executive's Remote and Rural Areas Resource Initiative. NHS Highland supplied staff time from CS and PH. Prof. David Gunnell of the University of Bristol, Mike Muirhead of ISD, Professor David Godden of the University of Aberdeen and Brodie Paterson of the University of Stirling provided helpful advice during the study.
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| 15496228 | PMC529267 | CC BY | 2021-01-04 16:28:47 | no | BMC Public Health. 2004 Oct 20; 4:49 | utf-8 | BMC Public Health | 2,004 | 10.1186/1471-2458-4-49 | oa_comm |
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BMC Musculoskelet DisordBMC Musculoskeletal Disorders1471-2474BioMed Central London 1471-2474-5-351548557810.1186/1471-2474-5-35Research ArticleA high prevalence of cumulative trauma disorders in Iranian instrumentalists Sadeghi Shahram [email protected] Behrooz [email protected] Seyed Mostafa Jazayeri [email protected] Ali [email protected] Peyman [email protected] Pain research group, academic centre for education, culture and research, Iran medical science branch. No 31, Karimkhan zand Avenue, shahid hosseini alley, multidisciplinary pain clinic. Tehran, Iran2 Department of physical medicine and rehabilitation, Shiraz medical school, Shiraz, Iran3 Emergency department, Hazrat-e-rasoul medical complex. Iran university of medical sciences, Tehran, Iran4 department of biostatistics, Shiraz medical school, Shiraz, Iran2004 14 10 2004 5 35 35 5 5 2004 14 10 2004 Copyright © 2004 Sadeghi et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Cumulative trauma disorders (CTDs) are common in musicians and their prevalence has been the subject of a number of studies in most western countries. Such studies are scarce in developing countries despite the possibility that CTDs may have a different prevalence in these countries, especially when considering traditional musical instruments and different methods of playing. Although not formally studied before, according to our experience the prevalence of CTDs seemed to be high among Iranian instrumentalists.
We proposed this study to determine the prevalence of CTDs in amateur music students playing one of the two traditional Iranian instruments: Daf and Setar.
Methods
In a prospective cross sectional study, we interviewed and examined the students of three music training centers in Iran. Seventy eight instrumentalists, who were playing Daf or Setar and twelve students who had not started playing yet were regarded as case and control groups respectively. Some of them also underwent electrodiagnostic studies.
Results
Forty-seven percent (17 of 36) of the Setar players and 57% (24 of 42) of the Daf players and fifty-three percent (41 of 78) of the instrumentalists as a whole had CTDs. None of them had carpal tunnel syndrome.
Conclusions
Our study revealed that the prevalence of CTDs in Iranian instrumentalists was unusually high. In addition to age, other variables may be contributory. This needs to be further studied.
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Background
Cumulative trauma disorders, also called repetitive stress injuries, overuse syndromes or repetitive motion injuries [1-3] are common in musicians [4,5] and are caused by repetitive motions. Nerve entrapments, stress fractures, tendonitis, bursitis and muscle strains have been labeled in this category [1,5].
To date no study has been performed about the prevalence of cumulative trauma disorders (CTDs) in players of Iranian instruments. According to our experience, it seemed to be unusually high when compared with related prevalence in nonprofessional players of classical instruments as reported by Fry [4].
This study was performed to determine the prevalence of cumulative trauma disorders in amateur music students playing two traditional Iranian instruments: Daf and Setar.
Daf is a percussion musical instrument that has a circular wooden frame covered with goat skin with or without metal discs around its edge. To play Daf the player shakes it and hits it with both hands (fig. 1).
Setar is a string musical instrument that has 4 strings. It is played by the index of right hand (fig. 2).
In comparison to classic musical instruments, Setar resembles the Guitar, but Daf doesn't have any similar equivalent.
Methods
In a prospective cross sectional study, we interviewed and examined the students of three music training centers, numbering 94. Twelve students who were at their first sessions and hadn't begun to play were selected as control group.
Age, sex and duration of playing (date of starting and daily playing time) as well as vocational and avocational risk factors for developing CTDs were recorded after a direct interview. Then the students were referred to a physician who did not know whether the student belonged to the case or control group. He then evaluated their upper limbs and necks. Specific attention was paid to pain, paresthesia, sensory changes, tenderness, range of motion, muscle power and muscle stretch reflexes. In addition, Phalen, Tinel and carpal compression tests [6] were performed to detect the presence of carpal tunnel syndrome (CTS); the most common neuropathy reported in instrumentalists [5].
Since the standard diagnostic test for CTS is electrodiagnostic study [7], all of the students were asked to attend our center for electrodiagnostic studies. In all of the participants, antidromic median sensory nerve action potential (SNAP) was obtained from the third digit at both 7 and 14 cm. Then the split times and amplitudes were compared. Also distal latency for the motor median nerve was obtained. We also compared the wrist versus midpalm compound muscle action potential (CMAP) amplitudes [8-10]. Electromyographic investigation was not performed.
The data were analyzed by SPSS software using Chi square and Fisher's exact tests.
Results
Ninety four students were included in this study. Four students were excluded from the study, two because of a history of musculoskeletal pain before attending the music center, one because of playing two instruments and one, serving as a typist.
Twelve of the students who had not started to play were assigned to the control group and the remaining 78 students; 42 in Daf and 36 in Setar groups; were considered as case group (table 1).
Mean age of students in the case group was 21.2 years (SD: 3.8) including 47 females and 31 males. Mean duration of instrument playing in this group was 7.9 months (SD: 5.4).
Mean age of students in control group was 25.2 years (SD: 9.2). This group consisted of 9 females and 3 males.
Mean Duration of daily playing in Setar students was more than Daf students (1.6 Vs 1.5 hours) which was not statistically significant (P value = 0.8).
Mean duration of daily playing in male and female students was 1.8 hours and 1.4 hours respectively. Which was not statistically significant (P value = 0.64).
Forty-one students in case group (53% of the total of 78) had musculoskeletal pain and there was a significant correlation between playing Daf and Setar and development of musculoskeletal symptoms.
The prevalence of pain among females was twice as much as males but the difference was not statistically significant (P value = 0.12) (table 2).
The prevalence of musculoskeletal pain in Daf players was more than Setar players (57%vs 47%, P value = 0.38); again, this difference was not significant (table 2).
Regarding the location of pain, hand was the most common site; it was painful in 65% of cases (table 3).
Twenty six students, all from the case group attended our electrodiagnostic center, none of them had carpal tunnel syndrome.
None of the students in control group had problems in their exams and none of them attended for electrodiagnostic studies.
Discussion
A large number of amateur Daf and Setar players with a history of playing of less than 1 year (7.9 months) and almost 1.5 hours a day had musculoskeletal pain (that is considered a form of CTDs). The prevalence of pain in this group was much greater than students in tertiary music schools who train for some years for 6 hours a day (53% vs. 9.3–21%, respectively) and almost equals professional orchestra players (73–75%) [5].
In a group of instrumentalists (Guitarists, Harpists, Pianists etc.) Bejjani et al found a 77.5% prevalence of upper extremity disorders serious enough to impair the performance or to cause the musician to stop playing at least temporarily [11]. similarly, it is possible that some of the students also had quit playing before they had chance to enter our study (case selection bias) this might have caused an underestimation of the prevalence of the CTDs observed in this study. So it may be reasonably concluded that 53% is the minimum prevalence of CTDs in the studied group.
How can we explain this high prevalence?
The most important cause of CTDs is repetitive motions and in fact multiplication of duration and intensity of exercise [4]. Since both the duration and the intensity of exercise in these players were much less than that of professional music students or music trainees, other factors should be considered.
According to Fry [4], other important factors that predispose to CTDs are genetics and student technique. Since the students were taught in certified centers and the music teachers were satisfied with the students' techniques, we assumed that playing method was not of primary concern.
The particular instrument has been shown to be a risk factor for developing CTDs [5,14]. On the other hand, Setar is not heavier than guitar, nor does its playing need awkward positions, so the instrument in itself may not explain this high prevalence.
Another risk factor is age [12]. It has been shown that adults who start playing, may be more vulnerable to developing CTDs. The extent to which this may have affected the results of our study is not clear so we additionally proposed that the studied group might have been inherently susceptible to develop CTDs because of some genetic factors such as joint hypermobility.
This hypothesis can explain, at least in part, the wide range (9–49%) [5-12] of the prevalence of CTDs in music students by different studies. However, genetic analyses and larger studies are needed for validating this hypothesis.
As mentioned, we did not find any case of symptomatic carpal tunnel syndrome or nerve conduction abnormalities suggesting subclinical median neuropathy at the wrist, implying that CTS is a more advanced form of cumulative trauma disorders when compared to musculotendinous unit CTDs.
Hand was the most common painful site in this study (65%); a finding which is in concert with other published studies (41–54 %)[4,11].
There are two other findings in the current study left to be explained:
First: Daf is played being held using both hands but Setar is being held like Guitar. So it can be postulated that playing Daf is more harmful than Setar and we expected more CTDs in Daf players. Although, the prevalence of CTDs was higher in Daf players, the difference was not significant (p value = 0.38). A significant difference may be found with a large scale study.
Second: it has been known that CTDs are more common in females [5,12,13]. In our study, we also found a higher occurrence of CTDs among females but the study failed to reveal a statistically significant difference (p value = 0.12), perhaps because of small sample size.
Conclusions
Our study revealed that the prevalence of CTDs in Iranian instrumentalists was abnormally high. This is an unusual finding that can't be fully explained by the difference in the instruments (classical versus traditional), playing method or intensity of the exercise. Other susceptibility factors such as age at the starting of playing or genetic predisposition may be contributory. Larger studies focusing on individual characteristics and genetic analyses are needed to delineate other important factors.
Competing interests
The authors declare that they have no competing interests.
Abbreviations
CTD: cumulative trauma disorders
CTS: carpal tunnel syndrome
Authors' contributions
SS: suggesting the proposal, examining the volunteers, writing the paper.
BK & SMJS: examining the volunteers
AB: great help in writing the paper and statistical analysis
PJ: statistical analysis
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The authors would like to thank:
Research deputy of Shiraz (Shiraz-Iran) university of medical sciences, for financial support.
And seyed Mohammad Hussein Modarresi, MD PHD, associate professor of genetics (Tehran medical university) for his useful comments.
(Written consent was obtained from the subjects for publication of figures)
Figures and Tables
Figure 1 Bijan Kamkar (one of the most famous Iranian Daf players/teachers) showing the playing technique for Daf. With thanks to Hana Kamkar (photographer).
Figure 2 Ghashang Kamkar (Setar player/teacher) showing the playing technique for Setar.
Table 1 Sex distribution of students
Instrument/Gender male female total
Daf 15 27 42
Setar 16 20 36
Table 2 cumulative trauma disorders regarding instrument and gender
Instrument/Gender female male Total
Daf 19 5 24
Setar 9 8 17
Table 3 Location of pain and its relation to different instruments (some students have had more than one painful area)
Instrument/pain location hand forearm arm Coracoid process
Daf 14 8 4 8
Setar 13 4 1 1
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| 15485578 | PMC529268 | CC BY | 2021-01-04 16:03:43 | no | BMC Musculoskelet Disord. 2004 Oct 14; 5:35 | utf-8 | BMC Musculoskelet Disord | 2,004 | 10.1186/1471-2474-5-35 | oa_comm |
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BMC BiotechnolBMC Biotechnology1472-6750BioMed Central London 1472-6750-4-241550068110.1186/1472-6750-4-24Methodology ArticleDetecting imbalanced expression of SNP alleles by minisequencing on microarrays Liljedahl Ulrika [email protected] Mona [email protected] Andreas [email protected]änen Ann-Christine [email protected] Molecular Medicine, Department of Medical Sciences, Uppsala University, Uppsala, Sweden2004 22 10 2004 4 24 24 3 3 2004 22 10 2004 Copyright © 2004 Liljedahl et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Each of the human genes or transcriptional units is likely to contain single nucleotide polymorphisms that may give rise to sequence variation between individuals and tissues on the level of RNA. Based on recent studies, differential expression of the two alleles of heterozygous coding single nucleotide polymorphisms (SNPs) may be frequent for human genes. Methods with high accuracy to be used in a high throughput setting are needed for systematic surveys of expressed sequence variation. In this study we evaluated two formats of multiplexed, microarray based minisequencing for quantitative detection of imbalanced expression of SNP alleles. We used a panel of ten SNPs located in five genes known to be expressed in two endothelial cell lines as our model system.
Results
The accuracy and sensitivity of quantitative detection of allelic imbalance was assessed for each SNP by constructing regression lines using a dilution series of mixed samples from individuals of different genotype. Accurate quantification of SNP alleles by both assay formats was evidenced for by R2 values > 0.95 for the majority of the regression lines. According to a two sample t-test, we were able to distinguish 1–9% of a minority SNP allele from a homozygous genotype, with larger variation between SNPs than between assay formats. Six of the SNPs, heterozygous in either of the two cell lines, were genotyped in RNA extracted from the endothelial cells. The coefficient of variation between the fluorescent signals from five parallel reactions was similar for cDNA and genomic DNA. The fluorescence signal intensity ratios measured in the cDNA samples were compared to those in genomic DNA to determine the relative expression levels of the two alleles of each SNP. Four of the six SNPs tested displayed a higher than 1.4-fold difference in allelic ratios between cDNA and genomic DNA. The results were verified by allele-specific oligonucleotide hybridisation and minisequencing in a microtiter plate format.
Conclusions
We conclude that microarray based minisequencing is an accurate and accessible tool for multiplexed screening for imbalanced allelic expression in multiple samples and tissues in parallel.
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Background
Single nucleotide polymorphisms (SNPs) are highly abundant in the human genome, appearing on average at 0.1% of the nucleotide positions [1]. Thus, each gene or transcriptional unit will contain multiple SNPs that potentially give rise to sequence variation between individuals and tissues on the level of RNA. Recent studies indicate that differences in the expression levels of the alleles of heterozygous SNPs may occur frequently for human genes [2-6]. Imbalanced allelic expression was detected in foetal liver or kidney tissues for more than half of 602 genes analysed, and one third of the genes displayed more than four-fold differences in allelic expression [3]. Another study detected lower levels of allelic imbalance for one fifth of 129 genes analysed in lymphoblastoid cell lines [4].
Non-synonymous SNPs in coding regions of genes may be functional by altering an amino acid, which in turn may affect the structure and function of the encoded protein, while synonymous SNPs may have functional consequences by affecting the stability or folding of mRNA transcripts. Intronic SNPs may give rise to alternatively spliced mRNAs, while SNPs in 5'- or 3'-untranslated mRNA regions may affect the stability or processing of the RNA. Moreover, SNPs in non-protein coding regions of genes that affect binding of regulatory factors may cause imbalanced expression of SNP alleles. This form of genetic variation has been suggested as a common cause of both normal and disease-related inter-individual variation in complex phenotypes [7]. Clearly, methods with high accuracy that can be used in a high throughput setting are needed for systematic surveys of expressed sequence variation and its molecular causes.
Owing to the high sequence specificity of nucleotide incorporation by DNA-polymerases, single nucleotide primer extension has proven to allow quantitative determination of SNPs in genomic DNA in several studies and assay formats (for a review, see Syvänen 2001 [8]). A frequently used quantitative application of the method is to determine SNP allele frequencies in pooled DNA samples [9-13]. The rationale for detecting imbalanced expression of the two alleles of a heterozygous SNP by minisequencing is to measure the ratio between the amounts of labelled nucleotides incorporated in the minisequencing reactions for the two SNP alleles in RNA (cDNA) samples from the tissue of interest. These ratios are then compared to the corresponding ratio measured in genomic DNA, where the two alleles are present in an equimolar ratio [2,4,14-16]. Imbalanced expression of the alleles of a SNP is revealed by a difference in the ratios measured in the RNA and DNA samples.
We are currently using microarray based minisequencing for multiplex genotyping of SNPs. Our custom-made microarrays permit the genotyping of up to 100 SNPs in 80 samples per standard microscope slide, either using immobilised minisequencing primers [17,18] or using a "tag-array" format [19,20] of the method [13]. The purpose of this study was to evaluate the performance of these two microarray formats in quantitative determination of SNP alleles on the RNA level as alternatives with higher multiplexing capacity than previously used primer extension methods in which the SNPs are analysed in individual reactions. Using these systems, we were able to detect significant differences in the amounts of the two alleles of heterozygous SNPs on the RNA level.
Results
We used a panel of ten coding SNPs in five genes to choose the optimal microarray based minisequencing strategy for multiplex, quantitative genotyping of SNPs in DNA and RNA samples. The selected SNPs were located in genes shown by reverse transcriptase PCR analysis to be expressed in one or both of two endothelial cells lines, HUVEC (human umbilical vein endothelial cells) and HAEC (human aortic endothelial cells) that served as our model cell lines in this study (data not shown). We evaluated two formats of microarray based minisequencing by performing five parallel assays with each method for each sample in the evaluation. The SNPs were analysed in both DNA polarities and the evaluation of the methods was based on the DNA polarity yielding the highest signal-to-noise ratio.
In Method I, immobilised minisequencing primers are extended with fluorescently labelled ddNTPs in reactions performed on the microarray surface after annealing of the multiplex PCR products to the primers [18,21]. In Method II, cyclic primer extension reactions are performed in solution in the presence of 5'-tagged minisequencing primers, PCR products and fluorescent ddNTPs [22,23]. After the cyclic reactions the extended primers are captured on a microarray surface carrying immobilised oligonucleotides complementary to the 5'-tag sequences on the minisequencing primers. Both these systems are performed in an "array-of arrays" format developed previously in our laboratory [24].
We analysed a dilution series with mixtures of DNA from two individuals with different genotypes for the panel of ten SNPs in both DNA polarities. The genotyping results from these mixtures of known amounts of the two SNP alleles are expressed as the signal ratio between the fluorescence signals corresponding to the two alleles of each SNP. The quantitative analysis of these ten SNPs is illustrated in Figure 1 by regression lines, in which the mean signal intensity ratios are plotted as a function of the known allelic ratios in the mixed samples. The coefficient of determination (R2), which describes how well the regression line fits the data points, was used to assess the accuracy of quantification of the SNP alleles by Methods I and II. As can be seen in Table 2, the R2 values are close to one for most of the SNPs analysed, demonstrating little scatter of the data points around the regression line. For Method I, six of the ten SNPs analysed have R2 values ≥ 0.95, while for Method II the R2 values are ≥ 0.95 for eight of the SNPs. Thus, accurate quantification of SNP alleles is possible by both methods. The slopes of the regression lines vary between the ten SNPs as well as between the two methods (Figure 1). A regression line with a steep slope usually corresponds to a high R2 value, as observed for the SNP rs5930 LDLR analysed by Method I and SNP rs5331 EDNRB analysed by Method II. A flat slope does not necessarily imply less accurate quantification, as exemplified by the SNP rs4331 ACE, where Method II yielded a flat slope with a higher R2 value than Method I.
We also determined the sensitivity of the methods for detection of a minority allele. The detection limit was defined as the percentage of the minority allele in the mixed sample, for which the signal ratio differed from the signal ratio in the corresponding homozygous sample with a p-value < 0.05 in a two sample t-test. Depending on the genotype of the DNA samples used for the dilution series, determination of the lower limit of detection was possible for seven of the ten SNPs with allele ranges 0–50% or 0–100% in the mixed samples (Table 1). For the remaining three SNPs with the allele range 50–100%, the smallest percentage of an allele that could be distinguished from a heterozygous genotype was identified by the same approach. Using Method I, we were able to detect less than 5% of the minority allele for two SNPs (rs1042713 ADRB2 and rs5925 LDLR) and less than 9% for rs4331 ACE, rs1042719 ADRB2, rs5351 EDNRB and rs5930 LDLR (Table 2). Method II allowed more sensitive detection of minority alleles than Method I. Less than 2% was detectable for the SNPs rs1042713 ADRB2, rs1042719 ADRB2 and rs5351 EDNRB, and less than 9% was detectable for the SNPs rs4331 ACE, rs5925 LDLR, rs5930 LDLR and rs1433099 LDLR (Table 2). For the SNPs rs1042714 ADRB2, rs1042718 ADRB2 and rs1799983 NOS3, we were able to measure 4–14% deviations from the heterozygous genotype (Table 2). These results show that the amount of SNP alleles can be accurately determined on the DNA level by Methods I and II using reference samples with the two SNP alleles present in known ratios.
Next, the performance of the two methods in quantitative analysis on the RNA level was assessed. The ten SNPs were first genotyped in genomic DNA (gDNA) from the HUVEC and HAEC cells to identify those SNPs that were heterozygous in either or both cell lines. Three SNPs in the low density lipoprotein receptor gene (LDLR; rs5925, rs5930 and rs1433099) were heterozygous in the HAEC cell line, and one SNP in each of the genes encoding angiotensin I converting enzyme (ACE rs4331), β2-adrenergic receptor (ADRB2 rs1042719) and endothelin receptor type B (EDNRB rs5351) were heterozygous in the HUVEC cell line. These SNPs were genotyped in cDNA produced from total RNA extracted from the cells with the corresponding gDNA as reference samples using both methods. Table 3 presents the mean fluorescence signals with coefficients of variation (CV) obtained in five parallel reactions for the six SNPs in cDNA and gDNA from the HUVEC and HAEC cells. For the heterozygous SNPs the largest difference in the variability between parallel reactions was observed between SNPs, with the lowest CV values (3.6 – 8.6 %) for the rs1042719 ADRB2 SNP, and the highest CV values (13 – 41%) for the rs1433099 LDLR SNP. No systematic differences in the variability of parallel reactions were observed between Method I and Method II, or between cDNA and gDNA. Table 4 shows the differences in mean signal intensity ratios between the cDNA and gDNA assays for the six SNPs that were heterozygous in HUVEC or HAEC cells, respectively, together with the corresponding normalized cDNA/gDNA ratios. The SNPs in the ACE, ADRB2 and EDNRB genes displayed significant imbalanced expression in the HUVEC cells using both methods. For the SNP rs4331 ACE, the signal intensity ratio based on the raw data obtained by Methods I and II differed from each other, but despite this large difference, both methods yielded similar levels of allelic imbalance for this SNP after normalisation against the signal ratio in gDNA (Table 4). Only for one of the three LDLR SNPs (rs5930), the difference in fluorescence intensity ratios between cDNA and gDNA from HAEC cells reached statistical significance by both methods. Allelic imbalance of the LDLR gene was detected for the LDLR SNP rs5925 using Method II only.
To test that the results on imbalanced allelic expression detected by the multiplexed microarray based methods represents the true biological situation in the cells, we analysed the heterozygous SNPs in five replicate RNA samples prepared from HUVEC or HAEC harvested at different time points from different cell culture flasks. We also analysed the three LDLR SNPs in five replicate reverse transcription reactions from the same RNA sample prepared from HAEC cells. For this analysis we used our first generation solid-phase minisequencing assay for individual SNPs in a microtiter plate format. The concordant cDNA/gDNA ratios from these control experiments from independent cell and RNA samples presented in Table 5 show that the detected allelic imbalance was not caused by the procedures for RNA extraction or cDNA synthesis. Finally, we verified the results obtained by microarray-based minisequencing for three of the SNPs by real-time PCR with allele specific hybridization probes (TaqMan). Table 4 shows these results together with the corresponding results by solid-phase minisequencing in a microtiter plate format. Allelic imbalance was detected with statistical significance for the SNP rs1042719 ADRB2 and the SNP rs1433099 LDLR by both methods. Particularly for the SNP rs1042719 ADRB2, the cDNA/gDNA ratios obtained by the two reference methods were highly similar to the results from the microarray-based methods presented in Table 4, as well as with each other. As for the microarray-based Method II, the difference in signal ratios between cDNA and gDNA measured by the TaqMan assay for the SNP rs5925 LDLR did not reach statistical significance due to large variation between parallel assays. Analysis of the SNP rs1433099 LDLR by the reference methods confirms the imbalanced expression of the LDLR receptor alleles.
Discussion
The purpose of our study was to evaluate microarray based minisequencing for multiplexed detection and quantification of imbalanced expression of SNP alleles, as a prelude to further large scale screening for allelic imbalance. We found no significant differences in the performance of our two "in house" methods, minisequencing with primers directly immobilised on the microarrays (Method I)[18] and the "tag-array" format, based on cyclic minisequencing followed by capture on microarrays using immobilised complementary "tag" probes (Method II) [23]. Both methods showed a linear relationship between SNP allele ratios and the signal intensity measured in the four-colour fluorescence minisequencing assay for all SNPs. With respect to accuracy assessed by coefficients of variation (CV) between five parallel assays both methods performed equally well, and the CV values between parallel assays were indistinguishable between genomic DNA and reverse transcribed cDNA samples. The sensitivity of detecting a SNP allele present as a minority in a sample was defined as the percentage for which the signal ratio differed from the signal ratio in the corresponding homozygous sample with a p-value < 0.05 in a two sample t-test. The sensitivity differed between SNPs, and range from 1% to 9%, with a trend to be slightly better using the "tag-array" system (Method II). In several cases the p-values were lower than 0.05 (Table 2), which indicates that in practice the sensitivity of detection would be lower than the stringent limit set here. The sensitivity of our multiplex microarray based minisequencing methods compares well with the sensitivity of other single nucleotide primer extension assays performed for individual SNPs in recent studies [4,25-27].
It is notable that the largest differences in accuracy and sensitivity were observed between SNPs. Some of the SNP-to-SNP differences are likely due to differences is the accuracy and efficiency of incorporation of the four different fluorescently labelled nucleotide analogues by the DNA polymerase [13,26] as well as to other sequence context dependent factors. The large variation between parallel assays for the SNP rs1433099 LDLR prevented detection of the allelic imbalance for the LDLR gene, while imbalance was detected by the SNP rs5930 LDLR using both methods. This result demonstrates that it is preferable to analyse more than a single SNP in each gene in systematic screening for allelic imbalance in gene expression. As more data from primer extension assays accumulate, it may be possible to improve the accuracy of the system by improving the SNP selection and assay design further with the aid of algorithms developed based on this data [28,29].
Comparison of the relative amounts of the alleles of six SNPs on the RNA (cDNA) level to heterozygote SNPs in genomic DNA revealed four SNPs with imbalanced expression of the two alleles. A three-fold increase in the expression of the T-allele for the SNP rs4331 ACE was the most pronounced difference observed. In our study, 1.4–1.5-fold differences in allelic expression levels were detectable. The sensitivity of detecting a minority allele in our system would allow the distinction between 10-fold reduction in the expression of an allele and monoallelic expression, for example as a result of imprinting. Owing to its potential for high throughput screening of large numbers of samples, we have also performed a preliminary evaluation of the commercial SNPstream genotyping system (GenomeLab, Beckman Coulter) that also utilises the "tag-array" primer extension strategy in a semi-automated 384-well microtiter plate format for detection of imbalanced allelic expression [30]. The same trend of imbalanced allelic expression was observed for each of the SNPs, which is encouraging for future studies of imbalanced allelic expression in a high throughput semi-automated way. Other studies that have used fluorescent single base primer extension assays report that 1.2 – fold to 1.5 – fold differences in allelic expression are detectable [2,4,5]. Primer extension methods based on direct measurement of fluorescent signals, including the microarray-based methods evaluated here, are likely to provide better accuracy and sensitivity for allele quantification than homogeneous primer extension based on fluorescence polarisation [31,32], in which the allele quantification relies on measurement of small differences between large polarization signals.
It is also reassuring for future large scale detection of imbalanced allelic expression that the accuracy of our methods seemed to be similar for cDNA and genomic DNA. Analysis of replicate RNA samples from different batches of both cell lines using a microtiter plate format of the minisequencing method evidenced for the biological authenticity of the allelic imbalance detected using minisequencing in the microarray format. The data obtained from independent cell samples also indicate an acceptable reproducibility of RNA extraction, RNA storage and cDNA synthesis. Another important factor besides sample to sample variation that may affect the accuracy of the relative allele quantification is the amount of mRNA subjected to the analysis. At a low copy number of mRNA, the stochastic distribution of the RNA templates may be a major source of variation [33]. The reason for the large variation between parallel assays for the LDLR receptor gene observed with all four methods used in our study may reflect a low expression level of the LDLR gene in the HAEC cells. Moreover, the amount of gene specific transcript in each RNA sample may vary which makes it difficult to perform balanced multiplex RT-PCRs to screen for allelic imbalances in several genes in one reaction.
A similar minisequencing strategy as the one used for determination of imbalanced expression between SNP alleles can also be used for determination of the relative expression levels of highly homologous genes [15] and for determination of alternatively spliced transcripts [34], a resolution that is beyond the capacity of traditional microarray based RNA expression profiling.
Conclusions
Here we demonstrated the applicability of two formats of microarray based minisequencing for detecting imbalanced expression of SNP alleles. The accuracy and sensitivity of both systems allow detection of 1.4- to 10-fold differences in the expression levels of the two alleles of heterozygous SNPs. The microarray-based minisequencing systems utilise widely available reagents and equipment, and can thus easily be established "in-house". Moreover, the system is flexible with respect to number of SNPs and samples to be analyzed. Systematic quantitative screening of genetic diversity on the RNA level in multiple individuals and tissues will be a future approach in the elucidation of the molecular mechanisms that regulate gene expression.
Methods
DNA and RNA samples
DNA samples from 30 volunteer donors were genotyped by Methods I and II to identify individuals of different genotypes for the panel of ten SNPs analysed. The SNPs are described in the section "SNPs and primers" below. DNA (10 ng/μl) from one individual was serially diluted 2:1 into DNA (10 ng/μl) from a second individual, to yield a series of DNA samples with different ratios between the SNP alleles. These mixed DNA samples were used for construction of quantification standard curves. Depending on the genotype of each SNP in the two individuals whose DNA was mixed, dilution series of samples with different allelic ranges were obtained for the ten SNPs, as specified in Table 1.
Human Umbilical Vein Endothelial Cells (HUVEC) and Human Aortic Endothelial Cells (HAEC) (Cascade Biologics, Inc., Portland, OR, USA) were grown in Medium 200 with Low Serum Growth Supplement (LSGS Kit, Cascade Biologics, Inc., Portland, OR, USA) at 37°C in a humidified atmosphere of 5% CO2. Cells from the cultures were harvested at 80% confluence according to the manufacturer's instructions. Total RNA was isolated from the cells using the TRIZOL®Reagent (GIBCO BRL, Paisley, Scotland) and the RNA samples were stored at -70°C until use. High quality RNA with A260/A280 ratio over 1.9 and intact ribosomal 28S and 18S RNA were used for cDNA synthesis. The RNA samples were treated with 1 U RQ1 RNase-free DNase (Promega, Madison, WI, USA) per μg RNA. Two to 2.5 μg total RNA was subjected to first strand cDNA synthesis using SuperScript™ II (RNase H- Reverse Transcriptase, Invitrogen, Carlsbad, CA, USA) reagents in a 20 μl volume. DNA was extracted from the cells using GenElute™ Mammalian Genomic DNA Kit (Sigma, St Louis, MO, USA) and stored at -20°C until use.
PCR
The fragments comprising the SNPs were PCR-amplified in individual reactions using 10–15 ng genomic DNA or one tenth of the cDNA products, 0.2 mM dNTPs, 1U AmpliTaq ® Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA), 1.5 mM MgCl2, and 0.2–0.3 μM of primers in 50 μl of 10 mM Tris-HCl pH 8.3 and 50 mM KCl. The PCR conditions were initial activation of the enzyme at 95°C for 10 min followed by 35 cycles of 95°C for 1 min, 56°C for 1 min and 72°C for 1 min and a final extension at 72°C for 7 min in a Thermal Cycler PTC225 (MJ Research, Watertown, MA, USA). The amplified fragments were combined and concentrated to 60 μl using Microcon ® YM-30 Centrifugal Filter Devices (Millipore Corporation, Bedford, MA, USA).
SNPs and primers
Ten SNPs located in coding regions of genes known to be expressed in HUVEC and HAEC cells were analysed. Information on the SNPs, including dbSNP [35] ID number and nucleotide variation is given [see Additional file 1] together with the sequences of the minisequencing primers. The primers for PCR and minisequencing were designed using the Oligo Primer Analysis software v6.65 (Molecular Biology Insights Inc., Cascade, CO, USA).
Preparation of microarrays
The minisequencing primers or the complementary tag-oligonucleotides were covalently immobilised on CodeLink™ Activated Slides (Amersham Biosciences, Uppsala, Sweden) by the mediation of a NH2-group in their 5'- or 3'-end, respectively. The oligonucleotides were applied in duplicates to the slides at a concentration of 25 μM in 150 mM sodium phosphate pH 8.5 using a ProSys 5510A instrument (Cartesian Technologies Inc, Irvine. CA, USA) equipped with one Stealth Micro Spotting pin (SMP3B, TeleChem International Inc., Sunnyvale, CA, USA) to minimise the variation between spots in different "subarrays". The oligonucleotides were spotted in an "array-of-arrays" configuration that facilitates analysis of 80 individual samples in parallel on each microscope slide [24]. In each "subarray" a fluorophore-labelled oligonucleotide was included as a control for the immobilisation process. A reference oligonucleotide, complementary to a synthetic template included in the minisequencing reaction mixtures to monitor the difference in incorporation efficiency of the four nucleotides by the DNA polymerase, was also included in each "subarray". Finally, an oligonucleotide designed not to hybridise to any of the oligonucleotides present in the reaction mixture was included in each "sub-array" to be used for background corrections. After printing, the slides were incubated in a humid chamber for at least 24 hours, followed by treatment with ethanolamine according to the manufacturer's instruction. The slides were then stored desiccated in the dark until use.
Minisequencing using immobilised primers (Method I)
Aliquots of 7.5 μl of the concentrated PCR products were analysed in five parallel "subarrays" for each sample, essentially as described previously [18]. The PCR products were allowed to anneal to the immobilised oligonucleotides. After washing, the extension reactions were performed with 0.75 U of Thermo Sequenase™ DNA polymerase (Amersham Biosciences, Uppsala, Sweden) and 0.35 μM Texas Red-ddATP, Tamra-ddCTP, R110-ddGTP and Cy5-ddUTP (Perkin Elmer Life Sciences, Boston, MA, USA) in Thermo Sequenase™ reaction buffer in a total volume of 15 μl, followed by washing of the slide.
Minisequencing using "tag-arrays" (Method II)
Five parallel reactions with a 4.5 μl aliquot of the concentrated PCR products were analysed for each sample, as described in detail in [23]. Excess of PCR primers and dNTPs were removed by treatment with 5 U of exonuclease I and 1 U of shrimp alkaline phosphatase (USB Corporation, Cleveland, OH, USA). The cyclic minisequencing reactions were performed in the presence of the 20 tagged primers at 10 nM concentration, 0.1 μM Texas Red-ddATP, Tamra-ddCTP and R110-ddGTP, 0.2 μM Cy5-ddUTP (Perkin Elmer Life Sciences, Boston, MA, USA) and 1 U of Thermo Sequenase™ DNA polymerase (Amersham Biosciences, Uppsala, Sweden) for 55 cycles of 95°C and 55°C for 20 s each. The extension products were allowed to anneal to the immobilised complementary tag oligonucleotides at 42°C for 2.5 hours followed by washing of the slide.
Solid-phase minisequencing in a microtiter plate format
PCR was run with one of the primers biotinylated. The biotinylated PCR products were immobilised in a microtiter plate coated with streptavidin (Combiplate 8, Labsystems, Helsinki, Finland) and the unbiotinylated strand was removed with alkali treatment [9,15]. The minisequencing mixture, containing the appropriate tritium labelled dNTP (Amersham Biosciences, Uppsala, Sweden), AmpliTaq ® DNA polymerase (Applied Biosystems, Foster City, CA, USA) and the minisequencing primer was added. The extension reaction was allowed to proceed for 10 min at 50°C. The extended primers were released with alkali and the amount of incorporated tritium labelled nucleotide was measured.
Hybridisation with allele-specific TaqMan probes
Primers and probes for the TaqMan assays were designed by Applied Biosystems as Assay-by-Design (rs1042719 ADRB2 and rs5925 LDLR) or Assay-on-Demand (rs1433099 LDLR) service. The probes for the two alleles were labelled with the reporter dyes FAM and VIC respectively. The sequences of the primers and probes for the SNPs rs5925 LDLR and rs 1042719 ADRB2 are found in [Additional file 1]. The primer and probe sequences for the SNP rs1433099 LDLR were not made available to us by ABI since this SNP is included in their Assay-on-Demand program.
Real time quantitative PCR was run in 25 μl TaqMan Universal PCR Master Mix (Applied Biosystems) with 200 nM of both labelled TaqMan probes, 900 nM PCR-primers and 10 ng genomic DNA or one tenth of the cDNA products. The PCR conditions were initial activation of the enzyme at 95°C for 10 min followed by 60 cycles of 95°C for 15 sec and 60°C for 1 min in a ABI7000 instrument (Applied Biosystems, Foster City, CA, USA).
The signal intensity ratios were calculated based on normalised ΔRn fluorescence values obtained from the assay during the exponential phase of PCR. The ΔRn values were retrieved from cycle 38 for the SNP rs1042719 ADRB2, cycle 42 for the SNP rs5925 LDLR and cycle 43 for the SNP rs1433099 LDLR. Imbalanced expression of the SNP alleles was determined by a t-test as described below.
Signal detection and data analysis
In Methods I and II fluorescence was measured using a ScanArray ® Express instrument (Perkin Elmer Life Sciences, Boston, MA, USA) with the excitation lasers Blue Argon 488 nm, Green HeNe 543.8 nm, Yellow HeNe 594 nm and Red HeNe 632.8 nm with the laser power set to 80% and the photomultiplier tube gain adjusted to obtain equal signal intensities from reaction control spots for all four spectra. The fluorescence signals were extracted using the QuantArray ® analysis 3.1 software (Perkin Elmer Life Sciences, Boston, MA, USA). The mean of the fluorescence signals for the duplicate spots was corrected for the average background in each "sub-array" separately. The data was handled and interpreted using the Microsoft ® Excel program.
The genotype for each individual SNP was assigned by calculating a ratio between the fluorescence signals for the two alleles. Coefficients of determination (R2) were assigned by linear regression analysis of the relationship between the signal intensity ratios determined from the minisequencing assay and the known allelic ratios in the mixed samples for the quantification standard curves. Two-sample t-tests with two-tailed significance levels assuming unequal variance were performed to determine the lowest level of detection of a specific allele for the quantification standard curves and to evaluate the imbalanced expression of the two alleles of the SNPs in the cell lines.
Authors' contributions
UL participated in the design of the study and in RNA and DNA extraction, and performed all the laboratory work involving "in-house" minisequencing methods, performed the statistical calculations and drafted the manuscript. MF cultured the cells, performed RNA and DNA extraction, performed the assays with the reference method, and provided input to the manuscript. AD performed the assays using the SNPstream system. A-CS conceived the study, participated in its design, coordination and in preparation of the manuscript. All authors read and approved the final manuscript.
Supplementary Material
Additional File 1
Additional file1 is a pdf-file with information on the SNPs, including dbSNP ID number, nucleotide variation and the sequences of the primers and probes used in the microarray based minisequencing and TaqMan assays respectively.
Click here for file
Acknowledgements
We acknowledge Raul Figueroa for array production, Ann-Christin Wiman for assistance with the SNPstream assays and David Fange for programming Microsoft ® Excel macros. Financial support was provided by the Swedish Research Council (VR) and the K&A Wallenberg foundation via Wallenberg Consortium North.
Figures and Tables
Figure 1 Regression lines displaying accuracy and sensitivity of quantitative genotyping of SNPs. The regression lines were obtained by analysing ten SNPs in a series of mixed samples with varying amounts of DNA from two individuals of different genotype. The signal intensity ratios from minisequencing using immobilised primers (Method I, black diamonds) and "tag-array" minisequencing (Method II, grey squares) are plotted as a function of the known allelic ratios in the mixed samples. The SNP names are given in the panels. The signal intensity ratios are mean values of five replicate reactions. The signal intensity ratios obtained in homozygous samples (allele ratios zero) are indicated as black diamonds and grey squares on the left vertical axis of each panel.
Table 1 SNP genotypes of the DNA samples used for preparing the dilution series for the quantification curves.
SNPa Sample 1 Sample 2 Allele rangeb
rs4331 ACE TT CC 0–100% T
rs1042713 ADRB2 AA GG 0–100% A
rs1042714 ADRB2 CC CG 50–100% C
rs1042718 ADRB2 CC CA 50–100% C
rs1042719 ADRB2 CG GG 0–50% C
rs1799983 NOS3 CC CA 50–100% C
rs5351 EDNRB GA AA 0–50% G
rs5925 LDLR TT CC 0–100% T
rs5930 LDLR TC CC 0–50% T
rs1433099 LDLR TC CC 0–50% T
a SNP name consisting of dbSNP ID number and gene name acronym.
b The percentages of the allele from Sample 1 in the dilution series with the 0–100% allele range were: 0%; 2.1%; 4.9%; 7.5%; 11.6%; 17.9%; 27.5%; 42.3%; 65%; 100%. The corresponding percentages in the 0–50% allele range were 0%; 1.0%; 2.5%; 3.8%; 5.8%; 8.9%; 13.7%; 21.1%; 32.5%; 50%, and in the 50–100% allele range they were 50%; 51.3%; 52.5%; 53.8%; 55.8%; 58.9%; 63.7%; 71.1%; 82.%; 100%.
Table 2 Results for the regression lines describing the accuracy and limit of detection for the methods when analysing mixed samples of a dilution series.
SNPa Methodb R2c Detection sensitivity (%)d p < 0.05 p-valuee
rs4331 ACE I 0.95 7.5 0.0076
II 0.97 4.9 0.0107
rs1042713 ADRB2 I 0.99 4.9 0.00051
II 0.98 2.1 0.000077
rs1042714 ADRB2 I 0.96 14 * 0.042
II 0.98 8.9 * 0.047
rs1042718 ADRB2 I 0.99 14 * 0.020
II 0.99 5.8 * 0.0011
rs1042719 ADRB2 I 0.90 5.8 0.0072
II 0.96 1.0 0.013
rs1799983 NOS3 I 0.99 8.9 * 0.0015
II 0.97 3.8 * 0.023
rs5351 EDNRB I 0.87 5.8 0.016
II 1.0 1.0 0.000016
rs5925 LDLR I 1.0 4.9 0.014
II 0.98 4.9 0.018
rs5930 LDLR I 0.94 8.9 0.00030
II 0.63 33 0.011
rs1433099 LDLR I 0.75 ND ND
II 0.88 5.8 0.017
a dbSNP ID number and gene name acronym.
b Minisequencing using (I) immobilised primers; (II) cyclic primer extension and "tag-arrays".
c Coefficient of determination describing the fit between the regression lines in Figure 1, and the data points.
d Level at which the minority SNP allele can be detected. The percentages correspond to the mixture with a signal intensity ratio significantly different (p < 0.05) from the signal intensity ratio of the corresponding homozygous or heterozygous (*) sample. ND: Not possible to determine due to scatter of the data points.
e p-value for difference between signal ratios at the detection level and at 0% of the minority allele.
Table 3 Fluorescence signals obtained by genotyping six SNPs in RNA (cDNA) and genomic DNA from the HUVEC and HAEC cells using the two minisequencing methods.
Fluorescence signal (CV %)b Fluorescence signal ratioc
cDNA gDNA cDNA gDNA
SNPa Method Allele 1 Allele 2 Allele 1 Allele 2
rs4331 I 17975 (31%) 4428 (20%) 17568 (12%) 10291 (10%) 4.1 1.7
ACE II 20447 (37%) 21796 (11%) 8928 (35%) 26913 (18%) 0.94 0.33
rs1042719 I 14368 (12%) 28538 (25%) 21179 (11%) 22192 (12%) 0.50 0.95
ADRB2 II 11475 (3.6%) 34346 (5.4%) 18495 (7.4%) 39569 (8.6%) 0.33 0.47
rs5351 I 11026 (10%) 1743 (15%) 7344 (16%) 2077 (17%) 6.3 3.5
EDNRB II 65257 (0%) 7484 (14%) 64360 (3.3%) 11552 (20%) 8.7 5.6
rs5925 I 13984 (25%) 16040 (5.7%) 9586 (32%) 9329 (20%) 0.87 1.0
LDLR II 3504 (27%) 9582 (14%) 1951 (10%) 7113 (8.2%) 0.37 0.27
rs5930 I 5680 (13%) 5028 (13%) 7207 (22%) 9244 (17%) 1.1 0.78
LDLR II 5410 (31%) 9965 (15%) 3261 (27%) 11183 (9.0%) 0.54 0.29
rs1433099 I 8806 (20%) 4594 (10%) 7307 (32%) 3646 (23%) 2.0 2.0
LDLR II 2550 (41%) 4507 (16%) 1727 (36%) 3743 (13%) 0.57 0.46
a rs4331 ACE, rs1042719 ADRB2 and rs5351 EDNRB were analysed in HUVEC and rs5925 DLR, rs5930 LDLR and rs1433099 LDLR were analysed in HAEC.
b Mean values and coefficient of variation (CV) of five parallel minisequencing assays from the same PCR product.
c The homozygous signal intensity ratios were 0.0027 for rs4331 ACE, 0.0094 for rs1042719 ADRB2, and 0.10 for rs5351 EDNRB in gDNA from HAEC, and 0.0069 for rs5925 LDLR, 0.0056 for rs5930 LDLR, and 0.014 for rs1433099 LDLR in gDNA from HUVEC.
Table 4 Fluorescence signal intensity ratios obtained by genotyping six SNPs in RNA (cDNA) and genomic DNA from the HUVEC and HAEC cells using the two microarray-based minisequencing methods and two reference methods.
Fluorescence signal ratio Difference in cDNA and gDNA ratios
(p-value) cDNA/gDNAc
SNPa Methodb cDNA gDNA
rs4331 ACE I 4.1 1.7 0.00095 2.4 (1.9–2.8)
II 0.94 0.33 0.0060 2.8 (1.8–4.0)
rs1042719 ADRB2 I 0.50 0.95 0.000025 0.54 (0.47–0.69)
II 0.33 0.47 0.0070 0.71 (0.67–0.75)
TM 0.086 0.19 <0.0001 0.47 (0.31–0.56)
MS 0.48 0.99 <0.0001 0.48 (0.34–0.62)
rs5351 EDNRB I 6.3 3.5 0.00014 1.8 (1.6–2.0)
II 8.7 5.6 0.0030 1.6 (1.4–1.8)
rs5925 LDLR I 0.87 1.0 0.33 0.85 (0.63–1.1)
II 0.37 0.27 0.029 1.4 (1.1–1.5)
TM 3.3 1.4 0.15 2.4 (1.4–5.6)
MS 0.36 0.28 0.027 1.3 (0.98–1.5)
rs5930 LDLR I 1.1 0.78 0.035 1.5 (1.1–1.9)
II 0.54 0.29 0.0030 1.8 (1.5–2.3)
rs1433099 LDLR I 2.0 2.0 0.66 0.96 (0.84–1.1)
II 0.57 0.46 0.33 1.2 (0.65–1.5)
TM 1.0 0.49 <0.0001 2.0 (1.9–2.1)
MS 0.47 0.32 0.060 1.5 (1.0–2.0)
a rs4331 ACE, rs1042719 ADRB2 and rs5351 EDNRB were analysed in HUVEC and rs5925 LDLR, rs5930 LDLR and rs1433099 LDLR were analysed in HAEC.
b Minisequencing using (I) immobilised primers; (II) cyclic primer extension and "tag-arrays", TaqMan (TM) and solid-phase minisequencing (MS). TM-assays were not possible to design using the Assay-by-Design and Assay-on-Demand service at ABI for the remaining three SNPs, MS was performed for the SNPs with working TM-assays.
c Mean and range is given for five parallel reactions.
Table 5 Test of allelic imbalance in replicate RNA-preparations and cDNA samples.
cDNA/gDNA ratios in replicate RNA preparations a
rs4331 ACE rs1042719 ADRB2 rs5351 EDNRB
HUVEC 2.8 (1.4 – 4.0) 0.48 (0.34 – 0.62) 1.2 (1.1 – 1.4)
rs5925 LDLR rs5930 LDLR rs1433099 LDLR
HAEC 1.3 (0.98 – 1.5) 1.6 (1.0 – 2.3) 1.5 (1.0 – 2.0)
cDNA/gDNA ratios in replicate cDNA samples b
rs5925 LDLR rs5930 LDLR rs1433099 LDLR
HAEC 1.5 (1.3 – 1.7) 1.9 (1.7 – 2.4) 1.1 (0.87 – 1.4)
Average cDNA/gDNA ratios from five replicate experiments with range of variation in parantheses.
a cDNA synthesised from RNA extracted from five different cell batches analysed in duplicate using solid-phase minisequencing assays in a microtiter plate format.
b Five replicate cDNA samples were reverse transcribed from the same RNA preparation and analysed in duplicate assays.
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The NCBI SNP database
| 15500681 | PMC529269 | CC BY | 2021-01-04 16:02:56 | no | BMC Biotechnol. 2004 Oct 22; 4:24 | utf-8 | BMC Biotechnol | 2,004 | 10.1186/1472-6750-4-24 | oa_comm |
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BMC BiotechnolBMC Biotechnology1472-6750BioMed Central London 1472-6750-4-261550713410.1186/1472-6750-4-26Methodology ArticleEfficient activation of gene expression using a heat-shock inducible Gal4/Vp16-UAS system in medaka Grabher Clemens [email protected] Joachim [email protected] Developmental Biology Program, European Molecular Biology Laboratory (EMBL), 69117-Heidelberg, Germany2004 26 10 2004 4 26 26 20 7 2004 26 10 2004 Copyright © 2004 Grabher and Wittbrodt; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Genetic interference by DNA, mRNA or morpholino injection is a widely used approach to study gene function in developmental biology. However, the lack of temporal control over the activity of interfering molecules often hampers investigation of gene function required during later stages of embryogenesis. To elucidate the roles of genes during embryogenesis a precise temporal control of transgene expression levels in the developing organism is on demand.
Results
We have generated a transgenic Gal4/Vp16 activator line that is heat-shock inducible, thereby providing a tool to drive the expression of specific effector genes via Gal4/Vp16. Merging the Gal4/Vp16-UAS system with the I-SceI meganuclease and the Sleeping Beauty transposon system allows inducible gene expression in an entirely uniform manner without the need to generate transgenic effector lines. Combination of this system with fluorescent protein reporters furthermore facilitates the direct visualization of transgene expressing cells in live embryos.
Conclusion
The combinatorial properties of this expression system provide a powerful tool for the analysis of gene function during embryonic and larval development in fish by ectopic expression of gene products.
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Background
The most widely used strategies to investigate the function of genes in medaka (Oryzias latipes) are the analyses of mutants, miss-expression of wild type genes or their variants by mRNA injection and gene specific translational inhibition by morpholino injections [1-3]. However, the phenotype of a given mutation mainly reflects the first temporal function of the affected gene in embryonic development, obscuring possible later functions. Similarly, mRNA and morpholinos exert their functions immediately following injection, providing information only on the early role of the gene of interest. A detailed analysis of gene function in a given process can thus be a difficult task. The Gal4/UAS system provides an alternative and more specific strategy to analyze specific functions of a gene [4,5]. The direct application of the Drosophila Gal4-UAS approach, by the generation of transgenic lines, has been established successfully in zebrafish [6,7]. However, the generation of different transgenic activator and effector lines may be a time- and space-consuming task, and expression levels in these transgenic lines are weak, probably due to a limited transactivation potential of Gal4 in fish. Gal4/Vp16, a fusion of the yeast Gal4 DNA-binding domain with the strong Vp16 transactivation domain of the herpes simplex virus [8] can be used to enhance transactivation efficacy. Yet, strong transcriptional activators can cause unspecific promoter squelching [9] resulting in retardation of embryogenesis [10]. Nonetheless, the Gal4/Vp16-UAS system has been used in zebrafish in transient approaches resulting in mosaic, but easily detectable transgene expression [11].
We have applied the Gal4/Vp16-UAS system for transient transactivation in a heat-shock inducible transgenic Gal4/Vp16 activator line. Generation of transgenic medaka lines, which allow the induction of the Gal4/Vp16 activator to 'physiological' (i.e. non-toxic) levels was achieved by using a 5' truncated version of the zebrafish heat-shock promoter HSP70 [12]. Using a heat-shock promoter to drive expression of the Gal4/Vp16 activator allows tight temporal control of activator and effector (reporter) gene expression. To trace transgene expression in cells of living embryos we have used the cyan fluorescent and yellow fluorescent proteins (CFP, YFP). Combination with the meganuclease (MN) transgenesis system [13] and the direct-inverted repeats (IR/DR) of the Sleeping Beauty (SB) transposon system [14] yielded high numbers of transgene expressing cells. Thus, in contrast to the entirely mosaic nature of a transient approach reported thus far, the combined use of a transgenic activator line with systems enhancing even DNA distribution or early integration allows uniform expression of injected effector genes upon induction by heat-shock treatment without an immediate need to generate transgenic UAS lines.
Results and discussion
Generation of a heat-shock inducible transgenic Gal4/Vp16 activator line (pCG6.0WCS/T)
DNA injection leading to mosaic expression in G0 allows in vivo tracing of transgene-expressing cells and observation of effects exerted by the transgene through application of fluorescent markers [11]. However, elucidation of biological questions sometimes requires ubiquitous expression of transgenes in a temporally controlled manner. While the MN protocol strongly reduces mosaicism, it does so only in a fraction of injected embryos ([13], Fig. 1F,1G,1H,1I,1J,1K and Table 1). This can be improved by the use of transgenic animals providing inducible and sufficient expression in all cells.
The idea is to combine stable heat-shock inducible expression of the Gal4/Vp16 activator with transient expression of effector genes upon microinjection. The effector constructs are uniformly distributed in the entire embryo due to the presence of the SB direct-inverted repeats [15]. We have designed two activator/reporter vectors containing Gal4/Vp16 under control of a 1.5 kb fragment of the zebrafish (zf) HSP70 promoter (pCG5.0WCS) or a 0.6 kb 5' truncated fragment of zfHSP70, respectively (pCG6.0WCS). Both vectors contain CFP downstream of several UAS elements as an internal reporter. The IR/DRs of SB and two I-SceI meganuclease sites flank this entire expression cassette (Fig. 1A). The internal reporter provides a direct read-out for activator expression. A third vector (pCG3.0Y), containing YFP downstream of several UAS elements and flanked by IR/DRs, was designed as an independent reporter (Fig. 1A).
It has been shown that the Gal4/Vp16 activator can interfere with general transcription by titrating the basal transcription machinery [16]. We observed developmental retardation and malformation in all embryos injected with Gal4/Vp16 driven by ubiquitous promoters. Similarly, co-injection of high concentrations of Gal4/Vp16 mRNA (50 ng/μl) with pCG5.0WCS always resulted in developmental malformations (not shown). However, DNA co-injections did not affect embryonic development in transient experiments when the HSP70 promoter was used to control Gal4/Vp16 expression (Fig. 1B,1B',1F,1G,1H,1I,1J,1K). Moreover, co-injections of low concentrations of Gal4/Vp16 mRNA (3.5 ng/μl) with the activator/reporter construct pCG5.0WCS also showed no effects on embryogenesis (Fig. 1C,1C'), suggesting that the toxicity of the activator depends on the expression level.
The truncated version of the zfHSP70 promoter fragment used in the activator/reporter construct pCG6.0WCS showed a moderate activation upon heat-shock treatment. This allowed adjusting the induction levels by varying the heat-shock duration. A transgenic medaka line was established (by co-injection of circular vector pCG6.0WCS with MN) in which expression levels directly correlated with the heat-shock duration. Extended heat-shocks resulted in very high expression levels, but also caused retardation phenotypes due to the strong transactivation potential of the Gal4/Vp16 fusion protein. Comparable phenotypes were not observed in heat-shock treated wild type embryos. Depending on the developmental stage at the time of induction, the duration of heat-shock treatment was adjusted to induce Gal4/Vp16 and reporter expression without interfering with embryonic development. Induction periods ranged from one minute of heat-shock at 37°C at early stages (~st16/21hpf) to 10 minutes at later stages (~st22/38hpf). On top of uniform CFP expression in the entire embryo and yolk upon heat-shock, some regions of the embryo showed additional responsiveness of the reporter (Fig. 1E,1E').
Microinjection experiments and RT-PCR revealed that reporter gene (CFP) expression in transgenic fish is mediated by Gal4/Vp16. Offspring of pCG6.0-WCS/T transgenic fish was injected with Gal4/Vp16 mRNA (3.5 ng/μl) at the one-cell stage without heat-shock treatment. Injected embryos exhibited uniform expression of CFP shortly after the onset of zygotic transcription at the mid-blastula transition [17], indicating that CFP expression was induced in response to Gal4/Vp16 (Fig. 1D,1D').
We applied RT-PCR for the dose/response analysis of activator and reporter mRNA (Fig. 2A). Transcripts of Gal4/Vp16 were detectable already 10 minutes after a heat-shock of 90 seconds at 37°C. Following a steady increase until about three hours after induction, Gal4/Vp16 messages were degraded between five and ten hours to undetectable levels after twenty hours. CFP mRNA was first detected after two hours and transcript levels were still increasing after 25 h. This indicates that the transcription of the reporter CFP is controlled by Gal4/Vp16 protein and that active Gal4/Vp16 is still present when the amount of its transcripts already dropped below detectable levels (Fig. 2A).
Activation of an independent reporter upon injection into the transgenic activator line pCG6.0WCS/T
We tested the Gal4/Vp16 activator line pCG6.0WCS/T as a tool to induce expression of an independent reporter upon injection of plasmid DNA (pCG3.0Y). Transgenic embryos were injected with different concentrations of the reporter pCG3.0Y (5–150 ng/μl). Injected embryos were subjected to heat-shock treatment at different developmental stages for various periods of time, kept at 28°C thereafter and monitored for activator and effector expression during the following days. Due to the SB IR/DRs flanking the expression cassette, the independent reporter was distributed equally in the entire embryo resulting in ubiquitous expression of YFP, entirely co-localizing with the internal reporter (CFP). Additional mosaic clones of cells expressing YFP at higher levels presumably reflect higher plasmid concentrations in these cells (Fig. 2B,2C,2D,2E,2F,2G,2H,2I,2J and Table 1). However, YFP expression levels appeared relatively independent from the DNA concentration, but were directly correlated to the expression levels of the activator or internal reporter, respectively.
Conclusions
Here we show that the Gal4/Vp16-UAS transactivation system can be efficiently used in medaka. By using fluorescent proteins as internal or independent reporter, cells co-expressing the activator and the gene of interest can be visualized directly. Transparency of these fish embryos allows the evaluation of the cellular fate and response to ectopically expressed genes by time-lapse analyses. The combination with inducible promoters permits temporal control of effector gene expression and enables the modulation of the response intensity by adjusting the duration of the heat-shock treatment. This inducible system can be used in transient experiments to study the behavior of transgene expressing cells in an otherwise wild type environment. The combination with the MN and SB system offers to tailor a range of different levels of mosaicism (Fig. 1F,1G,1H,1I,1J,1K). A transgenic Gal4/Vp16 activator line was generated, which provides a powerful tool to induce activator and effector gene expression in a ubiquitous manner at a given time-point (Fig. 1E,1E'). When used in microinjection approaches of reporter vectors containing IR/DRs, our transgenic activator line allows ubiquitous and uniform expression of the reporter gene without the need to generate transgenic effector (UAS) lines (Fig. 2B,2C,2D,2E,2F,2G,2H,2I,2J). In addition to temporal control mediated by the heat-shock promoter, induction using a focused laser-beam [12] could provide precise spatial control of the effector gene expression.
Methods
Plasmids
pCG3.0Y
A YFP/SV40pA cassette was cloned downstream of a 4xUAS/dHSP70 element (non responsive to heat-shock; kind gift of M. Gonzalez-Gaitan). This entire cassette was cloned into pCG1.1 containing the IR/DR sequences of the SB transposable element [14] resulting in a 5.1 kb plasmid containing the reporter cassette (4xUAS/dHSP70/YFP/SV40pA) and the pBSII backbone flanked by a left and right IR/DR of SB (Fig. 1).
pCG5.0WCS
A 1.5 kb zebrafish HSP70 promoter fragment [12] was subcloned upstream of Gal4/Vp16/SV40pA. The entire cassette was further subcloned into pCG3.0C (containing CFP instead of YFP, see above) resulting in a 8.7 kb plasmid (pCG5.0C) containing the expression cassettes zfHSP70/Gal4/Vp16/SV40pA followed by 4xUAS/CFP/SV40pA flanked by the IR/DRs of SB. Finally, the expression cassettes including the inverted repeats were cloned into a I-SceI backbone vector [13] and verified by sequencing (Fig. 1).
pCG6.0WCS
The 1.5 kb zfHSP70 promoter fragment in pCG5.0WCS was replaced by a truncated zfHSP70 promoter fragment lacking 900 bp 5' to the internal BamHI site resulting in a 7.8 kb plasmid that was verified by sequencing (Fig. 1). Further structural details of the activator and reporter vectors are available upon request.
pCGGal4/Vp16
A Gal4/Vp16 fusion construct (Gal4 DNA binding domain: amino acids (aa) 1–147 and Vp16 transactivation domain: aa 411–491) was designed from Clontech vectors pM (Gal4) and pM3-VP16 (Vp16) and cloned into pCS2+ [18].
Gal4/Vp16 mRNA was transcribed in vitro from pCGGal4/Vp16 using the mMessage mMachine kit (Ambion Inc.).
Microinjection and heat-shock treatment of medaka embryos
For microinjections, one-cell stage embryos of the Cab inbred strain were used. Microinjection capillaries were backfilled with the injection solution [DNA (5–150 ng/μl); Yamamoto buffer (1×) or DNA (5 ng/μl); Yamamoto buffer (0.5×); I-SceI buffer (0.5×, New England Biolabs); I-SceI meganuclease (0.35 u/μl, New England Biolabs) with or without Gal4/Vp16 mRNA (3.5–50 ng/μl)]. DNA was prepared using a Qiagen Maxiprep kit (Qiagen, USA) and dialyzed using nitrocellulose filters (#VSW01300; Millipore, USA). DNA was injected through the chorion into the cytoplasm of one-cell stage embryos. Heat-shock treatment was performed in small volumes (100–200 μl) using a waterbath at 37°C. Animals used in the study were kept according to national and international ethical provisions for animal husbandry as implemented at EMBL.
Microscopy
Embryos were observed and scored using a MZFLIII dissecting microscope with a 436/20 nm (EF); 480/40 nm (BF) filter set for CFP, a 510/20 nm (EF); 560/40 nm (BF) filter set for YFP and a 360/40 nm (EF); 420 nm (BF) filter set for UV/Brightfield. The stereomicroscope was equipped with a DC500 digital camera for imaging (Leica Microsystems, Germany).
RNA isolation and RT-PCR
Transgenic embryos were heat-shocked and subsequently kept at 28°C to recover for different periods of time. Total RNA was isolated from individual embryos as described [19]. Total RNA was subjected to reverse transcription (SuperscriptII, Gibco-BRL) using a mixture of random hexamer primers (25 μM, Amersham) and gene specific oligomeres for Gal4/Vp16 (25 μM; 5'-CCACGTCCAAAGCCCCATAC-3') and CFP (25 μM; 5'-GTTCATCCATGCCATGTGTAATCCC-3') in a 20 μl reaction. 2 μl of each RT reaction was used for PCR in a 50 μl reaction. The primer pairs used were Gal4/Vp16up2 (5'-GATAATGTGAATAAAGATGCCGTCA-3') and Gal4/Vp16low2 (5'-CCACGTCCAAAGCCCCATAC-3') to amplify a 420 bp fragment, and CFPup2 (5'-TCAAGGAGGACGGCAACATC-3') and CFPlow2 (5'-GTTCATCCATGCCATGTGTAATCCC-3') to amplify a 320 bp fragment. Amplification of a 580 bp fragment of c-actin was used as an internal control using the intron-spanning primer pair c-actinup2 (5'-GCCGCGACCTTACAGACTACCT-3') and c-actinlow2 (5'-CTGTTTAGAAGCATTTGCGGTGGAC-3'). An initial 1 min. denaturation step at 95°C was followed by an additional denaturation step for 30 sec. at 95°C, annealing for 30 sec. at 60°C and elongation at 72°C for 30 sec. The program was repeated for 30 cycles followed by a final extension step for 5 min. at 72°C.
Authors' contributions
CG designed and performed all experiments and drafted the manuscript. JW conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.
Acknowledgements
We thank J. R. Martinez-Moralez and F. Loosli for continuous discussions and helpful suggestions and all members of the Wittbrodt lab for critically reading the manuscript. We thank A. Krone and B. Wittbrodt for technical assistance and E. Grzebisz and A. Nowicka for fish husbandry. This work was supported by grants from the EC and the HFSP (J.W.).
Figures and Tables
Figure 1 Schematic representation of Gal4/Vp16-UAS expression vectors (A), comparison of induction levels between transient and transgenic approaches (B-E') and transient Gal4/Vp16 mediated reporter expression in medaka embryos induced by heat-shock (F-K). A; Gal4/Vp16 activator units driven by a 1.5 kb (pCG5.0WCS) or a 600 bp (pCG6.0WCS) heat-shock promoter fragment followed by a SV40 polyadenylation signal are shown on the left. Reporter units are separated from the activator units by the pBSII backbone. YFP (pCG3.0Y) or CFP (pCG5.0WCS/6.0WCS) open reading frames are placed downstream of 4 UAS elements and followed by a SV40 polyadenylation signal. Entire expression cassettes are flanked by the IR/DRs of the SB transposon system. Additionally, I-SceI meganuclease sites flank the expression cassettes of activator vectors. Abbreviations and actual sizes of each vector are given. B-E'; Wild type medaka embryos were injected with pCG5.0WCS and MN and subjected to heat-shock treatment (B, B') or with MN and Gal4/Vp16 mRNA without treatment (C, C'). Similarly, the transgenic activator line pCG6.0WCS/T was injected with Gal4/Vp16 mRNA (D, D') or subjected to heat-shock treatment (E, E'). Anterior is to the top (B-E'). DNA and RNA concentrations are indicated together with the developmental stage and the duration of heat-shock treatment. Microinjection of the activator/reporter plasmid pCG5.0WCS results in activation of CFP according to the distribution of plasmid DNA (B-C'). In contrast, induction of activator and reporter in the transgenic line by mRNA injection or heat-shock treatment results in ubiquitous and entirely uniform expression of CFP (D-E'). F-K: Activator and reporter vectors were injected into one-cell stage medaka embryos. Anterior is to the left (F-K). Developmental stage at heat-shock induction and duration of treatment is indicated. HS treatment of up to 2 hours did not interfere with embryonic development but yielded detectable transgene expression (F-K). CFP (internal reporter) shows the expression pattern of the activator Gal4/Vp16. YFP shows activation of the independent reporter. Co-injection of activator pCG5.0WCS and independent reporter pCG3.0Y (100 ng/μl each) resulted in mosaic activation of reporter gene expression (F-H) only. Co-injection of activator pCG5.0WCS (5 ng/μl) and independent reporter pCG3.0Y (100 ng/μl) with I-SceI resulted in a broad range of different levels of mosaicism. Notably, 16% of co-injected embryos showed highly uniform expression (I-K). Abbreviations: CFP, cyan fluorescent protein; HS, heat-shock; hpf, hours post fertilization; IR/DR, inverted/direct repeats; MN, meganuclease; pA, SV40 polyadenylation signal; pBS, pBluescriptII; st, developmental stage; UAS, upstream activating sequence; zf, zebrafish.
Figure 2 Kinetics of activator and reporter induction (A) and activation of an independent reporter in pCG6.0WCS/T (B-J). A; Transcriptional induction of the activator Gal4/Vp16 and the internal reporter (CFP) in pCG6.0WCS/T was analyzed by RT-PCR. Embryos were heat-shock for 90 seconds at st20/31.5hpf and were allowed to recover for the indicated periods of time. Activator transcripts were detectable already 10 minutes after induction and the levels increased up to 3 hours. Degradation to undetectable levels was complete after 20 hours. Activator dependent transcription of the internal reporter CFP was observed only after 2 hours and levels were still increasing after 25 hours. C-actin was used as an internal control. (B-J); The independent reporter plasmid pCG3.0Y was injected into the transgenic Gal4/Vp16 activator line. Anterior is to the left (B-J). Expressions of both internal and independent reporters were observed in a weak to strong ubiquitous manner (B-J). Occasional higher levels of internal reporter expression were observed in some parts of the embryonic body. These higher levels were paralleled by independent reporter expression (B-D, H-J). Mosaic clones exerting stronger YFP expression reflect locally higher plasmid concentrations (B-J). Abbreviations: h, hours; hpf, hours post fertilization; HS, heat-shock; M, size marker; st, developmental stage.
Table 1 Microinjection of reporter vector.
Reporter expression
Vector MN Host Fish before induction1 after induction2 (mosaic) after induction2 (uniform)
pCG5.0WCS pCG3.0Y + wildtype 74 52 61/61 (82%) 12/12 (16%)
pCG3.0Y - pCG6.0WCS/T3 162 29 -/- 73/73 (100%)
1...Leaky expression was mainly observed in the yolk only
2...Expression of internal (CFP) and independent (YFP) effector in the embryonic body was scored separately; CFP/YFP
3...Injection was performed in offspring of heterozygous carriers mated to wildtype fish, resulting in ~50% transgenic embryos
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| 15507134 | PMC529270 | CC BY | 2021-01-04 16:02:57 | no | BMC Biotechnol. 2004 Oct 26; 4:26 | utf-8 | BMC Biotechnol | 2,004 | 10.1186/1472-6750-4-26 | oa_comm |
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BMC Complement Altern MedBMC Complementary and Alternative Medicine1472-6882BioMed Central London 1472-6882-4-141549623110.1186/1472-6882-4-14Research ArticlePractice patterns of naturopathic physicians: results from a random survey of licensed practitioners in two US States Boon Heather S [email protected] Daniel C [email protected] Janet [email protected] Karen J [email protected] Bruce [email protected] Jennifer [email protected] Elaine H [email protected] Michael J [email protected] Richard A [email protected] David M [email protected] Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada2 Center for Health Studies, Group Health Cooperative, Seattle, Washington, USA3 Bastyr University, Naturopathic Medicine Program, Kenmore, Washington, USA4 Private Practice, Olympia, Washington, USA5 Centers for Disease Control, Vessel Sanitation Program, Atlanta, Georgia, USA6 Natural Health Products Directorate, Health Canada, Ottawa, Ontario, Canada7 University of Washington Departments of Medicine and Health Services, Seattle, Washington, USA8 Beth Israel-Deaconess Center for Alternative Medicine Research and Education, Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA2004 20 10 2004 4 14 14 24 6 2004 20 10 2004 Copyright © 2004 Boon et al; licensee BioMed Central Ltd.2004Boon et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Despite the growing use of complementary and alternative medicine (CAM) by consumers in the U.S., little is known about the practice of CAM providers. The objective of this study was to describe and compare the practice patterns of naturopathic physicians in Washington State and Connecticut.
Methods
Telephone interviews were conducted with state-wide random samples of licensed naturopathic physicians and data were collected on consecutive patient visits in 1998 and 1999. The main outcome measures were: Sociodemographic, training and practice characteristics of naturopathic physicians; and demographics, reasons for visit, types of treatments, payment source and visit duration for patients.
Result
One hundred and seventy practitioners were interviewed and 99 recorded data on a total of 1817 patient visits. Naturopathic physicians in Washington and Connecticut had similar demographic and practice characteristics. Both the practitioners and their patients were primarily White and female. Almost 75% of all naturopathic visits were for chronic complaints, most frequently fatigue, headache, and back symptoms. Complete blood counts, serum chemistries, lipids panels and stool analyses were ordered for 4% to 10% of visits. All other diagnostic tests were ordered less frequently. The most commonly prescribed naturopathic therapeutics were: botanical medicines (51% of visits in Connecticut, 43% in Washington), vitamins (41% and 43%), minerals (35% and 39%), homeopathy (29% and 19%) and allergy treatments (11% and 13%). The mean visit length was about 40 minutes. Approximately half the visits were paid directly by the patient.
Conclusion
This study provides information that will help other health care providers, patients and policy makers better understand the nature of naturopathic care.
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Background
The number of Americans using complementary and alternative medicine (CAM) rose from 34% to 42% from 1990 to 1997, with annual spending for CAM therapies in excess of $21 billion [1]. The majority of the research on the use of CAM services has focused on patients or consumers of CAM. Several patient characteristics have been found to be associated with increased CAM use including female gender, child-bearing age, above average income, above average education and diagnosis with a chronic or life-threatening condition [1-4]. Reasons why consumers seek care from CAM practitioners have also been identified. These include: the failure of conventional treatment to alleviate symptoms; psychological congruence between the individual's belief system and the CAM therapy; individuals' expectations that use of CAM will empower them by offering a greater sense of control over personal health care decisions; adverse effects of conventional therapies; and dissatisfaction with the care of conventional practitioners [1-3,5-8].
Although we now have some understanding of who seeks CAM care and why, relatively little is known about the actual practices of CAM practitioners including the type of patients they treat, the kinds of therapies they use, and the numbers of patients they see each day. In addition, it is unclear if the practice patterns of CAM practitioners vary by state. The objective of this paper is to describe and compare the practice of naturopathic physicians in two US states: Washington and Connecticut.
Naturopathic medicine
Naturopathic medicine is a system of health care, based on the teachings of Benedict Lust [9], with a primary goal of enhancing the individual's innate self-healing ability by employing a variety of natural and largely non-invasive healing modalities. In some states, naturopathic physicians are authorized to employ more invasive interventions (i.e., prescription drugs, and minor surgery). Naturopathic medicine is defined by the American Association of Naturopathic Physicians (AANP) as:
... a distinct system of primary health care – an art, science, philosophy and practice of diagnosis, treatment and prevention of illness. Naturopathic medicine is distinguished by the principles upon which its practice is based. These principles are continually reexamined in the light of scientific advances. The techniques of naturopathic medicine include modern and traditional, scientific and empirical methods [10].
Naturopathic physicians are trained as generalists with expertise in a variety of core treatment methods including nutrition, hydrotherapy, colonic irrigation, physiotherapy, naturopathic manipulation, botanical medicine, homeopathy, pharmacology and minor office surgical procedures. Some licensed naturopathic physicians are also trained in traditional Chinese medicine, acupuncture and Ayurvedic medicine as well as clinical specialties such as natural childbirth [9,11-14]. There are currently six North American schools of naturopathic medicine whose graduates are eligible to sit state licensing examinations: Bastyr University (Seattle, WA), the Canadian College of Naturopathic Medicine (Toronto, ON, Canada), National College of Naturopathic Medicine (Portland, OR), Southwest College of Naturopathic Medicine (Tempe, AZ), the University of Bridgeport College of Naturopathic Medicine (Bridgeport, CT) (has applied for accreditation candidacy with the Council on Naturopathic Medical Education (CNME)), and the West Coast Naturopathic Medical College (Vancouver, BC) (not accredited by the CNME) [9].
Accurate estimates of the number of naturopathic physicians practicing in North America are difficult to obtain. A recent estimate (based on data from licensing bodies) indicated that in 2000 approximately 1300 licensed naturopathic physicians were practicing in the United States and another 500 were practicing in Canada. Naturopathic medicine is a licensed health care profession in twelve US states (Alaska, Arizona, Connecticut, Hawaii, Maine, Montana, New Hampshire, Oregon, Utah, Vermont, Washington, California), Puerto Rico and four Canadian provinces (British Columbia, Manitoba, Ontario and Saskatchewan) [9,15]. In most states and provinces where naturopathic medicine is not regulated, individuals may practice similar therapeutic approaches and/or call themselves naturopaths (whether or not they have been trained at a school for naturopathic medicine) because the term naturopathic medicine is not a restricted term. The number of individuals practicing in unregulated jurisdictions is unknown. In South Carolina and Tennessee, it is illegal to practice naturopathy [9].
Legal regulation of naturopathic medicine varies by state and province. Connecticut and Washington have relatively similar (and representative of the other ten states) requirements for licensure including: Graduation from an accredited four-year naturopathic medical school, successful completion of licensing examination (normally NPLEX), the submission of completed application forms and paying the required fee. In addition, Washington State requires that individuals be of good moral character with no history of unprofessional conduct [9].
The legal scopes of naturopathic practice in Connecticut and Washington are similar and relatively broad compared to the other states that license naturopathic physicians. Naturopathic physicians in these two states can diagnose and treat disease, utilizing a wide range of modalities and specialties including: minor surgery such as suturing (limited in Washington); physical therapies including hydrotherapy, naturopathic manipulation, and physiotherapy; colonic irrigation; electrotherapy (for example TENs); diagnostic x-ray; venipuncture; obstetrics (in Washington a midwifery license is required); gynecology; botanical medicine; nutrition; and homeopathy. Naturopathic physicians in Washington may prescribe a limited range of drugs; however, this is not part of the scope of practice in Connecticut. The practice of acupuncture requires separate licensure in both states [9].
Methods
Study design
The data derive from a parent study of licensed acupuncturists, chiropractors, massage therapists and naturopathic physicians [16]. The study was conducted in two phases: 1) random samples of licensed naturopathic physicians were interviewed by telephone; 2) a sub-set of those interviewed were recruited to record detailed information on 20 consecutive patient visits. Data were collected for each practitioner group in two states (one in the West and one in the Northeast). The West and Northeast were selected because these are the regions where CAM practitioners are concentrated in the United States [14,17]. The data for naturopathic physicians were collected in Washington and Connecticut.
Sample
Licensing Boards provided contact information for all licensed naturopathic physicians in Washington (1998) and in Connecticut (1999) with in-state addresses. In total, 142 licensed practitioners were identified in Washington and 63 licensed practitioners were identified in Connecticut. Providers without identifiable telephone numbers, and those not currently practicing were excluded. The proportion of excluded providers was 11% in Connecticut and 29% in Washington. See Table 1 for additional sampling details.
Table 1 Selection and participation of naturopathic physicians (NDs) in Washington (1999) and Connecticut (1998)
Connecticut Washington
NDs licensed in state 71 286
NDs randomly selected for study 71 200
NDs found eligible for study* 63 142
Eligible NDs interviewed 59 111
Interviewed NDs eligible to collect visit data** 55 93
Eligible NDs providing visit data 34 65
Total patient visits reported 631 1186
* NDs confirmed to be practicing in state and to have verifiable and functioning phone number
** NDs who reported seeing 10 or more patients in a typical week
To maximize the accuracy of state-wide estimates, data collection efforts were concentrated on high-volume practitioners: those with at least 20 patient visits per week. About 60% to 70% of practitioners had a high-volume practice and accounted for roughly 85% to 90% of all visits to the profession. The remaining practitioners were categorized as low-volume providers. While all high-volume practitioners were asked to collect data on 20 consecutive patient visits, only the first 10 low-volume practitioners were asked to collect data. The rationale was to collect only enough data from low-volume practitioners to ascertain whether their practices differed markedly from those of high-volume practitioners. It was ultimately decided, however, to weight data for high- and low-volume practitioners in a manner that produced annual estimates of visits in each state.
Naturopathic physicians who agreed to participate were asked to complete one-page encounter forms for 20 consecutive patient visits. No financial incentives were provided for participation in the study and the protocol was approved by the Group Health Human Subjects Review Committee and the Harvard Pilgrim Health Care "Committee on Clinical Investigations, New Procedures and New Forms of Therapy" prior to the commencement of data collection.
Data collection
Research assistants conducted telephone interviews with the randomly selected providers in Washington State in 1998 and in all eligible practitioners in Connecticut in 1999. (Given the small number of eligible practitioners in Connecticut, all were contacted). Information about individual demographic characteristics (e.g., age, gender, race/ethnicity); training characteristics (e.g., duration, location); practice characteristics (e.g., length of time in practice, licensure in other health care professions, practice arrangement); and workload characteristics (e.g., number of weeks in practice per year; hours per week of direct patient care) was obtained for each provider who agreed to be interviewed. Among those eligible to be interviewed, the participation rate was 94% in Connecticut and 78% in Washington.
Naturopathic physicians who were eligible to collect visit data (i.e., those who saw at least 20 visits per week plus a sample of those who saw at least 10 – 19 visits per week) were asked to complete encounter forms for 20 consecutive patient visits. (Only 2% of all visits were provided by naturopathic doctors with less than 10 visits per week.) Those who agreed were mailed encounter forms that were modeled after those used by the National Ambulatory Medical Care Survey (NAMCS) [18]. Each naturopathic physician was instructed to return the encounter forms by mail to the study center after completing them. Those who failed to promptly return their forms received a reminder telephone call. Among those eligible to collect visit data, 62% of naturopathic doctors in Connecticut and 70% of naturopathic doctors in Washington provided visit data.
Analysis
Descriptive statistics (means, standard deviations, frequencies) were used to present the practitioner characteristics in Table 2. In the analyses of visit characteristics (Tables 4 and 5), each visit in the sample was weighted by the inverse of the sampling probability, reflecting both the chance that the particular provider participated and the estimated proportion of that provider's annual visits included in the study. Consequently, our results represent estimates of all visits made to naturopathic physicians in each state except for the roughly 2% of visits made to practitioners with the lowest volumes [16].
Table 2 Characteristics of naturopathic physicians licensed in Connecticut (1999) and Washington (1998)
Connecticut (n = 59) % Washington (n = 111) %
Demographics
Female 58 57
White 95 94
Mean Age (SD) 43.6 (8.7) years 44.1 (9.2) years
Education
Institution*:
Bastyr University 37 80
National College 59 20
Other 3 1
Specialty training# 69 42
Licensed in other Health Profession* 17 (acupuncture 10%; all others less than 2%) 33 (chiropractic 8%; nursing 7%; acupuncture 6%; midwifery 5%; all others less than 2%)
Practice solo 51 54
*significant differences between the two states, chi square, p < 0.05)
# significant differences between the two states, t-test, p < 0.05)
Table 4 Characteristics of visits to naturopathic physicians in Connecticut (1999) and Washington (1998)
Connecticut (n = 631 visits) % Washington (n = 1186 visits) %
Gender
female 76 74
Age
Median 43 years 42 years
0–15 years 12.8 10.9
16–34 years 16.4 20.9
35–64 years 63.0 58.5
65 years+ 7.8 9.7
Race
White 97 96
Smoking status:
non-smokers 95 94
Type of major problema
Acute problem 22 23
Chronic problem, routine 53 56
Chronic problem, flare up 20 18
Pre/post-surgery/injury 1 1
Non-illness care 5 6
Primary reason for visitb
Fatigue 6.1 6.0
Headache 4.4 3.8
Back symptoms 4.4 6.5
Skin rashes 4.2 2.5
Menopausal symptoms 3.7 2.0
Bowel function changes 3.5 2.0
Anxiety or Depression 3.2 5.1
Allergies to food, milk 3.0 3.6
Sinus symptoms 2.8 <2
Upper respiratory symptoms 2.6 <2
Cough 2.4 <2
Neck symptoms 2.3 3.3
Infectious disease 2.2 <2
Ear infection symptoms 2.2 <2
Abdominal pain/cramps 2.2 2.7
Menstrual problems <2 2.1
Diagnosis by naturopathic physicianc
Menopausal disorders 4.9 3.1
Allergies 4.6 6.3
Back conditions 3.5 5.8
Fibromyalgia 3.0 2.0
Sinusitis 2.9 <2
Fatigue 2.4 2.7
Headache 2.2 3.1
Upper respiratory infections 2.0 <2
Neck conditions <2 3.3
Depression <2 2.6
Asthma <2 2.2
Anxiety <2 2.1
Dermatitis <2 2.0
New patients 22 22
Payment
Private insurance 60 43
Worker's compensation 0 3
Personal injury 0 3
Self-pay 37 48
Other 3 3
a Because 3% to 4% indicated multiple reasons, total percentages exceed 100%
b Patient report; only reporting reasons totaling 2% or more of visits
c Based on ICD 9 criteria; only reporting diagnoses totaling 2% or more of visits
Table 5 Characteristics of visits to naturopathic physicians in Connecticut (1998) and Washington (1999)
Connecticut (n = 631 visits) % Washington (n = 1186 visits) %
Diagnostics/Screen Services:
Examinations
Vitals (BP, pulse, temp) 28 39
HEENT 18 15
Complete physical 13 9
Mental Status <5 6
Imaging
x-ray 1 2
ultrasound 1 1
Blood tests
Complete blood count 7 10
Serum chemistry 7 9
Thyroid 3 7
Lipids panel 4 5
Allergy 4 2
Additional Tests
Stool analysis 5 4
Urine analysis 4 2
Vitamin/Mineral 3 0
Endocrine 2 3
Allergy Skin test 1 1
TB skin test 1 0
Therapeutics and Preventive Services:
Naturopathic Therapeutics
Botanical medicine 51 43
Vitamins 41 43
Minerals 35 39
Homeopathy 29 19
Acupuncture 14 4
Allergy treatment 11 13
Glandular therapies 4 13
Physical Therapies
Naturopathic manipulation 8 15
Physiotherapy 1 13
Hydrotherapy 4 10
Ultrasound 2 9
Mechanotherapy 2 7
Counseling/Education
Therapeutic diet 26 36
Self-care education 17 23
Exercise therapy 9 12
Mental Health 4 6
Visit disposition *
No follow-up planned 5 4
Return if needed 18 21
Return at specified time 77 74
Referred (% to an MD) 5 (4) 6 (4)
Visit Duration
Mean, minutes 40 44
<15 minutes 1 3
15–29 minutes 14 20
30 to 44 minutes 49 31
45–59 minutes 15 18
>/=60 minutes 20 28
* Totals add to more than 100% because multiple responses were allowed
Given the large sample sizes (631 and 1186), the weighted percentages presented in the tables have small standard errors, generally between 0.5 and 2.5 percentage points and rarely exceeding 3 percentage points. As a result, moderate to large differences between the states are also statistically significant and we do not include standard errors in the tables.
Results
Naturopathic physician participants
The demographic characteristics of the naturopathic physicians in Washington State and Connecticut were remarkably similar (see Table 2). Just over half were female, with mean ages of about 44 years. Over 90% of naturopathic physicians identified themselves as White.
The majority of practitioners (80%) in Washington State trained at Bastyr University in Seattle, while most practitioners in Connecticut trained at National College of Naturopathic Medicine in Portland, Oregon. Although naturopathic doctors in Connecticut were more likely to report advanced specialty training (such as homeopathy), those in Washington were more likely to be licensed in another health profession. Washington naturopathic physicians were most often also licensed in chiropractic (8%), nursing (7%), acupuncture (6%) and midwifery (5%), while Connecticut naturopathic physicians were most often also licensed in acupuncture (10%). Almost one-quarter (22.5%) of naturopathic doctors licensed in Washington had completed at least one year of post-graduate residency training compared with only 8.5% of those in Connecticut. (Residency training is not required for licensure in either state.)
Both groups graduated from 4–5 year accredited full time programs of naturopathic medical education and the resulting naturopathic practice characteristics were reported to be very similar across the two states (Tables 2 and 3). Just over half of the practitioners in both states practiced solo, averaged approximately 25 hours per week providing direct patient care and saw an average of just over 30 patient visits per week. Practitioners who reported seeing less than 10 patients per week were excluded from this study.
Table 3 Practices of naturopathic physicians licensed in Connecticut (1999) and Washington (1998)
Connecticut (n = 59) Washington (n = 110)
Median number of years in practice 9 7
Patient care hours in a typical week
Mean (S.D.) 25.8 (10.6) 24.5 (12.2)
Percentage reporting: 1–14 hours 10.2 20.0
15–24 hours 32.2 30.0
25–34 hours 39.0 26.4
35–44 hours 15.2 17.2
45+ hours 3.4 6.4
Patient visits in a typical week
Mean (S.D.) 32.7 (18.9) 30.5 (24.7)
Percentage reporting: 1–19 visits 20.7 34.6
20–29 visits 31.0 21.8
30–39 visits 13.8 21.8
40–49 visits 13.8 9.1
50+ visits 20.7 12.7
Characteristics of patients who see naturopathic physicians
As was the case for the practitioners, patients of naturopathic physicians in the two states were remarkably similar (Table 4). About 75% of patients were female, about 95% were White and non-smokers, and the mean age of the patients was 41 years. Just over 50% of visits were for chronic conditions, followed in frequency by acute problems and flare-ups of chronic problems. There was extensive overlap in the most common presenting complaints (based on ICD 9 classification) in Connecticut and Washington with fatigue, headache, back symptoms among the top four in both states. However, there was a wide variety in the presenting complaints recorded; the most common presenting complaint was identified by less that 7% of patients in both states. More than one of every five visits was made by a new patient. Overall, approximately half the visits were paid for by private insurers and a similar percentage were paid directly by the patient. Visits in Connecticut were slightly more likely to be covered by insurance (60% vs 49% of visits).
Characteristics of naturopathic medical visits
More than 70% of visits included examinations or the ordering of diagnostic or screening tests (Table 5), most often: assessment of vital signs (including blood pressure, pulse and temperature), and Head, Eyes, Ears, Nose, Throat (HEENT) assessment (typically done for upper respiratory symptoms). The most common blood tests ordered in both states were: complete blood counts and serum chemistry. Imaging and other types of diagnostic tests were used for no more than 5% of visits in both states.
Therapeutic and preventive modalities were used in most visits (96%) in both states. The most common naturopathic therapeutics used in both states were: botanical medicines, vitamins, minerals, homeopathy and allergy treatments (Table 5). In addition, glandular therapies were prevalent in Washington (13% of visits) and acupuncture was prevalent in Connecticut (14% of visits). The only physical therapies that are part of the naturopathic scope of practice used in more than 5% of the visits were naturopathic manipulation, physiotherapy (Washington only) and hydrotherapy (Washington only). Twenty-six per cent of visits in Connecticut included counseling or education compared with 36% of visits in Washington. Most often this included discussion of a therapeutic diet.
Three-quarters of all visits in both states resulted in a recommendation that the patient return at a specified time (Table 5). The mean length of visit in the two states was similar (40–44 minutes), but there was greater variability in the length in Connecticut.
A wide range of generally similar commercially packaged products (including herbs, vitamins and minerals) were used by naturopathic physicians in the two states (Table 6). Connecticut providers recommended about 50% more than Washington providers. Almost all of the top 15 commercially packaged products used in both states were vitamins and minerals as single agents or as combinations. The most common commercially packaged products were about 1.5 to 2.0 times more likely to be used in Connecticut compared to Washington. In addition, zinc and vitamin B6 were more likely to be used by NDs in Connecticut, while protomorphogens and vitamin B12 were more commonly used in Washington. (A protomorphogen is thought to be that component of the cell chromosome that is responsible for morphogenic (forming of body and organs) determination of cell characteristics. Theories about what protomorphogens are, as well as their role in health and disease continue to be debated.)
Table 6 Most common commercially packaged products used by naturopathic practitionersin Connecticut (1998) and Washington (1999)
Connecticut (n = 631 visits) Washington (n = 1186 visits)
Number of Products Used per visit
Range 0 to 28 0 to 26
Median 3.0 2.0
Mean 4.1 2.7
Most Common Commercially Packaged Productsa (weighted percent of visits)
Multivitamins 26.7 9.1
Digestive treatments 22.3 13.2
Magnesium 16.3 9.1
Combination vitamin and mineral 16.2 10.0
Calcium 15.8 9.3
Vitamin C 15.1 10.2
Minerals, NOSb 9.4 7.7
Zinc 9.0 <5
Multiminerals 8.4 7.8
Vitamins, NOSb 8.4 10.5
Vitamin E 7.9 5.2
Bioflavonoids 7.1 5.9
Vitamin B6 5.9 <5
Coenzyme Q10 5.8 <5
Selenium 5.2 <5
Protomorphogen <5 9.0
Vitamin B12 <5 7.4
a Excluding diets and foods; reporting only those with a weighted per cent >5%
b NOS = Not Otherwise Specified
Discussion
Given the broad scopes of practice of naturopathic medicine in Washington and Connecticut, the similarities in the practitioner characteristics, patient characteristics and practice characteristics in Washington State and Connecticut are remarkable. The finding that naturopathic medicine is primarily a white and female profession is consistent with other recent studies of naturopathic physicians in the United States and Canada [19,20]. Substantial fractions of naturopathic doctors, especially in Washington, are licensed in other CAM and conventional professions. Practitioners spend about 25 hours per week in patient care and see approximately 32 patients/week, which is slightly more than was reported in a recent Canadian study [20]. In comparison, according to the American Medical Association, general practitioners in the United States spend approximately 51.3 hours/week providing direct patient care and see approximately 125 patients in a typical week [16].
Patients who visit naturopathic physicians are primarily female, white, middle aged, non-smokers. In many ways they are demographically similar to the naturopathic physicians themselves. Patients present to naturopathic physicians with a broad range of complaints and are diagnosed by the practitioners with a wide variety of conditions. It should be noted that much of naturopathic practice is focused on chronic problems, particularly those exclusively or primarily presented by women (e.g., fatigue, headache, symptoms associated with menopause, depression). In fact 75% of all visits are made by women. The most common "reasons for visits" identified in this study were very similar to those noted in a Canadian study [20], suggesting that this sample of provider visits may be representative of naturopathic patient visits across North America.
Other than physical examination, blood tests and stool analysis, naturopathic practitioners use few diagnostics tests. However, they commonly recommend commercially packaged natural products such as herbal medicines, vitamins and minerals. Naturopathic practice also involves significant amounts of patient counseling and education. Overall, naturopathic physicians spend more than twice as much time with patients as conventional physicians at each visit (40 minutes vs. 14 minutes) [16], permitting more time to discuss patients' concerns and counseling/education about lifestyle issues such as diet.
Data from this and previous studies [19,20] indicate that patients visit naturopathic physicians for a wide range of conditions and are given a correspondingly wide range of therapies and treatments as part of their naturopathic care. This diversity makes it difficult to describe a "typical" naturopathic medical consultation and to develop standardized research protocols for studies evaluating naturopathic treatments.
Our results also have implications both for naturopathic physicians and the American health care system. They indicate that naturopathic physicians treat a broad range of health problems. The results from our study provide baseline data for tracking changes as the practice of naturopathic medicine evolves over time across the US in response to increasing research into the safety, efficacy and cost-effectiveness of naturopathic treatments, as well as to changes in the social, economic and regulatory environment.
Strengths and limitations of the study
The major strengths of this study are its large sample size, relatively high participation rates, and the ability to compare naturopathic physicians from two geographically distant states. The main limitation is that despite the similarities of the states studied, it is not known if the results are representative of the other ten states where naturopathic medicine is licensed. These two states reflect typical licensing requirements prevalent throughout the US. Washington State has one of highest numbers of licensed naturopathic physicians in the US, while the number of naturopathic physicians in Connecticut is significantly smaller, and more representative of, most other licensed states.
Another limitation of the survey was its inability to accurately capture the use of office-compounded tinctures and powders. Our results highlight the use of bottled products as a major part of naturopathic practice; however, the practitioners' use of their own combination preparations was difficult to record on the data collection forms. This aspect of practice is likely under-reported in our data.
Conclusion
Naturopathic physicians and their practices in Washington and Connecticut, two geographically distinct states, are similar. This study provides baseline data for describing and tracking the practice patterns and scope of practice of naturopathic physicians over time and will help inform researchers and policy makers considering the regulation of this profession in other states. It also helps conventional physicians, other health care providers and patients better understand the nature of naturopathic care.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
HB conceptualized and drafted the manuscript. DC conceptualized the overall project, designed and directed collection of the original data and participated in the conceptualization of this manuscript, and analysis of this data. KS participated in the design of the overall project and this manuscript, and participated in the statistical analyses. JE helped to conceptualize the overall project, directed the data collection, quality control, and participated in the analyses for this paper. BM, JB, EC, and DE helped to conceptualize the overall project including the data collection. MS helped to conceptualize this paper. RD helped to conceptualize the overall project, design data collection procedures, and secure funding. All critically edited drafts of this manuscript and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
This project was supported by grants from the Group Health Foundation, Grants #HS09565 and #HS08194 from the Agency for Health Care Policy and Research and Grant #AR43441-04S1 from the National Institutes of Health. In kind support was provided by the Centers for Disease Control and Prevention.
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| 15496231 | PMC529271 | CC BY | 2021-01-04 16:31:44 | no | BMC Complement Altern Med. 2004 Oct 20; 4:14 | utf-8 | BMC Complement Altern Med | 2,004 | 10.1186/1472-6882-4-14 | oa_comm |
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BMC Health Serv ResBMC Health Services Research1472-6963BioMed Central London 1472-6963-4-291551859210.1186/1472-6963-4-29Research ArticleRecruiting pregnant smokers for a placebo-randomised controlled trial of nicotine replacement therapy Coleman Tim [email protected] Marilyn [email protected] John [email protected] Jim [email protected] Sarah [email protected] Kim [email protected] Division of Primary Care, University of Nottingham, Queen's University Medical Centre, Nottingham NG7 2UH England2 Division of Respiratory Medicine, University of Nottingham, Nottingham City Hospital NHS Trust, Hucknall Road Nottingham NG5 1PB England3 Division of Epidemiology and Public Health, University of Nottingham, Queen's University Medical Centre, Nottingham NG7 2UH England4 Division of Obstetrics, Gynaecology and Child Health, University of Nottingham, Nottingham City Hospital NHS Trust, Hucknall Road Nottingham NG5 1PB England5 Academic Division of Midwifery, University of Nottingham, Nottingham City Hospital NHS Trust, Hucknall Road NOTTINGHAM NG5 1PB England2004 1 11 2004 4 29 29 4 5 2004 1 11 2004 Copyright © 2004 Coleman et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Smoking in pregnancy is a public health problem and effective methods for reducing this are required. Although nicotine replacement therapy (NRT) is effective for smoking cessation in non-pregnant people, there is no direct evidence concerning its effectiveness in pregnancy. Despite this, clinical guidelines recommend the cautious use of NRT during pregnancy. Randomised controlled trials are needed to determine the safety and efficacy of NRT when used by pregnant women for smoking cessation, but the feasibility of recruiting women to such trials is unknown. Consequently, in this study we aimed to determine i) the feasibility of recruiting women to a RCT of NRT in pregnancy as they attend hospital antenatal ultrasound examinations, ii) the proportion of such women who are eligible for and interested in trial enrolment and iii) research staff perceptions of how one method of trial recruitment could be improved.
Methods
During a one month period, all women attending for antenatal ultrasound examination in an English teaching hospital were asked to complete a questionnaire which determined their eligibility to enrol in a proposed placebo controlled randomised trial investigating the effectiveness of NRT in pregnancy. Women who were eligible to participate were asked whether they would do so and those who accepted enrolment were offered an appointment with a smoking cessation advisor.
Results
Over 99% (851/858) of women agreed to complete a questionnaire about smoking habits whilst waiting for ultrasound examinations. 10.3% (88/851) of women attending for antenatal ultrasound fitted eligibility criteria for a proposed RCT of NRT in pregnancy, but only 3.6% [(31/851), 95% CI, 2.4 to 4.9%] indicated on the questionnaire that they would like to take part in a study involving randomisation to placebo or active patches. Researchers offered trial enrolment to 26 of these 31 women and 96% (25) accepted. Staff recruiting women believed that trial recruitment would be maximised if women attending the ultrasound department knew about trial recruitment before attending and greater staff resources were made available for this. It was also perceived that women generally under-reported the amount they smoked on questionnaires completed whilst waiting in ultrasound department areas.
Conclusions
It is feasible to recruit women for a trial of NRT in pregnancy as they wait for antenatal ultrasound examinations. Using similar recruitment methods, researchers can expect to recruit between 24 and 49 women per 1000 approached.
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Background
Smoking harms unborn children causing intra-uterine growth restriction, preterm birth, miscarriage and perinatal death [1,2]. Despite this over a quarter of pregnant women in the UK smoke [3] and although pregnancy motivates a minority of women to stop smoking for at least part of their pregnancy, most start again after delivery [3]. Reducing smoking in pregnancy is, therefore, a health priority and effective methods for promoting smoking cessation in pregnancy are needed. Behavioural support is effective for smoking cessation in pregnancy, [4] but drug treatment is not generally used because of concerns that this may harm the fetus [5]. Outside of pregnancy, however, drug therapy can be effective and nicotine replacement therapy (NRT) increases quit rates of smoking cessation interventions it accompanies by 1.5 to 2-fold [6]. Unfortunately, the efficacy and safety of NRT in pregnancy are unknown.
Two trials conducted in pregnant women have shown NRT patches to have no greater effect on smoking cessation than placebo [7,8]. but neither trial has been large enough to be definitive. Additionally, nicotine is metabolised more quickly in pregnancy [9] and this could reduce the efficacy of NRT, as conventional doses will provide less nicotine substitution. Consequently, for NRT to be effective in pregnancy, higher doses might be needed but this raises questions of safety because nicotine is a recognised fetal toxin [10] and higher doses could increase the risk of any fetal damage occurring. Any such risk is difficult to quantify, though, because there is little human-subject evidence on the effects of nicotine (or NRT) in pregnancy and most evidence about the impact of nicotine in pregnancy is derived from animal studies [10]. In humans, nicotine is known to increase maternal blood pressure and heart rate but to have lesser effects on the fetal heart rate and these changes are less pronounced than those caused by smoking [10]. More encouragingly, the one completed trial of NRT in pregnancy [7] found babies born to women in the NRT treatment group had significantly higher birth weight than those treated with placebo, suggesting that intrauterine growth restriction caused by smoking is not attributable to nicotine. Further evidence on the safety of nicotine in pregnancy would help to inform clinicians' decisions about prescribing NRT in pregnancy.
Despite uncertainties about the effects of NRT in pregnancy, there is an expert consensus that NRT use is safer than smoking [11] which is reflected in clinical guidelines [5,12] and other advice to clinicians [13]. NRT use in pregnancy is, therefore, being encouraged in the absence of any direct evidence for it's effectiveness or safety and placebo-controlled RCTs are needed to resolve these issues. In pregnancy, women are generally advised to avoid unnecessary drugs [14] and it is likely, therefore, that women's reluctance to use drugs (including NRT, which contains a known fetal toxin [10]) might hinder recruitment to RCTs of NRT in pregnancy. Consequently, this paper describes a pilot study which aimed to determine: i) the feasibility of recruiting women to an RCT of NRT in pregnancy as they attended hospital antenatal ultrasound examinations, ii) the proportion of pregnant women attending such clinics who are eligible for enrolment and interested in participation in such a trial and iii) research staff perceptions of how one method of recruitment could be improved for use in a definitive RCT.
Methods
Ethical approval was granted by the Nottingham Research Ethics Committee 2. For one month, one of three research assistants (RAs) was based in the Nottingham City Hospital antenatal ultrasound department for all scanning sessions. All women attending for ultrasound scans were approached by an RA and asked to fill in a self-completion questionnaire (see appendix) which asked their number of weeks gestation and whether or not they smoked at all. Respondents who answered that they smoked cigarettes or tobacco were then asked a series of questions about their smoking behaviour to ascertain whether or not they satisfied eligibility criteria for a proposed RCT (see Figure 2). Those smokers who were at an appropriate gestation, who smoked heavily enough for entry to the RCT and who were interested in stopping smoking completed the whole questionnaire. Before the final questionnaire item, they read brief information about the use of nicotine replacement therapy in pregnancy (which was on the questionnaire). The final item then asked women whether or not they would like to take part in a study which investigated the effectiveness of NRT in pregnancy. Those who did were subsequently offered enrolment into the study by the RA and their responses were noted. The enrolment offer included an explanation that no randomised study of NRT was currently taking place but that this was possible in the near future (which, at that time, the authors believed to be true). Women who agreed to enrolment were aware that they might participate in a study that would involve them being randomised to either active or inactive patches and, although nicotine was potentially harmful, NRT use was believed to be safer than smoking. RAs arranged appointments with the local NHS smoking cessation service [15] for those women who accepted the offer of enrolment and, if they attended these, women received counselling from midwives who specialised in smoking cessation counselling for pregnant women.
Enrolment criteria for the proposed trial (Figure 2) were constructed to ensure that only women who were addicted to nicotine were likely to be enrolled in the study. It was important to prevent any woman from entering a trial if it were possible that she might receive more nicotine via NRT dispensed in the trial than she otherwise would have received by smoking [11]. The antenatal ultrasound clinic setting was used because almost all pregnant women in the UK have an ultrasound scan performed between the end of the first and the middle of the second trimester of pregnancy. By recruiting in this setting, we were able to systematically approach all pregnant women residing in the local area who were at an appropriate stage of gestation and who were likely to deliver in the same hospital.
When the pilot was finished, the views of RAs and collaborating staff from the local NHS smoking cessation service were sought at a meeting. We asked their opinions of the utility of our method for detecting smokers (i.e. the questionnaire) and the practicality of recruitment within the ultrasound clinic setting. Where staff had criticisms, they were encouraged to suggest improvements in procedures. Key points made during this meeting were noted and issues described in the results section are those on which there was consensus.
Sample size and data analysis
We knew from routinely-collected clinic statistics that over 800 women would attend for antenatal ultrasound scans in a four week period and around 200 of these (25%) would smoke. We hypothesised that 20% of smokers (i.e. approximately 40 or 5% of clinic attenders) would agree to trial randomisation. We believed this a reasonable assumption because in a survey of women attending ante-natal clinics, Ussher found that, amongst women who smoked 10 or more cigarettes daily and who were interested in stopping smoking; 56% were also interested in using NRT [16]. Consequently, if our survey was conducted for one month, we calculated that would be able to detect the proportion of clinic attenders agreeing to randomisation with 95% CIs of 1–2%.
All data was entered into an SPSS database and quantitative findings are expressed as simple proportions of all clinic attenders, with 95% CI for the principal outcome.
Results
Figure 1 shows that 99.3% of women attending for antenatal ultrasound imaging during the study period consented to complete a questionnaire. Of questionnaire respondents, around 27% had smoked in the last week, but only 163 of these were within the appropriate gestation range for trial entry. The diagram shows that although 17% of attenders admitted to smoking on "at least most days", this number fell to around 13% who admitted smoking 10 cigarettes daily before they were pregnant and it fell further to 10% who admitted smoking both 10 cigarettes daily before pregnancy and 5 currently. Of these heavier smokers, 5.6% were interested in stopping smoking. Finally, 3.6% [(31) 95%CI, 2.4 to 4.9%] of women attending for ultrasound both met trial entry criteria as measured on the questionnaire and were interested in participation in a study involving NRT. RAs offered study enrolment to 26 of these 31 women and 25 (96%) accepted. The remaining 5 women left the ultrasound clinic before RAs could discuss participation with them. 21 women who agreed to the enrolment offer attended at least one appointment with the local NHS smoking cessation in pregnancy advisor.
Perceptions of research and smoking cessation service staff involved in pilot
There was consensus amongst RAs and NHS smoking cessation service staff that, if the following problems with study procedures were remedied, the proportion of women interested in trial participation who actually enrolled should increase:
• A number of women were interested in study participation but did not have sufficient time to discuss this with the RA in the clinic. These women might have been able to make time to discuss the study if they had known about the likelihood of being approached before attending the ultrasound department.
• On occasions, RAs failed to follow up women who had expressed an interest in study participation because they were busy with other duties (e.g. discussing enrolment with another woman).
• A number of women who had indicated on their questionnaire that they were interested in participation left the clinic before the RA could discuss enrolment with them. The pilot study did not have the resources to contact these women afterwards.
• Some women reported heavier smoking to smoking cessation advisors than they recorded on questionnaires. This apparent under-reporting of smoking behaviour may have resulted in some women who were actually eligible for study participation, not being offered study enrolment.
Discussion
We have demonstrated that it is feasible to recruit women to a trial of NRT in pregnancy as they attend antenatal ultrasound appointments. Although almost all women consented to complete a questionnaire enquiring about their smoking habits, only 10% of these were eligible to enrol in an RCT and less than 4% appeared likely to participate in one.
Our study provides useful, original information for researchers planning trials of NRT in pregnancy. The two published trials of NRT use in pregnancy give no information about numbers of women were asked to participate but declined. One trial took two years to enrol 250 pregnant women [7] and the other recruited 30 women, but the time taken to achieve this is not reported [8]. To determine the level of interest in using NRT amongst pregnant smokers, Ussher and West conducted a telephone survey of women who had reported themselves to be smokers during a "booking" appointment (i.e. their initial antenatal appointment) [16]. They found that around one half of women who admitted to smoking at their antenatal "booking" appointment (95/177) were interested in receiving help with stopping smoking, and of those who admitted to smoking 10 or more cigarettes daily, 56% were interested in using NRT. This survey, however, was conducted amongst an ante-natal clinic population with a smoking prevalence of around 12%, which is much lower than the national average, so the generalisability of study findings to other areas is questionable. Additionally, it gave no indication of the numbers of women who might agree to randomisation which included the possibility of being allocated a placebo treatment. Nevertheless, our findings are not necessarily inconsistent with the London antenatal clinic survey. We found that approximately 35% (31/88) of women currently smoking five cigarettes daily (who also admitted to smoking 10 cigarettes daily before pregnancy) were interested in trial participation.
Our findings compliment those of Ussher's survey by providing information about likely participation rates for any randomised controlled trial. Extrapolating our figure of 3.6% indicates that approximately 36 (95%CI, 24 to 49) women could be recruited annually to a RCT of NRT in pregnancy from a maternity hospital with 1000 births/pregnancies each year. Researchers planning trials of NRT in pregnancy can use this recruitment rate to inform the planning of studies. Trialists should note, however, that we have measured agreement to be randomised rather than actual randomisation. Additionally, we did not measure the numbers of exclusions amongst women who were interested in participation. Figure 2, however, lists exclusion criteria for a RCT of NRT in pregnancy that the authors have proposed and consideration of these suggest that few pregnant women agreeing to enrolment would be excluded from using NRT on medical grounds. Finally, pilot study findings are likely to be most applicable to countries which have policies on the use of NRT in pregnancy that are similar to those employed in the UK: the proportion of women agreeing to randomisation would probably be lower in any health care system in which health professionals generally advised pregnant women against using NRT.
Some modifications to the recruitment method we used might be needed to maximise trial recruitment rates. Recruitment from antenatal ultrasound waiting areas might be increased if women were sent information about the likelihood of being approached to join any trial before they attended (e.g. sending information out with appointments). Additionally, providing enough resources to ensure that a member of trial staff is always present to ask patients to join a trial is important. This would overcome the problem of missing potentially interested women when the researcher responsible for recruiting is busy enrolling other potential participants. Any recruitment method should also allow for the fact that some women who are interested in participating will not be able to wait in the clinic to discuss this further. Recruitment would, therefore, be enhanced if enough resources were provided to allow research staff to make contact with these women later, visiting them at home if necessary.
The observation by smoking cessation service staff that some women admitted smoking more heavily to them than they admitted on questionnaires is consistent with the literature. Aveyard investigated the relationship between urinary cotinine levels and reported numbers of cigarettes smoked during pregnancy and concluded that, at booking, some pregnant women probably minimise the numbers of cigarettes that they report smoking [17]. Similarly, Owen and McNeil's investigation of salivary cotinine levels in pregnancy indicated that a small number of pregnant women actually conceal the fact that they smoke [18]. These research findings probably reflect the fact that, in the UK, there is a social stigma attached to smoking in pregnancy which is likely to bias questionnaire respondents against an honest disclosure of their smoking habit. Some women in our pilot may have been happier to disclose the full extent of their smoking to a supportive health professional than they were to a questionnaire distributed by a RA in an out patient waiting area.
Trialists could reduce the impact that under-reporting of heaviness of smoking might have on trial recruitment by avoiding the use of questionnaires to quantify smoking habits. Questionnaires used in recruitment to trials of NRT in pregnancy might be more effective if they merely ask women to disclose whether or not they smoke and if they are interested in using NRT to stop. All who smoke and wish to stop using NRT could then be invited for further discussion of trial entry with a researcher or health professional and during this disclosure a more accurate assessment of heaviness of smoking might be obtained.
Conclusions
We have demonstrated that it is feasible to recruit pregnant women to a trial of nicotine replacement therapy in pregnancy in the antenatal ultrasound clinics. Using similar recruitment methods between 24 and 49 pregnant women per 1000 approached are likely to consent to participation in an RCT. RCT(s) of NRT in pregnancy are required and this data will help researchers planning such trials to determine the resources required to answer important questions concerning the effectiveness and safety of NRT in pregnancy.
Competing interests
TC has been paid for speaking at a conference by GlaxoSmithKline (GSK); he has also done consultancy work on one occasion for Pharmacia (now Pfizer). JB has been reimbursed by Glaxo Wellcome (now GSK) for attending 2 international conferences, has received a speaker honorarium from Glaxo Wellcome, has been the principal investigator in a clinical trial of nicotine replacement therapy funded by Pharmacia and has undertaken consultancy work for Novartis. All companies manufacture nicotine replacement products but none were involved in this study. All other authors have no conflicts of interest.
Authors' contributions
All authors were involved in various stages of study design. MA liaised with the NHS Stop Smoking and hospital antenatal services, collected data and supervised others in this. TC wrote the paper and all authors commented on drafts and approved final text.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgments
This study received no external funding. We acknowledge the support of midwives from Nottingham City Hospital and the collaboration of Alison Challenger, Jenneve Hutchinson and Vicky Milton from New Leaf NHS Stop Smoking cessation service, Nottingham. We are also grateful to Amanda Butler and Jenny Brooks for conducting data collection and to Laura Jones and Rose Henson for secretarial support.
Figures and Tables
Figure 1 Flow Chart of Study Participation
Figure 2 Eligibility criteria for proposed RCT
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| 15518592 | PMC529272 | CC BY | 2021-01-04 16:03:29 | no | BMC Health Serv Res. 2004 Nov 1; 4:29 | utf-8 | BMC Health Serv Res | 2,004 | 10.1186/1472-6963-4-29 | oa_comm |
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Malar JMalaria Journal1475-2875BioMed Central London 1475-2875-3-381551626910.1186/1475-2875-3-38ResearchImproving the accuracy of malaria-related laboratory tests in Ghana Bates Imelda [email protected] Veronica [email protected] Alex [email protected] Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK2 Public Health and Reference Laboratory, Korle Bu Teaching Hospital, Accra Central, PO Box 4456, Ghana3 Ghana Laboratory Service, Ministry of Health, Accra Central, PO Box 4456, Ghana2004 1 11 2004 3 38 38 24 8 2004 1 11 2004 Copyright © 2004 Bates et al; licensee BioMed Central Ltd.2004Bates et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Inaccurate malaria results can lead to patient mismanagement, misperceptions about malaria resistance patterns and public health misinformation. All laboratories need to be able to demonstrate that their results are accurate. Establishing and maintaining a system for monitoring test accuracy is a complex, expensive and technically demanding process, which very few poor countries have been able to implement. This study described the process and assessed the feasibility of establishing a nation-wide system for improving the accuracy of malaria-related tests in peripheral laboratories in Ghana.
Programme implementation
A baseline survey of all 693 laboratory staff in 205 sub-regional government and mission health laboratories in Ghana was conducted by a national network of laboratory supervisors. Survey results guided a training programme to improve test accuracy. Outcomes included changes in the quality of laboratory tests and the system was considered to be feasible if >50% of laboratory staff in each region received training and if test accuracy could be documented.
Programme indicators
74% (mean) of the 693 laboratory staff were assistants with no professional qualifications. There were marked differences between regions in the availability of essential resources for malaria diagnosis (e.g. microscopes). 93% of laboratory staff received training; in six months there were increases of 11% and 7% respectively in the number of laboratories producing haemoglobin and malaria microscopy results of acceptable quality.
Conclusions
It is possible to establish a system for improving and monitoring test accuracy in peripheral laboratories on a country-wide basis in a developing country using a model that could be adapted for use in other countries and for other components of health care provision.
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Background
Clinical laboratory services are a critical component of health systems. They are essential for patient management and for providing accurate public health data including early detection of malaria resistance. In many poorer countries, laboratory services have been neglected due to chronic under-investment. The emergence of drug-resistant malaria and the extra burden of supporting diagnosis and treatment of HIV/AIDS has increased the strain on laboratories. The widespread resistance of malaria to cheap, antimalarial drugs such as chloroquine and the increasing use of relatively expensive combination therapy, means that presumptive treatment of all fevers as malaria may no longer be a sustainable option for some countries [1]. It is, therefore, imperative that malaria diagnosis at peripheral level health facilities, where the greatest burden of malaria health care provision is focused, is accurate.
Establishing and maintaining an accurate and reliable laboratory service is a complex, expensive and technically demanding process, which very few poor countries have been able to implement. It depends on good laboratory management to oversee processes such as documentation, audit cycles, quality assurance and external validation, safety practices, and supervisory and accountability structures [2] and should be combined with improvements in clinical practice. Sub-standard laboratory services waste public and individuals' resources and result in clinical mismanagement and inaccurate health information. They also generate a culture of mistrust and communication breakdown between laboratory and clinical staff which contributes to low morale within the technical profession. Poor quality laboratory services have the greatest impact on the poorest people who use the service because they have the largest burden of ill-health [3]. To our knowledge there is no comprehensive nationwide system for monitoring the accuracy of malaria-related laboratory tests (i.e. malaria microscopy, haemoglobin estimation, tests associated with blood transfusion) currently operational in any sub-Saharan country.
In January 2000, the Ministry of Health in Ghana commenced a two-year programme to determine the feasibility of establishing a nationwide quality assurance system for common tests performed at peripheral (district and sub-district) laboratories. This programme was specifically designed to complement the Ministry's 5-year Programme of Work [4]. In Ghana, laboratory services exist at most levels of health facility, except smaller health centres. Ghana's decentralization policy means that regional and district health managers are responsible for delivering and evaluating health care provision, including laboratory services. Prior to this programme there were no local or national quality assurance systems and no functional supervisory network for laboratories in Ghana. This feasibility programme was built on established laboratory management structures and locally available resources and was implemented by a national network of senior laboratory technicians. The aim of the programme was to determine the feasibility of establishing a nationwide system for improving the accuracy of malaria and other common laboratory tests. All staff performing laboratory tests in all public sector peripheral laboratories in Ghana were included in the programme irrespective of their grade.
Programme implementation
Establishing a national network of laboratory 'supervisors'
Two senior technicians from each of Ghana's ten administrative regions were chosen by the Ministry of Health to constitute the national network of laboratory supervisors who would implement the programme. They were selected on the basis of their geographic location, seniority and commitment to improving laboratory services. All the supervisors had technical qualifications (2–3 years certificate or diploma course), but none had any higher technical or educational qualifications.
Programme objective and baseline survey
A workshop for the supervisors was held in Ghana at the beginning of the programme to define the objectives and to develop a workplan, timetable and monitoring processes. The agreed programme objective was to establish a system in all peripheral government laboratories in Ghana, for monitoring the accuracy of results of malaria microscopy, haemoglobin estimation and other commonly performed tests. The ability to train over 50% of laboratory staff in each of the ten regions and to monitor changes in the accuracy of results, were used as indicators of programme feasibility. As very little information was available about the state of Ghana's laboratories, supervisors initially carried out a nationwide baseline survey of all Ghana's peripheral laboratory facilities. They personally visited every government and mission laboratory in Ghana and collected first hand information about staff, equipment and tests offered. For each laboratory they also completed a safety checklist based on the World Health Organization's recommendations for laboratory safety [5].
Programme design and methods
Programme planning and training
Ideally training should be targeted towards the tests which are performed most poorly. However, as there were no monitoring systems in place in Ghana for laboratory tests and none of Ghana's laboratory staff had had any experience of these systems, it was not possible initially to identify the worst performed tests. The supervisors therefore started by providing training on seven of the most commonly performed tests. They used Ghana's Standard Laboratory Operating Procedures [6], which describe nationally standardized methods for individual tests, as their core teaching manual. Their choice of teaching methods, predominantly combinations of workshops and workplace training, varied between regions depending on local resources, needs and geography.
Monitoring test accuracy
Quality assurance systems used in industrialised countries and published in the literature are not appropriate for rural laboratories in poor countries such as Ghana. They are too complex for a workforce that is primarily made up of laboratory assistants and they are based on assumptions that the methods are generally automated and communication and transport networks are reliable. The supervisors therefore had to devise workable methods for externally monitoring test results from the peripheral laboratories. They distributed samples with known values to peripheral laboratories who processed them under normal working conditions. Once the methods for distributing, preserving and measuring test accuracy had been optimized for local use they were piloted in a small number of laboratories, before being introduced throughout each region. The ways in which the test accuracy was determined varied for each test. For example, for haemoglobin estimations a whole blood sample with known value (determined by repeated measurements in a regional laboratory) was distributed to peripheral laboratories and supervisors decided that laboratories with results outside the target value ±10% would receive priority for training. As the quality of results from peripheral laboratories improved, the supervisors reduced the acceptable range of results to ±5% of the target value. For malaria microscopy, the target malaria result of a whole blood sample or malaria smear was determined by consensus of several technicians from the regional hospital. Results from peripheral laboratories were considered 'accurate' if laboratories reported the presence (or absence) of malaria parasites and quantified them to within one grade (on a grading system of 0,+,++,+++) of the target result. For sickle cell tests a blood sample of know sickle status, determined by the regional laboratory, was distributed. Accurate results were those that correctly identified the sample as containing sickle haemoglobin or not. If the majority of results from peripheral laboratories for a single test varied from the target result, the supervisors re-evaluated their test target values. Whole blood samples were distributed in order to check the complete process of testing (e.g. pipetting accuracy, malaria smear preparation) rather than just an individual component. Supervisors used results of the quality monitoring to provide constructive feedback to the laboratory staff and to target their training towards tests and laboratories that performed particularly poorly.
Programme funding
Initially the programme was funded directly from Ministry of Health headquarters but subsequently, through collaboration with regional Ministry of Health administrators, several supervisors were able to access their own regional funds.
Programme indicators
Baseline survey
There were 205 laboratories in Ghana located in regional and district government hospitals (97), mission hospitals (53) or health centres (54). The total staff complement in these laboratories was 693. The percentage of staff in each of the ten regions with no professional qualifications (assistants or bench-trained with 6 weeks to 1 year training) varied from 60–83% (mean 74%). Even after correcting for differences in regional populations and excluding regions with teaching hospitals, there were wide variations between regions in the number of laboratories and the availability of essential laboratory equipment such as microscopes, (Table 1). The safety survey showed that over 50% of 62 laboratories surveyed lacked essential items such as automatic pipettes (necessitating mouth-pipetting) (74%), protocols for waste disposal and equipment maintenance (82% and 100% respectively), first aid kits (100%) and fire safety equipment (94%). Other unsafe practices included allowing patients inside the laboratory (89%) and eating and drinking within the laboratory (55%).
Table 1 Regional variations in laboratory resources corrected for population (year 2000) (excluding two regions with teaching hospitals)
Region 2 3 4 6 7 8 9 10 Mean Range
Population (millions) 2.10 1.55 2.65 2.10 1.25 0.70 1.80 1.85
Laboratories Total 16 15 40 7 26 9 57 32
per 100,000 pop. 0.76 0.97 1.51 0.33 2.08 1.29 3.17 1.73 1.48 0.33–3.17
Trained staff Total 13 9 21 11 11 7 19 24
per 100,000 pop. 0.62 0.58 0.79 0.52 0.88 1.00 1.06 1.30 0.85 0.52–1.30
Microscopes Total 30 20 51 NI 16 6 76 20
per 100,000 pop. 1.40 1.29 1.92 NI 1.28 0.86 4.22 1.08 1.72 0.86–4.22
Colorimeters Total 17 8 46 6 6 5 14 14
per 100,000 pop. 0.81 0.52 1.74 0.29 0.48 0.71 0.78 0.76 1.22 0.52–1.74
Centrifuges Total 20 15 46 12 6 6 54 21
per 100,000 pop. 0.95 7.42 1.74 0.57 0.48 0.86 3.00 1.14 2.02 0.48–7.42
Haematocrit centrifuge Total 9 4 39 7 8 2 5 10
per 100,000 pop. 0.43 0.23 1.47 0.33 0.64 0.29 0.28 0.54 0.53 0.23–1.47
Spectrophotometer Total 5 3 13 2 5 2 3 2
per 100,000 pop. 0.24 0.19 0.49 0.95 0.40 0.29 0.17 0.11 0.39 0.11–0.95
Blood mixer Total 5 3 20 0 0 1 10 0
per 100,000 pop. 0.24 0.19 0.75 0 0 0.14 0.56 0 0.24 0–0.75
NI = no information
Extent of training and impact on test accuracy
After 18 months, a mean of 93% (regional variation 58%–100%) of all Ghana's laboratory staff had been trained in malaria-related and other common tests. During the final six months of the programme the supervisors monitored the quality of haemoglobin estimations, sickle cell test and malaria microscopy results in 48% of all Ghana's 205 laboratories. In 4 regions the quality of up to 11 tests had been monitored. Tests that gave quantitative results were consistently the most poorly performed tests in all regions with only 78%, 78% and 84% of laboratories producing acceptable results for haemoglobin measurements, white blood counts and malaria microscopy respectively. Tests for HIV, hepatitis B, sickle-cell screen and ZN stain were consistently performed well with over 95% of laboratories meeting agreed target results. After a further six months training there were improvements in the accuracy of several tests, particularly in haemoglobin estimation and malaria microscopy with 89%, 83% and 91% of laboratories producing acceptable results for these tests respectively, (Table 2).
Table 2 Changes in test accuracy in six months
Mean number of laboratories with accurate results/total surveyed (%)
Test Month 1 Month 6
Haemoglobin 45/58 (78) 49/58 (89)
Malaria microscopy 49/58 (84) 49/54 (91)
Sickle screen 53/53 (100) 51/51 (100)
White blood count 31/40 (78) 40/48 (83)
Blood group 23/24 (96) 57/58 (98)
Cross match 7/7 (100) 7/7 (100)
HIV test 27/28 (96) 28/28 (100)
Hepatitis B antigen 22/22 (100) 40/40 (100)
ZN stain 38/39 (97) 39/39 (100)
Discussion
There is very little information available about the state and quality of laboratory services in peripheral health facilities in poorer countries. The baseline survey showed that in Ghana three quarters of public sector laboratory staff do not have any technical qualifications, and even the supervisory cadre only have Diplomas. Through this study we have shown that the lack of professional qualifications amongst the laboratory workforce and the inequitable distribution of laboratory equipment, are not obstacles to establishing a simple and far-reaching quality assurance process.
This study has shown that the worst performed tests at sub-regional level are those that generate quantitative or subjective results (such as haemoglobin estimation and malaria microscopy) rather than simple 'positive' or 'negative' results (such as HIV and hepatitis B screening tests). Further research is needed to examine the cost-effectiveness of potentially more accurate or less complex tests (e.g. malaria rapid diagnostic tests, HemoCue). Although these tests may be more expensive than those in use in most districts, their simplicity and accuracy may save downstream costs. One of the major limitations of this study is the lack of quality assurance at regional and supra-regional level for common laboratory tests. These were the laboratories responsible for determining the target values of tests distributed to peripheral laboratories. To overcome this problem, regional laboratories tested the samples several times before determining the target values and they also exchanged samples with each other as an inter-regional check on results. Plans are in place to expand this into an inclusive national quality monitoring system for higher level laboratories.
We have confirmed previous work showing that at peripheral health facilities haemoglobin using manual methods is the most inaccurate test [7]. Safety issues are often not a priority for laboratories in poorer countries but overcrowding and poor laboratory organisation are recognized to be associated with a significant risk of accidents and consequent infection of health workers. Many of the safety issues identified through this project could be easily and cheaply rectified but because there was no supervisory system in place they had been ignored.
To our knowledge this is the first external laboratory quality assurance programme in sub-Saharan Africa to demonstrate that it is feasible to achieve widespread coverage of peripheral health laboratories and staff. Through this programme an effective and practical model has been developed for improving the accuracy of common laboratory tests in Ghana. The model is based on a national network of senior technicians who implemented and supervised a continuous cycle of test monitoring and targeted training for all laboratory staff in their regions. The programme's success was dependent on the quality and commitment of the national network of laboratory supervisors. Participation in the programme was itself a strong motivational force for the supervisors. Because of the supportive attitude of the supervisors, the laboratory staff viewed the monitoring visits as educational rather than punitive and this ensured cooperation when remedial action was required. Health planners can use the lessons learnt from this programme to introduce measures to improve morale and job satisfaction of key health workers especially those in the neglected laboratory services. Components of this model have already been adopted by other countries for their laboratory programmes and could be adapted for other health disciplines in poorer countries.
Several steps are necessary before this model could be sustainably implemented. The network of supervisors needs to have pro-active long-term support from all stakeholders, combined with career and promotion packages. Health managers need to provide secure funding for laboratory quality assurance programmes at all levels. This includes establishing internal quality control measures and external validation systems that are linked into national and international external quality monitoring schemes. To be successful, this model will require prioritization of the laboratory service by policy makers at national and supra-national level and adequate representation of the laboratories in decision-making processes. Implementing a quality assurance system for laboratories in poorer countries is expensive and logistically complicated. Managers will need to balance cost against quality, taking into account that it is those who are most vulnerable to ill health who can least afford to bear the brunt of the consequences of inaccurate laboratory results.
Authors' contributions
IB and VB designed the outline framework for the programme. IB documented and collated results. VB was the local programme coordinator. AAA provided an overview and ensured the programme was compatible with Ministry of Health policies. All authors read and approved the final manuscript.
Funding sources
This study was supported by the Ministry of Health in Ghana and the Malaria Knowledge Programme (funded by the Department for International Development, UK) at the Liverpool School of Tropical Medicine. The Department for International Development had no role in study design, the collection, analysis and interpretation of data, in the writing of the report or in the decision to submit the paper for publication and accepts no responsibility for the information or views expressed.
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| 15516269 | PMC529273 | CC BY | 2021-01-04 16:37:27 | no | Malar J. 2004 Nov 1; 3:38 | utf-8 | Malar J | 2,004 | 10.1186/1475-2875-3-38 | oa_comm |
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Biomed Eng OnlineBioMedical Engineering OnLine1475-925XBioMed Central London 1475-925X-3-351549407210.1186/1475-925X-3-35ResearchImage fusion for dynamic contrast enhanced magnetic resonance imaging Twellmann Thorsten [email protected] Axel [email protected] Olaf [email protected] Martin O [email protected] Tim W [email protected] Applied Neuroinformatics Group, Faculty of Technology, University of Bielefeld, Germany2 Cancer Research UK Clinical MR Research Group, Section of Magnetic Resonance, Institute of Cancer Research, Royal Marsden Hospital, Sutton, Surrey, UK2004 19 10 2004 3 35 35 4 6 2004 19 10 2004 Copyright © 2004 Twellmann et al; licensee BioMed Central Ltd.2004Twellmann et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Multivariate imaging techniques such as dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) have been shown to provide valuable information for medical diagnosis. Even though these techniques provide new information, integrating and evaluating the much wider range of information is a challenging task for the human observer. This task may be assisted with the use of image fusion algorithms.
Methods
In this paper, image fusion based on Kernel Principal Component Analysis (KPCA) is proposed for the first time. It is demonstrated that a priori knowledge about the data domain can be easily incorporated into the parametrisation of the KPCA, leading to task-oriented visualisations of the multivariate data. The results of the fusion process are compared with those of the well-known and established standard linear Principal Component Analysis (PCA) by means of temporal sequences of 3D MRI volumes from six patients who took part in a breast cancer screening study.
Results
The PCA and KPCA algorithms are able to integrate information from a sequence of MRI volumes into informative gray value or colour images. By incorporating a priori knowledge, the fusion process can be automated and optimised in order to visualise suspicious lesions with high contrast to normal tissue.
Conclusion
Our machine learning based image fusion approach maps the full signal space of a temporal DCE-MRI sequence to a single meaningful visualisation with good tissue/lesion contrast and thus supports the radiologist during manual image evaluation.
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Background
In recent years, multivariate imaging techniques have become an important source of information to aid diagnosis in many medical fields. One example is the dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) technique [1,2]. After the administration of a gadolinium-based contrast agent, a sequence of d 3D MRI volumes is recorded from a certain part of the body (see Fig. 1). Thus, each spatial coordinate p = (x, y, z) in the volume can be associated with a temporal kinetic pattern vector which is regarded as a point in a signal space (see Fig. 2). The examination of these temporal kinetic patterns at different spatial coordinates in the volume allows the observer to infer information about local tissue types and states (see Fig. 3) [3].
Figure 1 Visualisation of contrast agent concentration as gray value images of the same volume slice at different points of time (Left to right: first precontrast, first postcontrast and fifth postcontrast image). The lesion is located near the centre of the right breast.
Figure 2 Alternative view on a temporal sequence of d 3D MRI volumes: Each spatial coordinate p in a 3D volume can be associated with a d-dimensional temporal kinetic vector xp consisting of measurements of the local intensity at d points of time.
Figure 3 Illustration of temporal kinetic patterns of contrast uptake for normal, benign and malignant tissue (left to right) measured during DCE-MRI with two precontrast and five postcontrast recordings. Especially the strong signal uptake between the two precontrast measurements and the first postcontrast measurement indicates suspicious tissue.
Today, much effort is spent on enhancing the capabilities of the imaging techniques e.g. increasing the spatial and temporal resolution. In contrast to these improvements in image acquisition, much less effort has been spent on effective visualisation methods. Even though several approaches for detection and classification of suspicious lesions in DCE-MRI data of the breast have been proposed (e.g. [4-8]), it is still common practice for the huge amount of data to be analysed manually using simple operations such as subtraction images of two volumes.
Obviously, these images can only comprise a small fraction of the information which is commonly spread over all volumes of the sequences. As a consequence, analysing multivariate images in radiology remains a time consuming and challenging task which potentially can be alleviated by the application of image fusion techniques.
Image fusion
Image fusion methods have been an area of research for several decades. According to Genderen & Pohl [9,10], image fusion 'is the combination of two or more different images to form a new image by using a certain algorithm' e.g. integration of a large number of multivariate images from a remote sensing process into one image. Because Genderen & Pohl already stated PCA as a standard technique for image fusion in remote sensing, we adopt the more general definition of the term image fusion from the remote sensing community. Whereas in the medical imaging community the meaning of the term image fusion is commonly restricted to fusion of multimodal images, the definition of this term used in this article also includes multivariate images such as multispectral or multitemporal images.
Pattern recognition methods such as artificial neural networks (ANN) have gained much attention from the remote sensing community [11-15]. From the point of view of pattern recognition, the problem of image fusion is strongly related to the task of dimension reduction: Ignoring the spatial order of the patterns x, the image data is an unordered set of patterns that forms a data distribution in the data space and image fusion or dimension reduction corresponds to a mapping
to a new low dimensional space which retains certain properties of the original data distribution. Subsequently, the mapped patterns can be spatially ordered according to the locations p of the corresponding sources, leading to the final fused images.
Well-known algorithms such as Principal Component Analysis (PCA) [16] or Self Organising Maps [17] have been successfully applied for various tasks of multispectral or multitemporal image fusion [11-15]. It is important to note that these methods are not bounded with limitations on the dimensionality of . Hence, they are especially suited if is high dimensional.
In this work, we investigate the application of machine learning algorithms to medical image fusion. We compare the results of the standard linear PCA with it's nonlinear extension, the so called Kernel PCA (KPCA) which was proposed by Schölkopf et al. in 1998 [18]. Our empirical observations are presented and discussed by means of DCE-MRI data sets from a breast cancer screening study [19]. Image material presented in this paper is also provided online in original size (PNG format) [20].
Methods
In the following, we briefly describe the theoretical background of the linear PCA and nonlinear KPCA algorithms and their application to the task of image fusion. Both methods determine a set of projection directions, referred to as principal directions (PDs), by optimising a certain criterion. The mapping M is defined by a subset of all possible PDs. Projecting each pattern xp on to one of these PDs associates each spatial position p with a new scalar value (the principal component) of which integrates information from the different components of xp, respectively. The resulting 3D image can be visualised as a gray value image or using perceptually optimised colour scales [21,22]. Alternatively, the low dimensional representation of the patterns can be displayed as RGB composite images, if M is defined by a set of three PDs.
Principal component analysis
Principal Component Analysis is one of the most frequently used dimension reduction method. Suppose the data are given by the set Γ = {xi}, xi ∈ , 0 ≤ i ≤ N, PCA is a transformation in a new coordinate system of uncorrelated and orthogonal principal axes ξ ∈ , |ξ| = 1 which can be derived from the eigenvectors of the covariance matrix
by solving the eigenvalue equation
λξ = Cξ (2)
for λ ≥ 0 and ξ ∈ \ {0}. The first eigenvector ξ1 (the one with the largest eigenvalue λ1) maximises the variance . Therefore, the set of the first n ≤ d eigenvectors or PDs carry more variance than any other n orthogonal projections.
Kernel principal component analysis
In recent years, kernel based methods have been the object of much research effort within the machine learning community. The concept of a subset of kernel methods is based on the combination of well-known linear algorithms such as Principal Component Analysis or Fisher Discriminant Analysis with nonlinear kernel functions [23,24]. While the application of these functions allows more powerful nonlinear solutions, the kernelised algorithms retain most properties of their linear versions.
Consider a nonlinear function
which maps the examples x ∈ Γ to some feature space [25]. Furthermore, assume that the mapped data are centred in . In order to perform the PCA in , one has to find the eigenvectors ξ of the covariance matrix
i.e. those vectors that satisfy with ξ ∈ \ {0} and λ ≥ 0. Substituting (3), it is easy to see that the eigenvectors ξ lie in the span of Φ(x1),...,Φ(xN). Therefore, Schölkopf et al. [26] define the equivalent eigenvalue problem
Nλα = Kα (4)
where α denotes the column vector of coefficients α(1),...,α(N) describing the dual form of the eigenvector by
and K is the symmetric Gram matrix with elements
Kij = K(xi, xj) = Φ(xi), Φ(xj). (6)
Normalising αk corresponding to the k-th eigenvalue λk of K ensures λkαk, αk = 1. Now, principal components can be extracted in by projecting an example x on ξk using
It is crucial to note that for extracting principal components using (4) and (7) the inner product Φ(xi), Φ(xj) is needed rather than the explicit images Φ(xi), Φ(xj) alone. Instead, one can use kernel functions fulfilling Mercer's Theorem such as the Gaussian Kernel
with bandwidth parameter σ or the Polynomial Kernel of degree d
K(xi, xj) = xi, xjd (9)
which allow the PCA in the corresponding to be performed implicitly with reasonable computational costs. For the Polynomial Kernel we have a clear interpretation of KPCA. In this case, is the space of all monomials of degree d of the pattern components. Thus, KPCA is a linear PCA of the corresponding high order statistical features. The KPCA algorithm can be summarised as follows:
1. Calculate the Gram matrix K of Γ using a suitable parameterised kernel function.
2. Transform K according
with . This transformation implicitly moves the centre of mass of the mapped data {Φ(xi)}, xi ∈ Γ to the origin of , i.e. centres the data in .
3. Calculate the eigenvector expansion coefficients αk, i.e. the eigenvectors of and normalise them.
4. Extract principal components using (7).
Compression vs. discrimination
Application of both image fusion techniques leads to a set of up to d PDs in case of PCA and up to N PDs in case of KPCA. In general, a compact visualisation of the complete data as a single image is desired. In this case, inspection of the fused image based on the PD corresponding to the first (largest) eigenvalue is optimal in terms of a general compression scheme: The projection on this PD retains most of the total data variance and leads to a reconstruction with least mean square error. Nevertheless, image fusion is commonly employed with a well defined intention e.g. in order to detect a specific phenomenon such as bushfires in multitemporal satellite images [11] or (as in this work) tumour lesions in DCE-MRI data. In addition to the general compression characteristics, the fused image has to show task-specific discriminative properties which do not necessarily reflect the total data variance. In this case, using a PD corresponding to one of the following eigenvalues may lead to more discriminative visualisations. If the image data are fused by KPCA, an additional degree of freedom can be exploited. In addition to the index of the selected PD, the type and parameterisation of the kernel K can be varied leading to alternative mappings to the feature space, changing the characteristic of the fusion image.
Experiments
In the following, the fusion results of both methods are discussed and illustrated with DCE-MRI sequences from six cases (referred to as S1,...,S6) which were taken during the the MARIBS breast screening study [19]. Each sequence consists of seven 3D MRI volumes of the female breast, recorded with a separation of 90 sec using a standardised protocol (A fast spoiled gradient echo sequence (FLASH) with TR = 12 ms, TE = 5 ms, ip angle = 35°, FOV = 340 mm and coronal slice orientation). Before recording the third volume, a gadolinium-based contrast agent was administered with a bolus injection. Therefore, each spatial position p in the 256 × 128 × 64 (1.33 mm × 1.33 mm × 2.5 mm) sized volume is associated with a pattern , d = 7 describing the temporal signal kinetic of the local tissue.
The images were manually evaluated by an expert who marked voxels of tumour with a cursor on an evaluation device. Below, the kinetic signals of the marked tumour voxels are labelled '+'. Signals corresponding to voxels of the complement of the marked region are labelled '-'.
For this kind of data, experiments of Lucht et al. [5] suggest recording a much longer temporal sequence of 28 images which makes the need for efficient fusion techniques evident.
Evaluation criteria
In order to provide an objective discussion of the value and drawbacks of both algorithms, we focus on the following requirements:
1. The marked region should be visualised with high contrast compared to unmarked regions in order to facilitate detection of kinetic signals which are similar to the marked signals.
2. The fusion image should follow the first criteria without time consuming manual manipulation by the observer (e.g. tuning of transfer functions such as windowing).
Following the first criteria, the purpose of the visualisation is specified implicitly by the voxel labels. In the present work, the expert marked regions of tumour tissues. Thus, optimal fusion images of an image sequence display locations of cancerous kinetic signals with high contrast to normal signals.
Next to the visualisation of the fusion images as gray value and RGB images, both methods are evaluated by means of a receiver-operating-characteristic (ROC) analysis [27,28]. To this end, pixel intensities of the fusion images are interpreted as confidence values for the existence of suspicious signals and are compared with the expert label as ground truth. The ROC analysis objectively measures the applicability of the fusion images for the task of lesion detection.
However, no conclusion can be drawn about how well other tissue types are distinguishable in the fusion images, i.e. how well the information of the entire signal space is represented.
Preprocessing
For numerical reasons, the voxel value range of each volume sequence is individually normalised to [0; 1]. In order to preserve the signal kinetics, the individual minimal and maximal intensity value is determined simultaneously on all d image volumes of each sequence. To ensure this normalisation is robust with respect to single outlier values, the values are calculated based on an application of a 3 × 3 × 3 median filter.
Since about 66% of each volume is covered by background, all images sequences are preprocessed with a full automatic tissue/background separation method. The histogram of the sum of local intensity differences (sod) feature
individually calculated for each sequence, has a bimodal shape and shows a clear separable maximum for the background voxels. The optimal threshold separating background from tissue can be computed automatically [29]. The resulting binary masks are postprocessed with a morphological closing operator [30] to ensure closed masks for the regions of tissue.
Adaptation
In order to automate and optimise the fusion process, a priori knowledge about the phenomenon to be visualised, given by the expert label, is used to find a suitable parameterisation of the algorithms as described in detail in the following section. In practice, these labels are not available for new image sequences. Thus, the algorithms have to be adapted on a small number of image sequences, e.g. from a subgroup of cases of a screening study, which were manually evaluated by a human expert and can be subsequently applied to the data of an arbitrary number of unseen cases.
To assure the experimental setup reflects the circumstances of a practical application, the data sets Γ used for adaptation consist of marked tissue signals from only five of the six image sequences and the sixth unseen image sequence is used for the evaluation of the algorithm's capabilities. This setup is repeated six times, each time using a different image sequence for evaluation. In case of KPCA, using all kinetic signals from the five image sequences is prohibitive due to the computational and memory complexity. Therefore, the KPCA is adapted with a reduced data set Γ consisting of all signals of the marked tumour regions and an equal number of signals randomly selected from non-tumour regions.
Parameter selection
An essential part of kernel methods is the mapping from the data space to the feature space by the kernel function. In this paper, we focus on the frequently used Gaussian Kernel (8) which is parameterised by the bandwidth parameter σ. Selection of this parameter is crucial for the fusion process. For the experiments, s is chosen by scanning the range [0.05,...,2.0] using a step size of 0.05. Because manual evaluation by visual examination of the fusion images of each parameterisation is time consuming, we apply an automatic selection heuristic for the bandwidth based on the component specific Fisher score
with class specific mean μ± and variance v±. The Fisher score is commonly used for ranking components x(k) of a set {(x, y)} of binary labelled (y = ±) examples according to their discriminative power. In a similar manner, the score can be evaluated for different PDs on a random subset of the training set Γ utilising the corresponding principal component values with their associated expert label and thus can be interpreted as a measure for the first evaluation criteria. Furthermore, the sign of the PCA/KPCA based PDs can be adjusted in order to obtain a high value for the average intensity of tumour voxels causing tumour lesions to appear as bright regions.
Thereby, the a priori knowledge of which region of the five image sequences used for adaptation should be visualised with high contrast can be utilised for selecting proper parameterisations which lead to discriminative visualisations tailored to the given task.
Fusion
For each method and image sequence, the first three PDs are used for calculating fused images, referred to as I1, I2 and I3. For the purpose of visualisation, the range of the voxel values is normalised to [0; 255]. Additionally, I1, I2 and I3 are composed in to an RGB image IRGB. For fusion images based on KPCA, the bandwidth for each Ik is chosen according to the individual maximum of the Fisher criterion as illustrated in Fig. 10.
Figure 10 Plot of Fisher score values for PD1 of the KPCA algorithm with varying bandwidth. The score indicates a varying magnitude of separation between the class of suspicious tissue signals and the class of normal tissue signals. Below, the fusion image I1 for S1 based KPCA with four different bandwidth values A, B, C and D is shown. Variation of the bandwidth leads to fusion images with varying imaging properties. The bandwidth B leads to a fusion image that displays the tumour with the highest contrast to the surrounding tissue and the Fisher score shows a peak at the corresponding position. For bandwidth values A, C and D, the Fisher score and the contrast in the fusion images decreases.
Results
Fusion results for the sequences S1,...,S6 based on the PCA algorithm are shown in the lower 2 × 2 block of Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8 and Fig. 9. For all six sequences, the fusion image I1 based on the PD with the leading eigenvalue does not lead to discriminative visualisations. The tumour lesions appear with the same intensity as fatty tissue, while glandula tissue is displayed as dark areas (S3, S4). In contrast to I1, the discriminative power of I2 is obviously much greater for all six image sequences. The display of the tumour lesions (high intensity values) differs significantly from areas of glandular tissues, blood vessels (medium intensity values) and fatty tissue (low intensity values). The contrast between tumour lesion and the surrounding tissue decreases in I3 of S2, S3 and S5. Additionally, the surrounding tissue is displayed less detailed (S1, S2, S4, S5). According to the weak discriminative characteristic of I1 and I3, the tumour lesions are coloured with shadings of green or cyan in the corresponding IRGB.
Figure 4 Fusion images I1, I2, I3 and corresponding colour composite image IRGB for sequence S1 based on KPCA (upper 2 × 2 block) and PCA (lower 2 × 2 block). The lesion is located near the centre of the left breast.
Figure 5 Fusion images I1, I2, I3 and corresponding colour composite image IRGB for sequence S2 based on KPCA (upper 2 × 2 block) and PCA (lower 2 × 2 block). The lesion is located in the lower left part of the left breast.
Figure 6 Fusion images I1, I2, I3 and corresponding colour composite image IRGB for sequence S3 based on KPCA (upper 2 × 2 block) and PCA (lower 2 × 2 block). The lesion is located near the centre of the left breast.
Figure 7 Fusion images I1, I2, I3 and corresponding colour composite image IRGB for sequence S4 based on KPCA (upper 2 × 2 block) and PCA (lower 2 × 2 block). The lesion is located near the centre of the right breast.
Figure 8 Fusion images I1, I2, I3 and corresponding colour composite image IRGB for sequence S5 based on KPCA (upper 2 × 2 block) and PCA (lower 2 × 2 block). The lesion is located near the implant in the right breast.
Figure 9 Fusion images I1, I2, I3 and corresponding colour composite image IRGB for sequence S6 based on KPCA (upper 2 × 2 block) and PCA (lower 2 × 2 block). The lesion is located near the centre of the left breast and is surrounded by glandular tissue.
Fusion images based on KPCA are shown in the upper 2 × 2 block of Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8 and Fig. 9. For S1, S2, S3 and S4, image I1 displays the tumour lesion with high contrast to the surrounding tissues. Adipose tissue appears in I1 and I2 with mediumin tensity. In I2 of S4 and S3, glandular tissue can be observed in addition to the tumour. These areas appear dark in I1. The fraction of glandular tissue regions in I2 of S1 and S2 is much smaller, since the tumour is located near the chest muscle where the breast mostly consists of fatty tissue and blood vessels.
An interesting detail can be observed in I3 of S4. The image clearly shows a ring structure as part of or around the tumour lesion. At positions inside the ring which are displayed with high intensity values in I1 and I2, the temporal kinetic patterns show a fast uptake with a following constant or slightly decreasing concentration of the contrast agent. In contrast to the signals inside the ring, all signals corresponding to the ring structure in I3 show as teadily increasing concentration. In all composite images IRGB except for S5, the tumour lesions are coloured white and can be easily discriminated from fatty tissue (shadings of blue to purple) and glandular tissue (shadings of blue to green). For image S5, only I1 shows a discriminative characteristic. The tumour is displayed as a small cluster of high intensity values in the lower right area of the right breast, next to the implant.
According to common practice, the curves obtained from the ROC analysis of the fusion images I1, I2 and I3 are compared by measuring the area-under-the-curve(AUC) values. The corresponding AUC values are listed in Tab. 1. The fusion image yielding the highest AUC value is printed bold for each sequence. For five of six sequences, a fusion image based on PCA yields the highest AUC value (column PCA in Tab. 1). The fusion image I2 based on the second PD of the PCA algorithm significantly outperforms the corresponding PCA based fusion images I1 and I3. A similar predominance of I2 can be observed for the KPCA based AUC values (column KPCA in Tab. 1). Here, I2 outperforms I1 and I3 in four of six cases (S1, S2, S3 and S5). Only for S4 and S6 the fusion image I1 yields the largest AUC value. Nevertheless for KPCA, the difference to the corresponding fusion images I1 and I3 is much less distinct. In particular I1 yields AUC values which are close to those of the corresponding fusion image I2. The predominance of the second component also decreases, if the PCA algorithm is trained with the reduced data set used for adaptation of the KPCA (column PCA (reduced) in Tab. 1). In comparison with the results of the PCA adapted with the entire data set, the AUC values of I2 decrease and increase for the fusion images I1 and I3.
Table 1 Area under ROC curve values for fusion images I1, I2 and I3 for series S1,...,S6 based on KPCA, PCA and PCA trained with the same reduced training set as KPCA. For each AUC value, the pixel intensities of the fusion images are interpreted as confidence values indicating the existence of suspicious signals at the corresponding positions. The largest AUC value for each case is printed bold.
Area-Under-ROC-Curve
KPCA PCA PCA (reduced set)
Sequence I1 I2 I3 I1 I2 I3 I1 I2 I3
S1 0.950 0.972 0.879 0.539 0.993 0.633 0.772 0.972 0.692
S2 0.918 0.945 0.728 0.727 0.993 0.712 0.852 0.948 0.547
S3 0.995 0.998 0.710 0.520 0.997 0.926 0.799 0.997 0.747
S4 0.996 0.985 0.259 0.926 0.999 0.919 0.992 0.985 0.963
S5 0.959 0.966 0.904 0.693 0.997 0.925 0.814 0.964 0.344
S6 0.994 0.986 0.706 0.926 0.999 0.919 0.785 0.986 0.802
The influence of the bandwidth σ on the fusion characteristic is illustrated in Fig. 10. For small values of the bandwidth σ only a small fraction of the tumour lesion appears with high intensities. If the bandwidth is chosen according to the maximum of the Fisher score, the lesion is visualised with high contrast to the surrounding tissue. In the shown example, the Fisher criterion decreases along with the contrast of the visualisation for further increasing bandwidth values.
Discussion
The results shown in the preceding section indicate that fusion of DCE-MRI data by PCA or KPCA leads to compact and meaningful visualisations. Lesions are correctly displayed as bright regions or with specific colouring and can be easily discriminated from surrounding tissue. Once a small subgroup of cases is evaluated, the obtained secondary information in the form of labelled tumour areas is utilised for automation of the data processing and presentation: (i) The sign of the PD is selected in a way that tumour lesions always appear with high intensities. (ii) The parametrisation of the kernel function of the KPCA is optimised in such a way that the fusion images show the desired discriminative characteristics. Thus, both evaluation criteria stated in the section Evaluation criteria are accomplished.
Although both methods are applicable for the task of image fusion, several properties should be discussed in more detail. According to the ROC analysis and visual appraisal, the fusion image I2 based on PCA shows for nearly all cases a discriminative characteristic which is superior to all other fusion images based on PCA or KPCA. While I1 based on PCA captures the slightly increasing elucidation of the major part of the breast, caused by minor accumulation of contrast agent in tissues such as fat, the fusion image I2 corresponding to the second PD of PCA shows the lesions with high contrast to the surrounding tissue. This can also be observed by means of the PDs itself. Figure 13 shows a plot of the components of the three PCA based PDs. The plot of PD1 shows anearly constant or slightly increasing curve, whereas the plot of the components of PD2 is similar to a typical temporal course of contrast agent concentration insuspicious tissue (see Fig. 3). The plot of PD3 shows increasing values for the components corresponding to the postcontrast measurements. From this follows that the major part of the signal variance is caused by voxels which exhibit signals at different intensity levels with only minor changes of intensity in the course of time. This fraction of data variance is captured by PD1 of PCA. The next major source of variance is the signal uptake between the precontrast and the first postcontrast measurement insuspicious tissue which is captured by PD2 and leads to the superior discriminative characteristics of the fusion image I2. PD3 is sensitive to signals which show a continuously increasing intensity for the postcontrast measurements. Hence, I3 is more discriminative than I1, but less discriminative than I2.
Figure 13 Plot of the components of the vectors PD1 (solid), PD2 (dashed) and PD3 (solid with crosses) based on the PCA algorithm. The plot of PD2 shows a typical signal of suspicious tissue (see Fig. 3) and therefore leads to discriminative fusion images with high intensity values at positions of tissue that exhibits a significant signal uptake after injection of the contrast agent.
The ROC analysis of the KPCA based fusion images indicates that the fusion images I2 show superior discriminative characteristics for four of six cases (S1, S2, S3 and S5). However, selection of a suitable kernel parametrisation leads to comparable AUC values for I1. For fusion images corresponding to PDs with smaller eigenvalues, KPCA based images still show more details than those based on PCA, if the bandwidth value is chosen according to the maximum of the Fisher score. Figure 11 shows the KPCA based (left column) and the PCA based (right column) fusion images I4, I5 and I6 for sequence S4. While KPCA distributes the total data variance on N PDs, the PCA method uses only d PDs. Therefore, the PCA based fusion images I4, I5 and I6 typically contain a large fraction of high frequent noise. It is important to note that the fusion images based on KPCA are not necessarily uncorrelated, if each image is calculated using PDs with different bandwidth values, and therefore may display redundant information. In five of six cases, RGB visualisations based on KPCA show the tumour lesion as white regions which are easy to discriminate from other tissue types. In contrast to subtraction images which also allow detection of lesions with high sensitivity (see e.g. [4]), the fusion images IRGB provide a more comprehensive display of the data. A single subtraction image displays only the information of a two dimensional subspace of the signal space , i.e. the information of two manually selected components of the signal vector. Without further manipulation of the transfer function and after selection of two suitable components, a subtraction image commonly shows the lesion as a cluster of high intensity values and other types of tissue are not displayed or indistinguishable. The fusion images are low dimensional representations of the entire signal space. Thus, the RGB composite images IRGB based on PCA or KPCA clearly display the lesion in combination with glandular or fatty tissue and major blood vessels.
Figure 11 Fusion images I4, I5 and I6 for S3 based on the PDs with the fourth, fifth and sixth largest eigenvalue. The left column shows the fusion images based on KPCA. Each fusion image was calculated with a bandwidth that was individually optimised according to the Fisher score. The right column shows the same images fused with PCA. In contrast to the KPCA based fusion images, these images show a significant fraction of high frequent noise and less details.
One drawback of KPCA is the increased computational and memory complexity in contrast to PCA. In case of KPCA, the complexity scales with the size N of the training set Γ. During the adaptation of KPCA, an N × N sized kernel matrix has to be stored and manipulated, whereas the covariance matrix for PCA is only of size d × d. Thus for KPCA, the computation time (LINUX system / 1.8 GHz Pentium IV / 2 GB RAM) for the adaptation, i.e. calculation of the kernel matrix and extraction of 3 PDs, increases significantly with the size of the training set Γ and takes 73 seconds for Γ consisting of 2700 training items which is comparable to the computation time of the PCA for the given setup (see Fig. 14). While even for large matrices, a subset of eigenvectors can be extracted in a reasonable time using efficient numerical software packages like LAPACK [31], the memory complexity obviously limits the size of Γ. One way to address this problem is to subsample the data. Instead of using a random sample of the whole data set, the chosen scheme assures the presence of tumour voxels in the training set. In the former case, the presence of a larger number tumour voxels is unlikely because of the unbalanced ratio between number of tumour voxels and the number of non-tumour voxels. Nevertheless, the reduction of the training data causes a degradation of the detection performance and changing fusion characteristics (see Fig. 12).
Figure 14 Computation time for adaptation of KPCA (solid line). The measured time includes calculation of the kernel matrix and the extraction of the first three PDs. Additionally, the time for adaptation of the PCA using the complete training data is shown (dashed line).
Figure 12 Image I1, I2, I3 and IRGB of S3(top block) and S4(bottom block) fused by the PCA algorithm which was adapted on the same reduced data set as KPCA.
More important for practical applications of both methods is the computational expense for calculation of the fusion images. Using PCA, the value of a fusion image voxel is equivalent to the inner product of two d-dimensional vectors and the calculation of the three fusion images I1, I2 and I3 of one volume slice takes approximately 1 second. In case of KPCA, the inner product has to be calculated in the feature space and the PD in is only implicitly given as an expansion of N kernel functions. Thus, computation of I1, I2 and I3 of one volume slice takes approximately 23 seconds for training sets Γ consisting of 1000 examples and increases linearly with the size of Γ (Fig. 15).
Figure 15 Computation time for the three fusion images I1, I2 and I3 of one slice using PCA (dashed line) and KPCA (solid line). The computation time of principal component values with KPCA increases linearly with the size of the training set. For PCA, the computation time depends only on the dimension of the signal pattern and is constant for the given setup.
In consideration of the fact that both methods are able to fuse the multitemporal DCE-MRI to single meaningful images which do not only show the lesion with high intensities, but also other types of tissue such as fatty or glandular tissue, the standard linear PCA seems to be most suitable for the given signal domain because of it's low computation time and superior detection performance. Only for PCA, the three fusion images can be calculated for a complete volume in a reasonable time and without delaying the diagnostic process. According to the ROC analysis, the introduction of nonlinearity by the kernel function did not improve the discriminative properties of the fusion images, but visual appraisal of the RGB composite images based on KPCA suggest a more comprehensive display of the different types of tissue. It is an open question whether fusion images of other data domains with more complex or higher dimensional signals might benefit more obviously from the nonlinearity of KPCA.
Conclusion
In this paper, we have demonstrated the integration of distributed information from DCE-MRI image sequences to meaningful visualisations by means of PCA and KPCA. Both methods were able to accentuate the regions marked by the expert as important in image sequences blinded to automatic analyses. By the employment of task-specific information, the parametrisation of the KPCA algorithm was optimised in order to accentuate the relevant characteristics of the visualisation.
List of abbreviations
PCA Principal Component Analysis
KPCA Kernel Principal Component Analysis
PD Principal Direction
DCE-MRI Dynamic Contrast Enhanced Magnetic Resonance Imaging
Authors' contributions
T. Twellmann, A. Saalbach and T. W. Nattkemper conceived the experimental setup. Implementation and realisation was done by O. Gerstung. Image acquisition was done under supervision of M. O. Leach.
Acknowledgements
The authors would like to thank the advisory staff, radiologists, geneticists and surgeons of the MARIBS Breast Screening Study:
Principal Investigator: M.O. Leach. Manchester: C. Boggis, S. Reaney, M. Wilson, J. Hawnaur*, J. Adams, D.G.R. Evans, A. Shenton. Sutton &St George's: P. Kessar*, G. Brown, J. Murfitt, R. Eeles*, R.S. Houlston, A. Arden-Jones, K. Bishop, S. Gray, F. Lennard, S. Shanley, N. Rahman, R. Houlston. Newcastle: J. Potterton, A. Coulthard, L. McLean, M. McElroy, W. Wotherspoon, F. Douglas. Cambridge: R. Warren, A.K. Dixon, P. Britton, R. Sinnatamby, A. Freeman, J. Mackay, J. Rankin. Edinburgh: J. Walsh, B.B. Muir, A. Murray, C.M. Steel, E. Anderson, J.M. Dixon. Leeds/Sheffield/Hull: L.W. Turnbull, G. Hall, P. Kneeshaw, A. Hubbard, G. Liney, C. Chu, O. Quarrell, J. Kumar. Guy's &St Thomas, London: S. Rankin, S. McWilliams, J. Pemberton, A. Jones, E. Sanderson, S.V. Hodgson. Aberdeen: F.J. Gilbert, G. Needham, H. Deans, K. Duncan, L. Gomersall, G. Iyengar, N. Haites, H. Gregory. Southampton: C. Rubin, M. Briley, S. Hegarty, G. Michaels, M. Reddy, D.M. Eccles. Dundee: D.G. Sheppard, J. Rehman, A. Cook, C. Walker, D. Goudie. Bristol: N. Slack, I. Lyburn, A. Jones, E. Kutt, J. Basten, M. Shere, S. Cawthorn, Z. Rayter. Birmingham: C.P. Walker, S. Bradley, M. Wallis, T. Cole, C. McKeown, J. Morton Glasgow: L. Wilkinson, J. Litherland, C. Cordiner, R. Davidson Liverpool: G. Whitehouse, D. Ritchie, F. White, I. Ellis Belfast: G. Crothers, McAllister, P. Morrison Northwick Park: W. Teh, B Shah, J. Paterson Portsmouth: P. Gordon, P. Buxton, J. Domsan UCH London: M. Hall-Craggs Barnet: G. Kaplan. Mount Vernon: A.R. Padhani, K. Raza. Study staff: L. Pointon, R. Hoff, D. Thompson, K. Chan, M. Khazen, C. Hayes, R. Fowler, M. Sydenham, E. Moore, J. Anderson, C. Levesley. Advisory Group members (not mentioned elsewhere): J. Brown (Bristol, MRC HSRC), D. Easton (Cambridge), L. Walker (Hull), S. Moss (Sutton).
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LAPACK – Linear Algebra PACKage
| 15494072 | PMC529274 | CC BY | 2021-01-04 16:37:31 | no | Biomed Eng Online. 2004 Oct 19; 3:35 | utf-8 | Biomed Eng Online | 2,004 | 10.1186/1475-925X-3-35 | oa_comm |
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Biomed Eng OnlineBioMedical Engineering OnLine1475-925XBioMed Central London 1475-925X-3-371550068710.1186/1475-925X-3-37ResearchAssessment of the dynamics of atrial signals and local atrial period series during atrial fibrillation: effects of isoproterenol administration Mainardi Luca T [email protected] Valentina DA [email protected] Leonida [email protected] Claudio [email protected] Massimo [email protected] Federico [email protected] Sergio [email protected] Department of Biomedical Eng., Polytechnic University of Milan, Via Golgi 39, 20133 Milano Italy2 Cardiologia, Dipartimento di Medicina, Chirurgia e Odontoiatria, Osp. San Paolo, Università di Milano, via A. di Rudini 8, 20142 Milan, Italy3 Electrophysiology Laboratory, S Ambrogio Hospital, Milan, Italy2004 22 10 2004 3 37 37 26 7 2004 22 10 2004 Copyright © 2004 Mainardi et al; licensee BioMed Central Ltd.2004Mainardi et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The autonomic nervous system (ANS) plays an important role in the genesis and maintenance of atrial fibrillation (AF), but quantification of its electrophysiologic effects is extremely complex and difficult. Aim of the study was to evaluate the capability of linear and non-linear indexes to capture the fine changing dynamics of atrial signals and local atrial period (LAP) series during adrenergic activation induced by isoproterenol (a sympathomimetic drug) infusion.
Methods
Nine patients with paroxysmal or persistent AF (aged 60 ± 6) underwent electrophysiological study in which isoproterenol was administered to patients. Atrial electrograms were acquired during i) sinus rhythm (SR); ii) sinus rhythm during isoproterenol (SRISO) administration; iii) atrial fibrillation (AF) and iv) atrial fibrillation during isoproterenol (AFISO) administration. The level of organization between two electrograms was assessed by the synchronization index (S), whereas the degree of recurrence of a pattern in a signal was defined by the regularity index (R). In addition, the level of predictability (LP) and regularity of LAP series were computed.
Results
LAP series analysis shows a reduction of both LP and R index during isoproterenol infusion in SR and AF (RSR = 0.75 ± 0.07 RSRISO = 0.69 ± 0.10, p < 0.0001; RAF = 0.31 ± 0.08 RAFISO = 0.26 ± 0.09, p < 0.0001; LPSR = 99.99 ± 0.001 LPSRISO = 99.97 ± 0.03, p < 0.0001; LPAF = 69.46 ± 21.55 LPAFISO = 55 ± 24.75; p < 0.0001). Electrograms analysis shows R index reductions both in SR (RSR = 0.49 ± 0.08 RSRISO = 0.46 ± 0.09 p < 0.0001) and in AF (RAF = 0.29 ± 0.09 RAFISO = 0.28 ± 0.08 n.s.).
Conclusions
The proposed parameters succeeded in discriminating the subtle changes due to isoproterenol infusion during both the rhythms especially when considering LAP series analysis. The reduced value of analyzed parameters after isoproterenol administration could reflect an important pro-arrhythmic influence of adrenergic activation on favoring maintenance of AF.
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Background
Atrial Fibrillation (AF) results from multiple, rapidly changing and spatially disorganized activation wavelets sweeping across the surface of the atria [1]. Among factors contributing to genesis and / or maintenance of circulating wavelets, Autonomic Nervous System (ANS) seems to play a major pro-arrhythmic role [2]. The arrhythmogenic influence of sympathetic and vagal mechanisms has been documented in several clinical and experimental studies [3,4]. In men, ablation of the major parasympathetic pathways to the atria drastically reduced vagally mediated atrial fibrillation [4]. It has also been reported that sympathetic stimulation by shortening atrial refractory periods, may increase vulnerability to atrial fibrillation in different experimental models [5]. The shortening of action potential duration and flattening of the restitution slope to cycle length changes induced by adrenergic activation, are two of the mechanisms favoring spiral wave induction and restraining spiral wave break-up [6]. Changes in action potential may also contribute to the perpetuation of atrial fibrillation [7].
In normal hearts, both vagal and sympathetic mechanisms have been associated with paroxysmal atrial fibrillation (PAF) initiation. Most of PAF episodes observed in patients with structural heart disease are triggered by sympathetic activation and vagal withdrawal [8].
Spectral analysis of heart rate variability before PAF episodes has further clarified the pro-arrhythmic role of the autonomic nervous system [9,10]. Bettoni [9] observed a primary increase in adrenergic drive occurring over at least 20 minutes before onset of PAF episodes followed by a shift towards a vagal predominance immediately before arrhythmia onset. Other authors described an increase in sympathetic modulation of sinus node (or a loss of vagal modulation) before PAF onset in the majority of patients [10-12]. More recently Lombardi [7] reported that signs of sympathetic activation characterized up to 70% of PAF episode onset, whereas in the remaining ones a vagal predominance was detectable. An increase in vagal modulation can also promote the stability of AF [13].
Even if AF has been classically described as a random process, a few studies have recently documented, using various signal processing methods, the existence of some determinism underlying AF. Linear analysis techniques documented relationships between intra-atrial recordings using both time-domain methods [14] and spectral-domain approaches [15,16], while the presence of non-linear patterns have been also recognized [17,18]. By using linear and non-linear indexes we have recently assessed [19] the dynamics of intra-atrial signal and local atrial period (LAP) series during different AF episodes. In particular, regularity (R) and synchronization (S) indexes [20], based on the estimation of the corrected conditional entropy and the corrected cross-conditional entropy respectively, were used to describe the dynamics in intra-atrial signals, whereas the LAP series were investigated using regularity and the level of predictability (LP). These parameters were suitable to describe the fine changing characteristic of atrial signals and LAP series [19] when passing from different atrial rhythms classified according to the Wells' criteria [21]. In the present paper, we evaluated whether changes in adrenergic control mechanisms could influence determinisms and dynamics of atrial signals and exploited the capability of linear and non-linear parameters (R and S indexes for intra-atrial signals, R and LP indexes for LAP series) to capture them. Adrenergic activation was mimicked by isoproterenol infusion. The effects of this sympathomimetic drug was evaluated in a small group of patients with a history of PAF during sinus rhythm and atrial fibrillation: four experimental conditions were analyzed (sinus rhythm (SR), sinus rhythm during isoproterenol administration (SRISO), atrial fibrillation (AF) and atrial fibrillation during isoproterenol administration (AFISO)).
Experimental protocol
Patient population
Nine patients (8 males and 1 female; mean age 60 ± 6 years) selected to sustain a left atrial ablation with encirclement of the pulmonary veins by transeptal approach were included in the study. All subjects were suffering from atrial fibrillation (AF) and were non responsive to anti-arrhythmic therapy (pharmacological therapy and electrical cardioversion). Paroxysmal and persistent AF episode were present in, respectively, 5 and 4 subjects. A history of AF was present for an interval ranging from 2 months to 10 years. The mean left ventricular ejection fraction was > 40% in all patients; the mean left atrial diameter was 37 ± 3 mm in 7 patients and 51 ± 8 mm in 2. Structural heart disease was present in 4 patients. Reported symptoms included palpitations (6 subjects), fatigue after effort (9 subjects) and syncope (2 subjects). Arterial hypertension was the most common comorbidity in our study group (4 patients). All the patients were in anti-arrhythmic drug wash-out at the time of the study. Flecainide, propafenone, metoprolol, cordarone and methyldigoxin were ceased ≥ 5 half-lives before ablation. Transoesophageal echocardiography was performed the day before the procedure to exclude atrial thrombus.
The Medical Ethical Committee approved this study and all subjects gave their written consent.
Study design
We investigated the effect of adrenergic activation induced by infusion of isoproterenol on atrial electrical activity. The electrophysiological procedure was performed in the Electophysiology Laboratory of the "Istituto Clinico Sant'Ambrogio" of Milan, Italy. Intracavitary electrocardiograms were recorded during the ablation procedure in which arrhythmic foci inside the pulmonary veins of the left atrium were electrically isolated.
The research project protocol included an intracavitary recording of multiple atrial electrograms during sinus rhythm and after induction of atrial fibrillation. In both experimental conditions, the recording was repeated during intravenous infusion of isoproterenol (0.01–0.02 mcg/kg/min) tiered to determine a 30% increase of heart rate. The four clinical experimental conditions were defined as sinus rhythm (SR), sinus rhythm during isoproterenol administration (SRISO), atrial fibrillation (AF) and atrial fibrillation during isoproterenol administration (AFISO). Details on the four epochs of the study can be found in Figure 1.
Figure 1 Timing and sequences of the experimental protocol epochs. The experimental protocol included four recording periods during: I) sinus rhythm (SR); II) sinus rhythm during isoproterenol infusion (SRISO); III) atrial fibrillation (AF); IV) atrial fibrillation during isoproterenol infusion (AFISO). The recordings during SR lasted at least 5 minutes (range 5 – 8 minutes), those during AF 90 seconds on average (range 60 – 120 seconds). The recordings during infusion of isoproterenol started after the drug had determined a 30% increase of heart rate. Induction of AF started 15 minutes after the end of SRISO, to guarantee the correct isoproterenol wash-out.
In all the patients, AF inducibility was obtained at twice diastolic threshold by burst atrial pacing (5'-second bursts at an output of 20 mA) from the mid coronary sinus beginning at a cycle length of 250 ms and reducing by 10 ms intervals until atrial refractoriness. All the nine patients were inducible. AF was considered inducible if it persisted for more than 1 minute. If AF terminated after less than 1 minute, induction was repeated until a maximum of 3 times. If AF became sustained (lasting > 10 minutes), ablation was performed after external DC cardioversion. All our patients underwent the procedure in spontaneous SR.
The duration of registration was an important parameter for the reliability of the analysis, because an insufficient number of atrial potentials (less than 250 – 300) could give errors in the estimation of conditioned probability. Therefore at least 5 minutes (range 5 – 8 minutes) of sinus rhythm and 90 seconds (range 60 – 120 seconds) of atrial fibrillation were registered.
The electrophysiological study was carried out using a deflectable 20 pole St Jude catheter (length 95 cm, 7 F, interelectrode spacing 2 – 10 mm), a deflectable decapolar catheter with a distal ring configuration, Lasso-Cordis Biosense Webster catheter (length 115 cm, 7 F, interelectrode spacing 2 – 5 mm) and 4 mm distal electrode catheter, Medtronic Sprinklr, with irrigated tip (for ablation, length 115 cm, 7 F, interelectrode spacing 2 – 5 mm). The St Jude catheter was in contact with the right atrial wall and inserted in the coronary sinus below the left atrium. The Lasso-Cordis Biosense Webster catheter and the Medtronic Sprinklr catheter were positioned in the superior pulmonary veins at the inside of the atrium.
For the purpose of this study, one surface ECG tracing and nine intracavitary atrial electrograms were stored on digital memory for subsequent off line analysis. In all patients, electrograms labeled 2 – 3 – 4 – 5 corresponded, respectively, to the superior, middle, middle inferior and inferior wall of the right atrium; electrogram 6 to coronary sinus ostium; electrograms 7 – 8 – 9 indirectly corresponded to the inferior and the left wall of the left atrium whereas electrogram 10 to the left superior pulmonary vein.
Electrograms 2 – 9 were recorded with 20 pole St Jude catheter, electrogram 10 with Lasso-Cordis Biosense Webster catheter.
Methods and Data analysis
Regularity
Conditional entropy (CE) may be used to estimate a regularity index, defined as the degree of recurrence of a pattern in a signal. CE represents the amount of information carried by the most recent sample x(i) of a normalized realization of x when its past L - 1 samples are known. CE is defined as [22]:
where p(xL - 1) represents the probability of the pattern xL - 1 (i - 1) of length L - 1 (xL - 1 (i - 1) = {x(i - 1),..., x(i - L + 1)}) and p(x(i) / xL - 1) the conditional probability of the sample x(i) given the pattern xL - 1. In (1) the first summation is extended to all the possible xL - 1 patterns, the second one is extended to all the different Lth samples of the pattern xL (i) (xL (i) = {x(i), xL - 1 (i - 1)}).
CE is maximum if x is complex and unpredictable and it reaches zero as soon as a new sample can be exactly predicted from the previous L -1 ones.
Using CE over short data series can cause an unreliable estimate of CE (CÊ) : when the conditioning pattern xL - 1 (i - 1) is found only once in the series x (i.e. p(x(i) / xL - 1) = 1), CÊ decreases to zero with L. As a consequence both periodic and completely unpredictable signals exhibit CÊ equal to zero when L increases. Therefore the corrected conditional entropy (CCE) must be introduced to perform a reliable measure over short data series:
CCE(L) = CÊ(L) + perc(L)·Ê(1) (2)
where perc(L) is the percentage of length L patterns found only one time in the data set and Ê(1) is the estimate of Shannon entropy of the process x. perc(L)·Ê(1) represents the corrective term that compensates the null information associated to the pattern found only once and it increases with L, while CÊ(L) decreases with L.
The minimum value of the CCE is the best estimate of CE and it's taken as an index of complexity: the larger the index, the less predictable the processes. The CCE is normalized by the Shannon entropy of the process in order to derive an index independent of the different probability distribution of the processes, thus obtaining:
An index of regularity (the opposite of complexity) may be defined as:
Rx = 1 - min(NCCE(L)) (4)
Rx tends to zero if x is a fully unpredictable process, it tends to one if x is a periodic signal and it assumes intermediate values for those processes that can be partially predicted by the knowledge of the past samples [20].
Synchronization
The cross-conditional entropy is introduced to define an index of synchronization, related to the repetition of a complex pattern involving two signals.
Given two normalized signals, the cross-conditional entropy of x given a pattern y is defined as [20]:
where p(yL - 1) represents the probability of the pattern yL - 1 (i) and p(x(i) / yL - 1) the conditional probability of the sample x(i) given the pattern yL - 1.
CEx/y represents the amount of information carried by the most recent sample of the signal x when L - 1 past samples of y are known. Over short data series, this definition suffers from the same limitations as conditional entropy, so analogously corrective terms and normalization are introduced. The uncoupling function (UF) is defined as:
UFx,y(L) = min(NCCEy/x(L), NCCEx/y(L)) (6)
in order to measure the amount of information carried by one signal that can't be derived from the knowledge of past samples of the other signal. In this way both causal directions are tested and it is taken the one that leads to the best prediction. For every length L pattern, UF chooses as input the signal that can be the best predictor of the other one. Besides, the joint pattern does not take into account past samples of the output signal to prevent to have a high coupling strength only because one signal has a large index of regularity.
The minimum of UF is taken as an index of uncoupling between x and y, therefore an index of synchronization (the opposite of uncoupling) can be defined as:
Sx,y = 1 - min(UFx,y (L)) (7)
and it quantifies the maximum amount of information exchanged between the two signals. Sx,y tends to zero if the two processes are uncoupled, it tends to one if they are perfectly synchronized and it assumes intermediate values when the two signals are able to exchange a certain amount of information [20].
Level of predictability
A discrete time series x(n) can be modeled as the output of an autoregressive model of p order
where n is the discrete-time index, the ak are the model coefficients and w(n) is a Gaussian white noise process of variance feeding the model. The actual sample differs from its model prediction, thus generating the prediction error
An index of the level of predictability (LP) may be defined as follows
LP = (1 - σe / σx) (10)
where σe is the standard deviation of e(n) and σx is the standard deviation of the process x. LP measures the percentage of power which may be predicted by the autoregressive model. In the case of a purely random signal (σe is quite close to σx) LP tends to zero, while in the case of a linearly predictable signal (σe tends to zero) the index tends to one and it assumes intermediate values for those processes that may be partially predicted from the model.
Signal pre-processing
All signals were appropriately recorded and digitized to a 1000 Hz sampling rate at 16-bit resolution. All bipolar electrograms were band-pass filtered (40 – 250 Hz) to remove baseline shift and high frequency noise. In order to cancel the possible effects of ventricular interference (affecting especially recordings during sinus rhythm), the averaged ventricular interference complex was computed and subtracted from each atrial signal [16]. In details, from the surface ECG, the occurrences of QRS were determined, and a template of the ventricular interference in each atrial electrogram was constructed by signal-averaging windows of 140 ms around each QRS (windows were positioned 40 ms before and 100 ms after the R wave). The template was then subtracted from the atrial signals at each occurrence of QRS.
After detecting time-instants of local atrial depolarization using a derivative / threshold algorithm, the detected depolarizations were visually scored and missed / erroneous detections were corrected by an expert operator using an interactive software. Then, the local atrial period (LAP) series were derived as the sequence of temporal distances between two consecutive local atrial activations [19] (the procedure is shown in Figure 2).
Figure 2 Extraction of local atrial period (LAP) series from intra-atrial signal. (a) Example of a recorded electrogram during sinus rhythm before pre-processing; (b)-(c) the related LAP series, obtained as the sequence of temporal distances between two consecutive local atrial activations, after detecting time-instants of local atrial depolarization using a derivative / threshold algorithm. a.u. = arbitrary unit
In analogy with previous studies [14,23], after canceling the ventricular interference, the absolute value of the output of the band-pass was low-pass filtered (50 Hz) and then sub-sampled (100 Hz) principally to reduce signal length and computation time.
Considering the number of recorded signals and patients, we analyzed 79 recordings in SR and in SRISO and 77 recordings in AF and in AFISO. Twelve recordings were disregarded for the low quality of electrograms. For each recording, atrial signals were divided into six-second segments and then analyzed. Regularity index was estimated for each six-second segment in each recording site and for each patient, while synchronization index was estimated for each pair of close recording sites (interelectrode distance equal to one).
Statistical analysis
The statistical analysis was carried out using Student's t-test for paired data, comparing each rhythm before and after isoproterenol administration, and between organized (SR) and not organized rhythm (AF).
Results
Atrial signals
Figure 3 shows an example of the distribution of regularity index (R) computed in one patient and in a single recording site (electrogram 8) during the four experimental conditions. The R values, computed in the six-second segments, are superimposed to their mean value. A significant reduction (p < 0.001) of the index passing from sinus rhythm to AF was detectable. Comparing the results obtained from the same rhythm with and without isoproterenol, a reduction of R was observed after drug administration in both sinus rhythm and atrial fibrillation. In this particular case, the decrease during sinus rhythm was statistically significant (p < 0.001). In particular, considering all patients recording sites, we observed 59 reductions (42 with p < 0.05) over 79 recordings passing from SR to SRISO and 40 (17 with p < 0.05) over 77 passing from AF to AFISO. This result reflects the global tendency of entire dataset, as illustrated in Table 1, where the mean value obtained from all patients recording sites is showed, underlining a statistically significant reduction passing both from SR to AF and from SR to SRISO.
Figure 3 Values of the R index in a single subject. Example of regularity (R) index computed for a patient in the electrogram 8 during the four phases of the analysis (SR, SRISO, AF, AFISO). Performance of the R index in the various six-second segments (circle) is superimposed to its mean value. A significant reduction of the R index can be observed passing both from sinus rhythm to atrial fibrillation and from sinus rhythm to sinus rhythm after isoproterenol administration. *p < 0.001
Table 1 Mean ± SD values of the proposed indexes in the four analyzed phases
SR SRISO AF AFISO
AS
R 0.49 ± 0.08 0.46 ± 0.09† 0.29 ± 0.09* 0.28 ± 0.08
S 0.28 ± 0.02 0.28 ± 0.03 0.20 ± 0.06* 0.20 ± 0.06
LAP
R 0.75 ± 0.07 0.69 ± 0.10† 0.31 ± 0.08* 0.26 ± 0.09†
LP 99.99 ± 0.001 99.97 ± 0.03† 69.46 ± 21.55* 55 ± 24.75†
Mean ± SD values of the proposed indexes in the four analyzed phases: sinus rhythm (SR), sinus rhythm during isoproterenol infusion (SRISO), atrial fibrillation (AF) and atrial fibrillation during isoproterenol infusion (AFISO) for Atrial Signals (AS) and LAP series.
† p < 0.0001 the comparison of a same rhythm before and during isoproterenol infusion.
The t-test comparing organized (SR) to not organized (AF) rhythms always results significant (* p < 0.0001).
Concerning the synchronization index (S), a significant decrease was observed only when comparing sinus rhythm to atrial fibrillation (Table 1). Evaluating results separately for every recording pair, we observed 35 decreases (7 with p < 0.05) over 69 values passing from SR to SRISO and 32 (9 with p < 0.05) over 66 passing from AF to AFISO.
Local atrial period
LAP series were analyzed using the level of predictability and the regularity index. Figure 4 illustrates an example of the LAP series during SR, SRISO, AF, AFISO and the corresponding NCCE function. The regularity (R = 1 - min(NCCE)) decreases visibly passing from sinus rhythm to atrial fibrillation. A decrease in both SR and AF after isoproterenol administration is also observed. The mean values showed in Table 1 are obtained as all patients mean and they underline an analogous tendency. In particular, regularity reductions are found in 7 patients after isoproterenol administration during both sinus rhythm and atrial fibrillation (5 with p < 0.05 passing from SR to SRISO; 2 passing from AF to AFISO).
Figure 4 LAP series in the four analyzed phases and the corresponding NCCE functions. Example of LAP series during (a) SR, (b) SRISO, (c) AF, (d) AFISO; (e)-(h) the corresponding NCCE functions (solid lines) depicted as sum of two terms: the decreasing one (CE, dotted line) and the increasing one (the corrective term, dash-dotted line). A clear increase in the minimum value (*) can be observed passing from SR to SRISO to AF to AFISO; therefore the R index = 1 - min(NCCE) (see text for more details) decreases passing from SR to SRISO to AF to AFISO.
Figure 5 shows the local atrial period series during SR, SRISO, AF, AFISO and the corresponding prediction errors. The prediction error increases passing from SR to SRISO to AF to AFISO. All patients mean reflects this tendency as shown in Table 1. In particular, considering single patients, reductions of level of predictability is observed in 8 of them after isoproterenol administration during both sinus rhythm and atrial fibrillation; the reductions are statistically significant (p < 0.05) in 6 patients passing from SR to SRISO, and only in 4 passing from AF to AFISO.
Figure 5 LAP series in the four analyzed phases and the corresponding prediction errors. Example of LAP series during (a) SR, (b) SRISO, (c) AF, (d) AFISO; (e)-(h) the corresponding prediction errors. A prediction error increase can be observed passing from SR to SRISO to AF to AFISO, showing the inability of the autoregressive model to predict the LAP series as the rhythm becomes less organized. This is equivalent to a reduction of the LP index = (1 - σe / σx) passing from SR to SRISO to AF to AFISO.
Discussion
The autonomic nervous system plays an important role in the genesis and maintenance of atrial fibrillation, but characterization and quantification of its pro-arrhytmic effects are extremely complex and therefore difficult to define. Aim of this study was to evaluate the capability of linear and non-linear parameters to capture the fine changing in the dynamics of atrial signals and LAP series during adrenergic activation induced by the injection of a sympathomimetic drug. The existence of determinism and of an underlining order during AF has recently been shown. In particular both linear [14-16] and non-linear [17,18] patterns have been recognized. In the present study, where a relative small population is considered, we observed a reduction of spatial organization after isoproterenol administration both in sinus rhythm and in atrial fibrillation. In particular analysis of LAP series showed a significant decrease of both LP and R indexes within the same rhythm after isoproterenol administration (see Table 1). This reduction could be related to an increase of atrial wave fronts disorganization. In fact in previous studies [19], both indexes were demonstrated to decrease passing from SR to AF-I and AF-II Wells' classes. Therefore it can be argued that the reduction observed after isoproterenol infusion may be a sign of a high disorganization induced by the drug: atrial activation patterns become less periodic, less predictable and less regular. In fact, in agreement with our previous findings [19], the higher is the regularity and predictability of sequence of atrial activation, the fewer are the circulating 'mother' wavelets according to Jalife's model [24]. This finding is in agreement with known effects of sympathetic activation at atrial level [25] and may provide additional insights to the understanding of the pro-arrhythmic role of ANS in patients with AF. In addition, as previously reported [19], a marked reduction of R and LP indexes was observed passing from SR to AF.
Concerning results obtained from atrial signals, both synchronization and regularity indexes showed a marked reduction passing from SR to AF well in keeping with previous findings [19] that documented the capability of these indexes to discriminate between organized and not-organized rhythms. However the two indexes were not able to evidence any differences after isoproterenol infusion. Only the R index was found significantly decreased in SR after drug administration. Nevertheless, the parameters revealed a tendency toward organization reduction after isoproterenol administration in both rhythms. In particular, responses to isoproterenol were more homogenous during sinus rhythm than during AF. This is maybe due to the fact that patients with a clinical history of AF, could already present alteration in atrial electrical properties likely to be involved in their predisposition to develop AF. Accordingly isoproterenol effects on dynamics of atrial signals are more evident in an organized rhythm (SR) than in AF, where an already disorganized rhythm can not be further fragmented by drug infusion. During AF in fact it has been suggested [1] that several wavefronts of electrical activity propagate through the atria in an irregular manner; this activity may partly obscure isoproterenol effects. Nevertheless, it has also been reported that atrial electrical activity may vary not only in relation to arrhythmia duration but also in relation to the structural characteristics of the atria [26]. In conclusion, the proposed set of linear and non-linear parameters is able to capture subtle changes in atrial dynamics during AF and drug infusion. These indexes could be employed to provide new insight into the mechanisms leading to initiation and maintenance of AF episodes.
==== Refs
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| 15500687 | PMC529297 | CC BY | 2021-01-04 16:37:31 | no | Biomed Eng Online. 2004 Oct 22; 3:37 | utf-8 | Biomed Eng Online | 2,004 | 10.1186/1475-925X-3-37 | oa_comm |
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Biomed Eng OnlineBioMedical Engineering OnLine1475-925XBioMed Central London 1475-925X-3-381550068910.1186/1475-925X-3-38ResearchDecrease of resistance to air flow with nasal strips as measured with the airflow perturbation device Wong Lily S [email protected] Arthur T [email protected] Biological Resources Engineering, University of Maryland, College Park, MD, 20742, USA2 Biological Resources Engineering, University of Maryland, College Park, MD, 20742, USA2004 22 10 2004 3 38 38 16 3 2004 22 10 2004 Copyright © 2004 Wong and Johnson; licensee BioMed Central Ltd.2004Wong and Johnson; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Nasal strips are used by athletes, people who snore, and asthmatics to ease the burden of breathing. Although there are some published studies that demonstrate higher flow with nasal strips, none had directly measured the effect of the strips on nasal resistance using the airflow perturbation device (APD). The APD is an inexpensive instrument that can measure respiratory resistance based on changes in mouth pressure and rate of airflow.
Method
This study tested forty-seven volunteers (14 men and 33 women), ranging in age from 17 to 51. Each volunteer was instructed to breathe normally into the APD using an oronasal mask with and without nasal strips. The APD measured respiratory resistance during inhalation, exhalation, and an average of the two.
Results
Results of a paired mean t-test comparing nasal strip against no nasal strip were statistically significant at the p = 0.05 level. The Breathe Right™ nasal dilator strips lowered nasal resistance by an average of 0.5 cm H20/Lps from an average nasal resistance of 5.5 cm H20/Lps.
Conclusions
Nasal strips reduce nasal resistance when measured with the APD. The effect is equal during exhalation and during inhalation.
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Background
Nasal dilator strips (NDS) are used by athletes, people who snore, and asthmatics to ease the burden of breathing. The nasal strips are used as a mechanical means of reducing nasal airflow resistance [1]. By lowering nasal resistance, they reduce the work of breathing and the supply of oxygen into the body could increase [2,3].
The size of the nostril limits the amount of air entering into the body. The NDS is placed along the nasal valve of the nose. The adhesiveness of the strip binds to the creases of the nasal valve to prevent the outer wall tissue of the nose from collapsing inward during nasal breathing. This mechanism thus dilates the nose and allows more air to flow into the nose [3].
The primary effect of the NDS could be either to dilate the air passage of the nose or to stiffen the nasal wall. Either mechanism would reduce nasal resistance and allow higher flow of air, but they can be distinguished over a range of air flows. Stiffening the nasal wall would have its most profound effect at higher flows where the Bernoulli effect would decrease internal nasal pressures and tend to constrict nasal passage diameter. Air passage dilation would tend to decrease nasal resistance more uniformly over a range of air flows.
Recent studies on the effectiveness of Breathe Right™ nasal strips tested participants under various rest and exercise conditions. Some found that the strips neither improve or diminish airflow [2,4-9], which contradict results found by others [1,3,10-14]. Various techniques were used to assess NDS effectiveness. Some measured the amount of airflow, others the area of the nostrils, and still others the nasal airflow resistance.
The Airflow Perturbation Device (APD) is a small, light weight, and easy to use instrument that measures respiratory resistance [15]. A segmented rotating wheel in the air flow path changes air flow and mouth pressure as the wheel momentarily partially obstructs the flow passage (Figure 1). The magnitude of these perturbations depends on the resistance of the wheel and respiratory resistance. Measurement of wheel resistance allows respiratory resistance to be calculated directly (Figure 2). Resistance values appear on a computer screen within a minute from starting the measurement. Thereafter they are updated as they occur.
Figure 1 The APD Sensor consists of a rotating wheel in the air path, a pneumotach, and pressure transducers [15]
Figure 2 The APD system consists of pressure and flow transducers, analog-to-digital conversion, and a computer display of results [1].
People breathe normally through the APD. No special breathing maneuvers are required. For this reason, the APD can be used with young children, older adults, unconscious patients, and animals. Respiratory resistance can be separated into inhalation and exhalation components, and resistance can be displayed against lung volume and air flow rate.
Respiratory resistance is normally measured through the mouth, with a nose clip and hands pressed against the cheeks. An oronasal mask may be used to obtain combined mouth and nose resistance, or nose resistance by itself if the mouth is closed. The APD used with an oronasal mask should be an ideal instrument to assess the effect of NDS.
This study is as much a test of the capabilities of the APD as it is a study of NDS. Objectives of this study were to: 1) determine if APD measurements of respiratory resistance measured with an oronasal mask matched those with breathing through a mouthpiece, and 2) measure the effects of NDS on respiratory resistance made with the APD. This is not a clinical study.
Methods
This study tested forty-seven volunteers (14 men, 33 women; age 17–51 yr; height 147–188 cm; weight 38–105 kg). Some had symptoms of nasal congestion such as asthma, allergies, and snoring. A written informed consent was obtained from each subject and the protocol was approved by the University of Maryland Institutional Review Board (IRB).
The nasal strips used in this study were a commercial product called "Breathe Right" (CNS, Inc., Minneapolis, MN, clear, medium/large nasal strips). According to the manufacturer's instructions, the nasal strips should be placed halfway down the nose along the nasal valve. The two end regions of the nasal strips should cover the left and right nasal creases.
This study contained three phases: 1) to determine the ability of the APD to measure oral resistance using either a mouthpiece or an oronasal mask, 2) to determine the ability of the APD to measure nasal resistance, and 3) to determine the effect of nasal strips on nasal resistances. Phase I consisted of two tests that measured oral breathing resistance. In the first test, the subject's nose was occluded with two layers of Durapore surgical tape (3 M, St. Paul, MN) while the subjects were sitting in an upright position. The subject was instructed to breathe normally into a cardboard mouthpiece. The second test repeated the same procedure as the first test, except the subject was breathing into an oronasal mask (Adult Mask 4–5+, Laerdal Medical, Wappingers Falls, NY). The subject was instructed to press their face against the mask while he/she breathed normally. The size of the mask was large enough that it contacted only the hard tissue on the bridge of the nose and did not compress the soft nasal septum.
The second phase of this study consisted of a test to measure nasal breathing resistance. The subject was instructed to breathe normally through the nose with the mouth closed and with no NDS. For the third phase, the subject placed a NDS across the nasal valve on his/her nose as shown on the instructions provided by the manufacturer. In both of these tests, the oronasal mask was used.
Air flow perturbations with the APD occur at a rate of about 10 per second [15]. Measurements were obtained in these experiments over approximately 100 perturbations. It has been previously found that measurements made over that time are relatively stable and reproducible [15]. Several time-averaged resistance values are displayed: resistance during inhalation, 2) resistance during exhalation, and 3) the average of inhalation and exhalation resistances. All three of these have been found to be useful.
Primary comparisons for this study were made using the average respiratory resistance. Secondary comparisons in the second phase of this study investigated the effects of NDS on inhalation and exhalation respiratory resistances. Statistical comparisons were made using a paired mean t-test with significance at the p = 0.05 level.
Results
Subject data appear in Table 1. Average resistance measured during mouth breathing with mouthpiece ranges from 1.90 to 5.03 cm H2O/Lps. In the past, average respiratory resistances for healthy adults have generally fallen in the range of 2.5 - 3.5 cm H2O/Lps, and such is the case here. Also, as expected, most respiratory resistance values during exhalation exceed those measured during inhalation.
Table 1 Subject data for APD Measurements of Respiratory Resistance when Measured Through the Mouth and Nose. Resistances are given in cm H2O/Lps.
Subject No. Sex Mouth Piece Mask Mouth Mask Nose Mask NDS
Inh Avg Exh Inh Avg Exh Inh Avg Exh Inh Avg Exh
1 F 3.15 3.06 2.97 2.86 3.08 3.30 4.65 5.13 5.61 4.08 4.13 4.18
2 F 3.00 3.26 3.52 2.98 3.29 3.59 5.84 5.72 5.59 3.64 3.47 3.30
3 F 2.69 3.19 3.68 2.84 3.38 3.92 4.94 5.73 6.52 4.55 5.26 5.97
4 F 4.00 4.62 5.25 3.97 4.46 4.95 5.40 5.87 6.31 5.11 5.45 5.79
5 F 3.13 4.04 4.95 3.58 4.11 4.63 5.33 5.59 5.85 4.62 5.30 5.98
6 M 2.14 2.41 2.69 2.10 2.34 2.58 3.76 4.15 4.53 3.71 4.16 4.61
7 F 2.73 3.15 3.85 2.43 3.11 3.79 3.92 4.16 4.39 3.54 3.62 3.69
8 M 2.03 2.33 2.64 2.12 2.24 2.36 4.32 4.50 4.69 3.65 3.85 4.06
9 M 1.87 1.96 2.06 1.83 1.82 1.80 3.16 3.53 3.91 3.26 3.47 3.68
10 M 3.49 3.95 4.41 3.27 3.90 4.52 5.96 6.27 6.57 6.13 6.20 6.28
11 F 2.89 2.97 3.04 2.76 2.91 3.07 7.59 7.33 7.07 7.48 7.05 6.61
12 F 3.67 4.24 4.82 4.18 4.38 4.58 6.36 7.14 7.92 6.75 6.82 6.90
13 M 2.20 2.54 2.89 2.13 2.49 2.84 5.27 5.50 5.73 4.80 5.04 5.27
14 F 3.08 2.98 2.88 2.98 3.07 3.17 5.12 5.25 5.38 5.34 5.36 5.38
15 M 2.37 2.45 2.53 2.64 2.77 2.90 5.44 5.63 5.82 5.33 5.57 5.81
16 M 3.48 3.58 3.68 3.16 3.47 3.77 5.71 5.30 4.90 4.71 4.88 5.04
17 M 2.40 2.85 3.30 2.22 2.74 3.26 4.54 4.90 5.25 4.38 4.70 5.07
18 F 2.96 2.96 2.95 2.95 3.04 3.12 6.84 7.19 7.54 6.71 6.89 7.07
19 M 2.00 2.30 2.59 2.54 2.10 1.67 3.82 4.25 4.68 3.20 3.39 3.57
20 F 2.40 2.85 3.30 2.81 2.77 2.73 4.12 4.16 4.20 3.61 3.73 3.85
21 F 4.22 4.20 4.17 4.34 4.42 4.49 6.76 6.97 7.10 6.36 6.75 7.14
22 F 2.73 3.12 3.50 3.02 3.12 3.22 6.60 6.74 6.89 5.90 6.08 6.26
23 F 4.87 5.03 5.19 4.49 4.96 5.42 5.96 6.23 6.70 5.06 5.30 5.54
24 F 4.38 4.79 5.20 4.41 4.64 4.87 6.35 6.68 7.01 5.70 6.05 6.39
25 F 2.61 3.02 3.44 2.51 2.94 3.36 3.98 4.03 4.08 3.56 3.42 3.28
26 M 2.12 2.42 2.71 1.80 2.26 2.71 5.87 5.84 5.81 5.31 4.38 5.44
27 F 3.22 3.77 4.31 3.48 3.58 3.68 4.51 4.76 5.01 4.24 4.58 4.93
28 F 2.85 3.01 3.17 2.74 2.94 3.14 4.71 4.64 4.56 3.57 4.17 4.76
29 F 2.84 3.26 3.67 3.04 3.39 3.75 4.94 5.56 6.18 4.40 5.04 5.68
30 F 2.76 3.08 3.40 2.81 3.05 3.29 6.24 6.40 6.56 5.57 5.76 5.94
31 F 2.90 3.21 3.51 2.68 2.88 3.09 4.71 4.88 5.04 5.03 5.16 5.29
32 M 1.76 1.90 2.03 1.54 1.87 2.20 4.02 4.49 4.97 4.00 4.18 4.35
33 F 3.65 3.94 4.23 3.76 3.92 4.09 4.90 5.30 5.71 4.90 5.26 5.61
34 M 1.94 2.15 2.36 1.97 2.17 2.36 5.19 4.90 4.61 4.37 4.36 4.36
35 F 2.78 2.93 3.09 2.81 3.05 3.29 3.81 4.45 5.10 3.75 4.20 4.65
36 F 5.21 5.09 4.97 4.46 4.81 5.71 4.81 5.19 5.56 4.00 4.04 4.08
37 F 3.18 3.44 3.69 3.02 3.34 3.66 5.49 5.70 5.91 5.50 5.58 5.65
38 F 2.77 3.48 4.20 2.62 3.31 4.00 6.70 6.60 6.51 5.97 6.12 6.26
39 F 3.66 3.57 3.48 3.53 3.54 3.55 7.62 7.73 7.83 6.94 7.33 7.72
40 F 2.57 3.05 3.52 2.58 2.97 3.36 3.64 4.13 4.63 3.58 3.78 3.98
41 F 3.76 3.74 3.71 3.17 3.68 4.18 4.70 5.11 5.51 4.13 4.54 4.95
42 M 3.27 3.39 3.58 3.43 3.61 3.80 6.08 6.44 6.79 5.90 6.25 6.60
43 F 3.27 3.65 4.03 3.89 3.68 4.47 4.47 4.78 5.09 3.69 3.98 4.27
44 M 2.23 2.51 2.79 2.17 2.34 2.51 6.48 6.42 6.35 5.87 6.01 6.15
45 F 2.09 2.95 3.02 2.98 3.07 3.16 6.10 6.65 7.20 5.86 6.20 6.55
46 F 2.98 3.12 3.26 3.15 3.23 3.30 5.70 5.65 5.60 4.57 4.68 4.78
47 F 2.67 2.64 2.61 2.56 2.56 2.57 4.35 4.74 5.12 3.29 3.64 3.99
Average 2.96 3.24 3.51 2.96 3.21 3.48 5.24 5.50 5.74 4.80 5.00 5.25
Std dev 0.76 0.75 0.82 0.73 0.76 0.88 1.09 1.02 1.03 1.11 1.10 1.12
Breathing through the mouth into the oronasal mask yielded almost the same values. Means of values with the mouthpieces and oronasal mask are 3.24 and 3.21, respectively. The difference was not statistically significant using a paired-t test at p = 0.05.
Figure 3 shows the graph of average respiratory resistance of mask vs. mouthpiece while breathing through the mouth. The graph has a slope of nearly 1.0 and an intercept of nearly 0.0, indicating a nearly perfect correspondence between the two methods of measurement. Both slope and intercept were tested statistically and the line was found to be identical to y = x at the p = 0.05 level. Comparison of inhalation resistance between mouthpiece and oronasal mask yielded the following equation:
Figure 3 Average oral respiratory resistance measured with a mouthpiece and an oronasal mask. There is almost perfect agreement between the two methods.
y = 0.9624 + 0.1041 R2 = 0.8435 (1)
where y = mask value of resistance and x = mouthpiece value of resistance
This equation was tested to be statistically equivalent to y = x. This indicates that the oronasal mask had no effect on the inhalation values.
A similar comparison of exhalation resistances gave the following:
y = 0.874bx + 0.4594 R2 = 0.8828 (2)
This equation did not pass the statistical test for equivalence y = x. The oronasal mask may have affected the measurement of respiratory resistance in the exhalation direction.
Figure 4 shows the relationship of average respiratory resistance when breathing through the nose measured with and without the nasal strips. The NDS data have a slope of nearly 1.0 and a y-intercept is approximately -0.4. This signifies a reduction of nasal breathing resistance using the nasal strip. Nasal resistances with no nasal strip range from 3.53 to 7.73 cm H2O/Lps, while nasal resistance with the NDS ranges from 3.39 to 7.33 cm H2O/Lps. This demonstrates the expected resistance reduction with NDS.
Figure 4 Average respiratory resistance while breathing through the nose in 47 subjects. Nasal strips showed a decrease of nasal resistance of 0.43 cm H2O/Lps.
All subjects except three showed a decrease in nasal resistance when breathing with the NDS. Average value of nose breathing without NDS was 5.50; with NDS it was 5.00. These means were highly statistically significantly different.
The effect of NDS on resistance during exhalation was also statistically highly significant. There was an average reduction of 0.45 cm H2O/Lps in resistance, and only six out of 47 subjects failed to demonstrate a decrease in resistance with NDS.
The effect of NDS on resistance during inhalation tested to be statistically highly significant, as well. The average resistance reduction was 0.49 cm H20/dps. Again, six subjects failed to demonstrate a decrease in resistance with NDS. These were not the same subjects that increased resistance in the exhalation direction.
Resistance differences with and without NDS in the inhalation and exhalation directions were tested to determine if NDS had a larger effect while breathing in one direction or the other. Means of the differences for inhalation and exhalation directions were tested with a paired t-test, and found to be statistically nonsignificant. It appears, therefore, that NDS affect nasal resistances equally during inhalation and exhalation.
Discussion
This study confirmed the results of other studies that showed a reduction of about 10% in nasal breathing resistance, as well as supported the claim of the manufacturer that the nasal strips provide nasal relief. Several subjects who had nasal congestion reported some relief in nasal breathing when using the nasal strip. Exactly which subjects these were was not recorded.
There was one surprise, though, in the results. The reduction in respiratory resistance due to NDS was a constant amount and not proportional to the resistance level present without NDS. This result was not expected, and no adequate explanation for it can be given at this time. It is not clear why this should be so, but we do not doubt that measurements made with the APD are correct, based on previous studies [15,16].
We cannot comment on the clinical significance of the resistance reduction with NDS use. It seems likely that some benefit could be obtained from such a resistance change, but whether it is actually detectable is not clear. Other reports in the literature [17,18] have concluded that the minimum detectable external resistance is about a constant 25–30% proportion of the resistance already present. The resistance change measured in this study is about 10% of the baseline resistance. If the use of NDS does result in a detectable change, then it may be that a different detection mechanism is operating. It is possible that the subject could detect nasal resistance only, rather than total respiratory resistance. Based on that supposition, NDS reduce nasal resistance by about 17%.
The APD has been shown to be able to measure respiratory resistance with either a mouthpiece or an oronasal mask. This may be a significant advantage of the instrument, especially because respiratory resistance measurement on unconscious or uncooperative patients would be much more easily made with a mask than with a mouthpiece. Equations (1) and (2) show the close correspondence between measurements made with both techniques, although the presence of an intercept and a slope different from unity indicate that the correspondence between mask and mouthpiece is not perfect. Resistances with a mask are both higher than resistances with a mouthpiece. The reason for this seems to be different mouth positions in both cases. We have laboratory experiences (not published) that demonstrate that tongue position can influence measured resistance. Breathing through the mask is probably done with the mouth closed more than when breathing through the mouthpiece. The measured difference between inhalation and exhalation resistances could reflect the effect of a pressure difference across the distensible smaller airways, which is greater inside than outside during inhalation, but smaller inside than outside during exhalation. This would lead to a dynamic compression of the small intrathoracic airways during exhalation. Another possible explanation is natural movement of the vocal chords such that they are closer during exhalation than during inhalation.
This study was a good test of the capabilities of the APD measuring device. Testing confirmed that the APD can detect resistance changes, and that measurements are easy to obtain. Results in this study are generally more consistent than other studies using other techniques [1-11]. The fact that the APD directly measures respiratory resistance, and is not an indirect measurement may be one reason for this consistency. Then, again, our subject population exhibited some homogeneity in age, social class, and racial makeup.
Conclusions
Nasal strips reduce nasal resistance by about 0.5 cm H2O/Lps. Thus, nasal strips do have a measurable effect on nasal resistance. The effect of NDS appears to be equal during exhalation and during inhalation.
The APD can be used to measure nasal resistance, and can detect resistance levels.
The APD can consistently measure oral resistance with either a mask or a mouthpiece.
Authors' Contributions
LW conducted the testing as an undergraduate student. ATJ provided the APD and mentored LW. All authors have read and approved this manuscript.
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| 15500689 | PMC529298 | CC BY | 2021-01-04 16:37:31 | no | Biomed Eng Online. 2004 Oct 22; 3:38 | utf-8 | Biomed Eng Online | 2,004 | 10.1186/1475-925X-3-38 | oa_comm |
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Int J Equity HealthInternational Journal for Equity in Health1475-9276BioMed Central London 1475-9276-3-101549407710.1186/1475-9276-3-10ResearchMusculo-skeletal pain among 40- and 45-year olds in Oslo: differences between two socioeconomically contrasting areas, and their possible explanations Brekke Mette [email protected] Per [email protected] Department of General Practice and Community Medicine, University of Oslo, Box 1130 Blindern, N-0317 Oslo, Norway2 Per Hjortdahl, Department of General Practice and Community Medicine, University of Oslo, Box 1130 Blindern, N-0317 Oslo, Norway2004 19 10 2004 3 10 10 27 5 2004 19 10 2004 Copyright © 2004 Brekke and Hjortdahl; licensee BioMed Central Ltd.2004Brekke and Hjortdahl; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The objective of the study was to compare the prevalence and severity of musculo-skeletal pain between two socioeconomically contrasting areas in Oslo, Norway, and to explore possible explanatory factors.
Methods
Questionnaire survey, carried out as part of The Oslo Health Study in 2000–2001. Data from 821 persons (40 and 45 year old) living in a less affluent inner city area (called east) were compared with 854 persons living in an affluent area of the city (called west). Bivariate comparisons (chi square test) and multiple regression analyses were performed to investigate differences between the samples.
Results
61 % in east and 56 % in west (p < 0.05) reported pain/stiffness in muscles/joints during the last four weeks. 30 % in east versus 19 % in west (p < 0.001) reported extensive pain. The between area difference in extensive pain was partially explained by physical inactivity, mental health problems and being of non-Western origin.
Conclusion
Musculo-skeletal pain is reported by 55–60 % of middle aged persons in Oslo during a four week period, and must be considered a normal phenomenon. Poor social conditions, inactivity, mental health problems and being an immigrant imply increased risk of more severe symptoms with a concomitant demand of health care.
population surveysocial inequalities in healthmuskulo-skeletal disordersmental healthimmigrants
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Background
In affluent societies like Norway, living conditions as well as general health status have improved during the last decades. In spite of this, social health inequities still exist, and recent analyses from Oslo even indicate an increase during the last 30 years [1]. Life expectancy for men living in the least affluent city area is 69 years, compared to 76 for men in the most affluent area [2]. Well-known risk factors like smoking, physical inactivity and overweight, as well as the incidence of atherosclerotic disease and several forms of cancer show similar correlation [3].
Some claim that future research on this topic should concentrate exclusively on interventions [4]. In Great Britain as well as Holland this has been highlighted for some time [5], and in Holland a national strategy for tackling health inequities has been developed [6]. Nevertheless, we need to continuously keep an eye on trends, as well as on causal and maintaining factors. And even if we have ample data on socioeconomic inequity regarding mortality and morbidity, we know far less about the dimensions of disease severity and patients' coping ability, in Oslo as elsewhere [7]. The aim of the present study was to investigate differences in prevalence and severity of musculo-skeletal pain between middle aged inhabitants of two socioeconomically contrasting areas in Oslo, based on a recent and comprehensive data collection, and to examine some possible explanatory factors. We chose to study musculoskeletal pain, as this is a major cause of disability in the industrialised world [8]. In Norway, musculoskeletal pain generates 15–20 % of consultations in primary care, and is one of the main reasons for sick leave and social security [9].
Methods
The data collection was part of the Oslo Health Study, a joint collaboration between the Oslo City Council, the University of Oslo and the Norwegian Institute of Public Health, which was conducted from May 2000 to September 2001. All residents born in 1924/25, 1940/41, 1955, 1960 and 1970 (n = 41353) received the three-page main questionnaire by mail, as an invitation to participate in a health screening. At the screening station a simple clinical examination and a blood test were performed, and the questionnaire was handed in. Two supplementary questionnaires were given out: one identical for all age groups, and one in four different versions. Participants were asked to fill in the supplementary questionnaires at home and return them by mail. Two reminders were sent to non-respondents. An overview of all topics covered in the questionnaires (in English) can be obtained from .
In the present study we analysed data from persons born in 1955 and 1960, who lived either in the inner eastern part of Oslo or in the outer western part (see below). We used data from the main questionnaire as well as from the age specific supplementary questionnaire.
The variables included from the main questionnaire were: marital status, educational level, employment status, disability pension, social assistance, country of origin, physical exercise, alcohol intake, smoking habits, general health status, mental health problems, and musculo-skeletal disorders. Country of origin was recoded as Western (Western Europe, North America, Australia) or non-Western (Eastern Europe, North Africa, Sub-Saharan Africa, Middle East, Indian subcontinent, Eastern Asia, Pacific, Middle America, South America) [10].
Mental health problems were assessed by the following question: Below is a list of various problems: Have you suffered from any of the following during the last week, including today? Put a cross for every problem. Choices: Not troubled, slightly troubled, quite a lot troubled, much troubled (values 1–4). The values were summarised and divided by the number of answers, and a mean value of 1,85 or more was used as a marker of mental health problems [10].
Musculo-skeletal pain was explored by the following question: Have you suffered from pain and/or stiffness in muscles and joints in the course of the last four weeks? Choices: Not troubled, somewhat troubled, very troubled (values 1–3) for the alternatives neck/shoulders, arms/hands, upper back, lower back, hips/legs/feet and elsewhere. The values were summarised and divided by the number of answers. A mean value of 2 or more was used as an indicator of extensive pain/stiffness in muscles or joints [10].
The variables from the age specific supplementary questionnaire included were: own income, household income, muscular pain/stiffness last 4 weeks, duration of muscular pain/stiffness, satisfaction with health care, and belief in own coping ability.
The east and west areas
Oslo's local authority districts can be ranked according to: level of income, education, employment, disability pension, housing standard, number of non-western immigrants, and mortality [11,12]. According to this ranking, three districts in the western part of the city are on top, indicating the best socioeconomic conditions. These are the districts Vindern, Røa and Ullern, here called west. Three districts in the inner eastern part take on the least favourable positions: Sagene-Torshov, Grünerløkka-Sofienberg and Gamle Oslo, here called east. Per January 1st 2000, west had 67296 inhabitants and east 80 668. (Since the study was done, the city of Oslo has reorganized the local authority districts. Vindern and Røa are joined under the name Vestre Aker, and the names of two others are changed to Sagene and Grünerløkka). We have chosen to compare these two areas, because they are strongly contrasted regarding living conditions.
Statistical analyses
Statistical analyses were performed using SPSS version 11.0. Bivariate comparisons of categorical variables were examined by the chi square test. Multiple regression analyses (stepwise) were performed to estimate the explanatory power of independent variables. A 5 % level of significance was chosen.
Results
The main questionnaire was completed by 821 forty- and 45 year olds living in east (50.7 % women) and 854 living in west (62.9% women), corresponding to a response rate of 39.0 % in east and 43.9 % in west. Some returned the questionnaire without attending the health screening, meaning that 1348 persons completed the supplementary questionnaires.
There was no significant difference regarding full time employment, and frequent use of alcohol was more common in west. For all other socioeconomic and lifestyle variables, as well as general and mental health, east came out poorer (Table 1).
Table 1 Oslo Health Study 2000–2001, 40- and 45- year olds Demographic variables, lifestyle, and self-reported health in east, west, and the whole city (percent).
East West City
Education =< 9 years 14.0 2.0 8.3
Education > 12 years 57.3 87.8 65.8
Single status 15.4 8.0 9.5
Employment full time 67.4 71.21 72.8
Disability pension 10.5 2.7 5.6
Social benefit 6.9 0.5 2.6
Own income < 200 000 30.2 19.6 24.6
Own income > 400 000 8.5 30.0 20.0
Household income < 200000 21.0 4.0 12.0
Household income > 500000 22.7 66.9 46.2
Non-western country of origin 24.5 5.4 16.8
No hard exercise 35.1 19.5 28.7
Alcohol at least once a week 49.4 66.9 52.9
Daily smoking 41.4 22.4 32.5
Less than good health 29.3 11.7 21.2
Mental health problems 23.1 9.4 12
1Not significant difference east versus west All other variables: significant difference, p < 0.001 (chi square test)
The proportion having experienced muscular pain/stiffness during the last four weeks, being very troubled by muscular pain/stiffness in various body parts, or reporting extensive pain/stiffness was higher in east. No difference was found regarding pain duration. Participants in west were more satisfied with health care and more confident in own coping ability (Table 2).
Table 2 Oslo Health Study 2000–2001, 40- and 45- year olds Musculo-skeletal disorders in east, west, and the whole city (percent).
East West City
Pain/stiffness in muscles/joints last four weeks 61.4 55.9* 58.4
Very troubled by pain/stiffness in arms/hands 6.7 4.0* 5.3
Very troubled by pain/stiffness in neck/shoulders 13.6 7.5++ 10.5
Very troubled by pain/stiffness in upper back 7.6 3.1++ 5.3
Very troubled by pain/stiffness in lower back 11.4 4.8++ 8.0
Very troubled by pain/stiffness in hips/legs/feet 10.6 4.8++ 7.6
Very troubled by pain/stiffness elsewhere 3.8 1.3+ 2.4
Extensive pain/stiffness in muscles and/or joints 30.0 18.9++ -
Duration > 3 years 44.2 42.5 43.4
Satisfied with health care (quite satisfied/very satisfied) 28.5 38.9+ 32.2
Confident in coping ability (quite sure/very sure) 74.9 86.2++ 76.7
* significant difference east versus west (chi square test) p < 0.05
+ p < 0.01
++ p < 0.001
Female gender, living in east, low education, low own income, non-Western country of origin, no hard exercise and mental health problems were all correlated to extensive muscular pain/stiffness (Table 3, left column). Female gender, no exercise, non-Western origin and mental health problems still implied increased risk of extensive muscular pain/stiffness when the other variables were adjusted for. Low education and living in east no longer showed an independent correlation with extensive pain/stiffness after adjustment (Table 3, right column).
Table 3 Oslo Health Study 2000–2001, 40- and 45- year olds living in areas east and west Odds ratio for much pain/stiffness in muscles and/or joints, related to demographic variables, lifestyle and mental distress. Logistic regression analyses.
Odds ratio (95 % CI), unadjusted Odds ratio (95 % CI), adjusted1
Female sex 1.45 (1.15 – 1.83) 1.85 (1.40 – 2.45)
Single status 1.02 (0.82 – 1.29)
Area east 1.88 (1.49 – 2.38) 1.22 (0.92 – 1.62)
Low education 2.11 (1.43 – 3.16) 1.09 (0.67 – 1.78)
Low income 1.34 (1.01 – 1.79)
Low household income 1.1 (0.72 – 1.68)
Non-western country of origin 4.41 (3.25 – 5.99) 3.17 (2.17 – 4.63)
Daily smoking 0.81 (0.64 – 1.04)
No hard exercise 2.00 (1.55 – 2.59) 1.37 (1.01 – 1.85)
Mental health problems 3.87 (2.92 – 5.14) 2.70 (1.94 – 3.76)
1Variables included into analyses: sex, area, education, country of origin, exercise, mental health problems.
We performed the logistic regression analyses for men and women separately (data not shown). Non-western origin was the most important predictor of extensive pain/stiffness in men (OR 3.36, 1.93 – 5.83) and mental health problems in women (OR 3.04, 95 % CI 1.99 – 4.66). When we performed the analyses for respondents of Western and non-Western origin separately (data not shown), mental health problems were the most important independent predictor for extensive pain/stiffness for both groups (OR 2.87, 95 % CI 1.97 – 4.19 for Western, OR 2.2, 1.1 – 4.5 for non-Western).
Discussion
In both areas around 60 % reported pain/stiffness in muscles/joints during the previous four weeks: 61.4 % in east and 55.9 % in west, a statistically significant difference of little clinical relevance. We do not know the prevalence among non-respondents, but as The Oslo Health Study implied a comprehensive data collection on many topics, it is unlikely that muscular problems in particular should influence response rate extensively. In a questionnaire survey we carried out in the same areas in 1994 (870 respondents in east, 892 in west, mean age around 40 years) approximately 55 % in both areas reported musculo-skeletal pain during the last four weeks [13]. In another Norwegian survey from 1991, only 15 % reported no muscular pain during the previous year, 58 % had experienced pain the last week, and 15 % reported pain every day during the previous year [9]. Periodic muscular pain or stiffness in one or more body regions should probably be considered a normal phenomenon among adults. Only when symptoms are strong, they imply disease and demand of health care [14].
It is thus important that the proportion reporting to be very troubled was significantly higher in east regarding all body regions. This might be due to a higher prevalence in east of specific musculo-skeletal diseases, like rheumatoid arthritis, fibromyalgia, etc. The Oslo Health Study asked about fibromyalgia and osteoporosis: In east 49 persons reported fibromyalgia and nine osteoporosis, compared to 19 and five in west. In previous studies we found no difference in prevalence of rheumatoid arthritis [15] or osteoarthrosis [13] between the two areas. The higher level of extensive pain in east corresponds, however, with our earlier results, as higher pain intensity, more widespread pain and higher disability scores were found among residents in east compared to west [13].
Blank and Diderichsen found social inequities in both frequency and intensity of a variety of common symptoms in a Swedish population [16]. Their results led to the hypothesis of "double suffering" also promoted by Eachus [7]: That lower classes both have more illnesses and experience these illnesses with greater intensity. Their lesser resources to cope with the consequences of disease also contribute to the suffering. Our present study lends support to this hypothesis: Physical and mental ill-health are more frequently reported in east, musculo-skeletal disorders are more common, the proportion reporting to be very troubled by pain is higher, and fewer respondents believe that they can continue their daily activities and fewer express satisfaction with health care.
The response rate in our material is low (39 % in east, 43.9 % in west). Total response rate in The Oslo Health Study was 46.5 % for 45- year olds, 43.7 % for 40- year olds and 46 % for all age groups. Non-attendance does not occur randomly. Analyses of the impact of self-selection on the Oslo Health Study have shown that the following sub-groups were under-represented among the attendees: unmarried or divorced, males, persons with low education, low income groups, receivers of disability benefit, inner city dwellers and those not born in Norway [17]. But when response rate is low, it also turns out that some healthy, highly educated and busy people have chosen not to participate [18]. We may suggest – but can not know for sure – that non-respondents in east belong mainly in the first group and in west mainly in the second. The implication would be that the differences observed between the areas would increase with increasing response rate.
We consider it a strength to use geographical area as a marker of socioeconomic position, and not for example individual education or income. Residential areas are distinct and easy to handle for authorities and politicians, and the majority of health care resources are allocated at area level. That inhabitants in affluent areas are healthier than in less attractive areas, is hardly a surprise, but which are the mechanisms behind the differences? There may be a certain amount of selection: The financial disadvantage of disabled people make it more likely that they live in poorer areas. In our material, far more people of non-western origin lived in east compared to west. As being of non-Western origin showed a strong independent correlation with severe muscular pain, this selection contributed significantly to the between area difference observed. A less healthy physical environment, less healthy lifestyle, and the psychological impact of being poorer than other people, are also possible explanatory factors [19]. Some authors have found that geographical variations in self-reported illness persist even after allowing for socio-structural individual characteristics [20,21]. This was not the case in our study, as area of living did not show any independent correlation with musculo-skeletal pain after adjustment for individual explanatory variables.
As our study is cross-sectional, causal interpretations cannot be made, we can only describe associations between socioeconomic measures and the health inequities observed. Several studies have shown that education and income can not explain the difference in self-reported health between socioeconomic contrasting areas [20-22]. Our results support this, and support the theory that psychological factors are important [23]. According to Wilkinson, socioeconomic inequality influences health through perception of place in the social hierarchy [24]. Such perceptions produce negative emotions like shame and distrust that are translated inside the body into poorer health via psycho-neuro-endocrine mechanisms [25].
Conclusion
The present study shows that even in Norway today the perception and impact of a health problem (musculo-skeletal pain) is related to a person's socioeconomic situation. Self-reported health status is known to correlate with mortality, and it is a person's perceived health problems which influence the demand for health care. Significantly more persons living in a non-affluent area of Oslo reported extensive pain, compared to persons in an affluent area. Inactivity, poor mental health, and being a non-Western immigrant implied increase risk of severe symptoms.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
The two authors planned and carried out the data collection together. MB carried out the data analyses and drafted the manuscript. Boyh authors approved the final manuscript.
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Int J Equity HealthInternational Journal for Equity in Health1475-9276BioMed Central London 1475-9276-3-111550069610.1186/1475-9276-3-11ResearchThe development of a strategy for tackling health inequalities in the Netherlands Mackenbach Johan P [email protected] Karien [email protected] Department of Public Health, Erasmus MC, University Medical Center Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, the Netherlands2 Department of Social Medicine, Academic Medical Centre, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, the Netherlands2004 23 10 2004 3 11 11 1 6 2004 23 10 2004 Copyright © 2004 Mackenbach and Stronks; licensee BioMed Central Ltd.2004Mackenbach and Stronks; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Over the past decade, the Dutch government has pursued a research-based approach to tackle socioeconomic inequalities in health. We report on the most recent phase in this approach: the development of a strategy to reduce health inequalities in the Netherlands by an independent committee. In addition, we will reflect on the way the report of this committee has influenced health policy and practice.
A 6-year research and development program was conducted which covered a number of different policy options and consisted of 12 intervention studies. The study results were discussed with experts and policy makers. A government advisory committee developed a comprehensive strategy that intends to reduce socioeconomic inequalities in disability-free life expectancy by 25% in 2020. The strategy covers 4 different entry-points for reducing socioeconomic inequalities in health, contains 26 specific recommendations, and includes 11 quantitative policy targets. Further research and development efforts are also recommended.
Although the Dutch approach has been influenced by similar efforts in other European countries, particularly the United Kingdom and Sweden, it is unique in terms of its emphasis on building a systematic evidence-base for interventions and policies to reduce health inequalities. Both researchers and policy-makers were involved in the process, and there are clear indications that some of the recommendations are being adopted by health policy-makers and health care practice, although more so at the local than at the national level.
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Introduction
Before 1980, socioeconomic inequalities in health were a non-issue in public health (research) in the Netherlands. This changed in the early 1980's as a result of the publication of the Black Report in England [1], and a report on inequalities in health between neighborhoods in the city of Amsterdam [2]. Gradually, interest in health inequalities rose, first among researchers and then among policy-makers. Interest among policy-makers was further strengthened by the "Health For All by the year 2000" targets of the World Health Organization that the Dutch government officially endorsed in 1985 [3]. In 1986, the Ministry of Health published its Health 2000 report which was the first government document to include a paragraph on socioeconomic inequalities in health [4]. This was followed in 1987 by a conference organized by the prestigious Scientific Council for Government Policy, the outcome of which was a recommendation to start a research program on health inequalities [5] (see Table 1).
Table 1 Summary of policy developments from 1980 to 2000
1985 The Dutch government adopted the WHO Health For All policy targets
1986 Publication of the Health 2000 Report [15] by the Ministry of Welfare, Health and Cultural Affairs, including a paragraph on socioeconomic inequalities in health
1987 National conference on socioeconomic inequalities in health, organized under the aegis of the Scientific Council for Government Policy, resulting in a proposal for a national research programme (1989–1993) funded by the ministry of Welfare, Health and Cultural Affairs
1991 National conference, again organized under the aegis of the Scientific Council for Government Policy, resulting in an agreement among several parties involved to implement activities to reduce inequalities in health
1994 Results of the first national research programme were reported to the Minister of Public Health
1995 Publication of an important policy document by the Ministry of Public Health, Welfare and Sport (Health and Wellbeing). Reduction of socioeconomic inequalities in health was mentioned as one of the policy goals. Initiation of second national research programme (1995–2000)
1996 Publication of a second document on Public Health Status and Forecasts, by the National Institute of Public Health and Environmental Protection. Socioeconomic inequalities in health were stressed as a major public health problem
2000 Report of the Lemstra committee on the enforcement of public health. The reduction of socioeconomic inequalities was mentioned as an important policy aim.
Growing demand by the Ministry of Public Health and parliament for information on effective interventions to reduce inequalities in health
2001 Results of the second national research programme, and recommendations based on these results, reported to the Minister of Public Health
Since then, the Dutch Ministry of Health has followed a systematic, research-based approach to tackling socioeconomic inequalities in health. An initial five-year research program mapped the nature and determinants of socioeconomic inequalities in health in the Netherlands [6]. A second six-year program launched in 1994 sought to gain systematic experience with interventions and policies designed to reduce socioeconomic inequalities in health. We report on the final phase of the second program: the development of a strategy to tackle health inequalities, and the production of a report containing recommendations for health policy making [7]. These recommendations were partly based on the results of the evaluation studies included in the second program. In addition, we will reflect on the way this report has influenced health policy and practice.
The report deals with socioeconomic inequalities in health, defined as systematic differences in health status between people with higher and lower socioeconomic status, as indicated by educational level, occupational class, and/or income level. Like other European countries, the Netherlands has substantial inequalities in health between socioeconomic groups. Differences in life expectancy at birth between socioeconomic groups are in the order of 4 years, and differences in healthy life expectancy have recently been calculated to be a staggering 14 years [8]. Inequalities in health care utilization, on the other hand, are quite modest, not only in an absolute sense [9], but also in comparison with other European countries [10]. In addition to socioeconomic health inequalities there are other important variations in health as well, e.g. between genders, regions, ethnic groups, and other socio-demographic variables [11]. Some of these are interwoven with socioeconomic inequalities in health, but the two programs mentioned above have tried to separate out the socioeconomic dimension from the other dimensions, in order not to dilute attention across too wide an area.
Case study
The research and development program
The main focus of the program was on developing and evaluating interventions and policies, but a number of other activities (monitoring of health inequalities, longitudinal explanatory study, research seminars, publications, documentation centre) were undertaken as well. Table 2 lists the evaluation studies that were commissioned after two calls for proposals and assessment by peer review. All interventions were aimed at tackling well-known determinants of socio-economic inequalities in health, such as poverty, smoking, working conditions, and accessibility of health care. Evaluation studies started between 1997 and 1999. The majority had a quasi-experimental design and compared health outcomes (e.g. school absenteeism) or intermediate measures (e.g. folic acid use) between an experimental and a control group. Positive results were reported for seven interventions: integrated program to prevent school children to start smoking, teeth brushing at primary school, adapted working methods and equipment for brick-layers, rotation of tasks among dustmen, formation of local care networks, peer education for Turkish diabetics, and introduction of nurse practitioners for asthma/Chronic Obstructive Pulmonary Disease patients. The other evaluation studies either failed because of an inadequate evaluation design or produced negative results [12]. [see Table 2].
Table 2 Intervention studies undertaken within the second national program on socioeconomic inequalities in health
Interventions targeting socioeconomic disadvantage
• Supplementary benefits to parents living in poverty, identified during preventive health screening of children (no evidence on effectiveness collected)
Interventions targeting health-related selection
• Counselling of secondary school children with frequent school absence due to illness (evaluation design failed)
Interventions targeting factors mediating the effect of socioeconomic disadvantage on health
• Tailored mass media campaign to promote periconceptional folic acid use (intervention did not reduce socioeconomic gap in folic acid use)
• Community-based intervention to improve health-related behavior in deprived neighborhoods (evaluation results will become available in 2002)
• Integrated program (including social skills teaching and monetary rewards) to prevent school children in lower general and vocational education to start smoking (intervention reduced smoking initiation rate)
• Teeth brushing at primary schools (intervention eliminated socioeconomic gap in teeth brushing)
• Adapted working methods (raised brick-laying) and equipment (lifting machine) for brick-layers (intervention reduced physical workload and sickness absenteeism)
• Rotation of tasks (driving and minicontainer loading) among dustmen (intervention reduced physical workload and sickness absenteeism)
• Introduction of self-organising teams in various production organisations (evaluation design failed)
Interventions targeting accessibility and quality of health care services
• Formation of local care networks among general practitioners, housing corporation staff and police officers to prevent homelessness among chronic psychiatric patients (intervention reduced house evictions and forced admissions to psychiatric hospitals)
• Peer education to diabetic patients of Turkish origin (intervention improved glycaemic control and healthy behaviour, but only in women)
• Introduction of nurse practitioners for asthma/COPD patients to general practice in deprived areas (intervention increased treatment compliance and reduced exacerbations)
When the results of the evaluation studies became available, meetings were held in 2000 with scientific experts and representatives from policy makers and from practice in six different areas (income, education, health promotion, working conditions, housing conditions, health care). During these meetings possible recommendations for new policies and interventions were tested and refined [13]. The input for the meetings not only included the results of the evaluation studies, but also added two additional papers. The first paper, drawn up by a scientist, gave an overview of effective interventions to reduce socioeconomic inequalities in health in that area. In the second paper, the implications of this overview for policy were analysed by an author with experience in that specific policy area (e.g. former secretary of state for educational affairs and the former minister of social affairs). The meetings contributed to a better understanding of current policy initiatives, and the major obstacles and promoting factors for a policy aimed at reducing inequalities in health.
The government advisory committee
Subsequently, the committee overseeing the program held a number of plenary meetings to develop a comprehensive strategy to reduce health inequalities. Committee members were appointed by the Minister of Health, and they included former and active politicians of various political backgrounds, as well as a representative of the ministry of health and researchers. A conscious attempt was made to represent the whole (relatively narrow) political spectrum in the Netherlands. Members ranged from left (represented by the social-democrat mayor of the fourth largest city in the country) to right (represented by a former chairman of, and current House of Lords member for, the conservative party, who was later succeeded by another House of Lords member for the same party), and the committee was chaired by a former christian-democrat Minister of Social Affairs. Researchers had an important influence on the whole process: JM was secretary of the committee, and KS acted as co-ordinator of the program, and both were involved in writing draft versions of the final report. The committee reported directly to the Minister of Health.
The rationale for the strategy
The committee started from the assumption that existing inequalities in health at least partly rank as unjust and that the government is responsible for achieving a reduction of these health differences. This assumption was based on the argument that health should be seen as a condition for the options open to individuals to structure their own life as far as possible according to their own ideas. Those health differences that are the consequence of an unequal distribution of living conditions over which individuals have no control, were thus seen as health inequities, to be tackled by the government. It was argued that this would require a comprehensive strategy, given the persistent and widespread character of socio-economic inequalities in health.
The committee wanted its strategy for reducing health inequalities to be based on sound evidence. Ideally, factors targeted by the strategy should be known to contribute to the explanation of health inequalities, and interventions and policies should be known to diminish exposure of lower socioeconomic groups to these factors. While the first requirement could be met relatively easily (and documentation was provided, with references, in the final report of the committee), the second requirement was more difficult to meet. Although the program produced evidence on effectiveness of interventions and policies and showed some positive results, this left important gaps in the knowledge base, both in terms of coverage of various policy options and in terms of strength of evidence. This problem was also encountered in other countries [14]. The committee considered that one cannot expect further evidence to become available unless large-scale measures to reduce inequalities in health are taken. It therefore decided to recommend a combination of implementation of 'promising' interventions with continued evaluation efforts. For each of the interventions and policies that were recommended for implementation, it carefully listed the available evidence, plus references.
In addition, the committee also paid attention to the political feasibility of possible policy recommendations. This aspect was discussed during the plenary meetings, in the light of the (political) experience of the committee members as well as the outcome of the working conferences that were mentioned before.
Targets
The committee decided to base its strategy on a number of quantitative targets, because these can aid in plotting a clear policy course and can function as milestones for interim assessments of the strategy. It took the World Health Organization target as its starting point [15], and reformulated it for the Netherlands as: "By the year 2020, the difference in healthy life expectancy between people with a low and people with a high socioeconomic status should be reduced from 12 to 9 years, due to a (stronger) increase in healthy life expectancy in the lowest socioeconomic groups."
In order to attain such an ambitious goal, major efforts are required, if only because during the last decades inequalities in health in the Netherlands have increased rather than decreased [16]. Although it was considered unwise to give up on the ambition laid down in this 'inspirational' target, the strategy focused on a set of 'intermediate' targets that seem feasible today or in the near future. These targets were chosen to represent each of the main entry-points for reducing socioeconomic inequalities in health, and were limited to intermediate outcomes for which quantitative data for the Netherlands are currently available.
Package of policies and interventions
Table 3 lists the interventions and policies constituting the strategy recommended by the committee. The strategy covers all four entry-points and spans the entire range between 'upstream' measures targeting socioeconomic disadvantage and 'downstream' measures targeting accessibility and quality of health care services. Where current policies were expected to contribute to reducing health inequalities (education policies, income policies, work disability benefit schemes, health care financing schemes), the committee explicitly recommended continuation. This is by no means trivial, because none of these achievements of the past can be considered safe for the future. For example, the Dutch government is considering a reform of the health care financing system that could lead to reduced coverage of health care for those insured under the current public scheme, and then would jeopardize equal financial accessibility.
Table 3 Recommended interventions and policy measures
Interventions and policies targeting socioeconomic disadvantage
• Continuation of policies that promote educational achievement of children from lower socioeconomic families.
• Prevention of an increase of income inequalities through adequate tax and social security policies.
• Intensification of anti-poverty policies, particularly policies that relieve long-term poverty through special benefit schemes and assistance with finding paid employment.
• Further development and implementation of special benefit schemes for families whose financial situation threatens the health of their children.
Interventions and policies targeting health-related selection
• Maintaining benefit levels for long-term work disability, particularly for those who are fully work disabled and those who are partly work disabled due to occupational health problems
• Adaptation of working conditions for the chronically ill and disabled in order to increase their work participation.
• Health interventions among long-term recipients of social assistance benefits in order to remove barriers for finding paid employment.
• Further development and implementation of counselling schemes for school pupils with regular or long-term absenteeism because of health problems.
Interventions and policies targeting factors mediating the effect of socioeconomic disadvantage on health
• Adapting health promotion programs to the needs of lower socioeconomic groups, particularly by focusing on environmental measures including the introduction of free fruit at primary schools and an increase of the excise tax on tobacco.
• Implementation of school health promotion programs that target health-related behaviour (particularly smoking) among children from lower socioeconomic families.
• Introduction of health promotion efforts into urban regeneration programs.
• Implementation of technical and organisational measures to reduce physical workload in low-level occupations.
Interventions and policies targeting accessibility and quality of health care services
• Maintaining good financial accessibility of health care for people from lower socioeconomic groups
• Relieving the shortage of general practitioners in disadvantaged areas.
• Reinforcing primary health care in disadvantaged areas by employing more practice assistants, nurse practitioners and peer educators, e.g. for implementing cardiovascular disease prevention programs and better care for chronically ill persons.
• Implementation of local care networks aiming for the prevention of homeliness and other social problems among chronic psychiatric patients.
In a number of other areas, the committee recommended intensified or new policies. These recommendations were partly based on reported positive results of intervention studies. This applies to the recommendations relating to school health promotion programs, technical and organizational measures to reduce physical workload, reinforcement of primary care in disadvantaged areas by employing practice nurses and peer educators, and local care networks to prevent social problems among chronic psychiatric patients. The results of some of the other intervention studies led to recommendations for further development of those interventions, as in the case of special benefit schemes for families living in poverty and counseling schemes for school absenteeism. Most of the other recommendations, however, are primarily based on an understanding of the factors that have been shown to contribute to health inequalities, and of the best way to deliver interventions targeting these factors.
The committee did not attempt to estimate the costs of the recommended interventions and policies.
Implementation
As experience has taught that implementing effective interventions should not be taken for granted, the committee advised that a steering group be formed to drive and control the process of implementing effective interventions. On the one hand, this should function as a highly visible focal point at which the expertise available in the Netherlands is made accessible to all relevant policy areas. On the other hand, the steering group should be able to act on its own initiative to capture and retain attention for socio-economic inequalities in health and to promote the implementation of policy proposals. Given these two functions, the committee advised including experts as well as representatives from the main relevant policy areas in the steering group.
Research and development
Given the fact that research has not yet fully disclosed the origins of socioeconomic inequalities in health, the committee considered continuation of explanatory research to be vital because it may lead to new entry-points for intervention. The same applies to further development of effective interventions and policies. The committee therefore recommended evaluation of all recommended interventions and policies during and after their implementation.
Presentation of the report
The committee published its main report in March 2001 [7]. The report was launched at a press conference, and presented to both the minister of health and the minister of the 'Major Cities policy'. It received wide media coverage. All major newspapers wrote extensively about the findings and recommendations, and these were also presented and discussed in various national television and radio programmes. Some criticism was heard as well. These include the argument that any (shared) responsibility on the part of the government for reducing socio-economic inequalities in health is at odds with the social trend towards stimulating individuals to take responsibility for themselves. This was discussed in the context of health related behaviour (smoking, nutritional pattern etc.) in particular.
A closing conference took place in October 2001. During that conference, the results of the evaluation studies as well as the proposed policy strategy were presented to a broad public, and reflected upon by, among others, Sir Donald Acheson from the UK. In addition, policy implications were discussed. Participants included researchers, policy makers and representatives from practice, not only from the public health and health care field, but also from other policy areas (social security, working conditions etc.).
Follow-up
The official cabinet reaction to the recommendations presented to parliament in November 2001 was positive but further elaboration of the recommendations as well as decision-making was deferred to the next cabinet [17]. A new cabinet was formed after turbulent elections in spring 2002 but fell within 3 months, and did not make decisions on a strategy to reduce socioeconomic inequalities in health. New elections were held in January 2003.
The delay in political decision making does not seem to have hindered the implementation of specific interventions that were evaluated within the programme. So far, at least a few of the interventions that have been proven to be effective have been implemented on a larger scale. These include the integrated programme to prevent school children from starting smoking, and the local care networks for chronic psychiatric patients.
Discussion
While many countries, including the UK, Sweden and Finland have had national research efforts in the field of socioeconomic inequalities in health during the second half of the 1990's, the Dutch program is unique for its emphasis on evaluation of interventions. More generally, the main distinguishing feature of the Dutch approach is its focus on commissioning evaluations of interventions. Although this was done in a systematic way, using an explicit conceptual and methodological framework, the program also had its obvious limitations. It had a modest budget (totalling 3 million Euro over a period of 6 years) and funded not more than 12, rather small-scale intervention studies targeting relatively easily modifiable factors. The latter is not only due to the small budget of the program, but also to strict methodological requirements which in practice made it nearly impossible to study the effectiveness of broader policy measures [18]. In hind-sight, we consider this the most important limitation of the program: the lack of studies on the possible impact of broader policy measures, mainly related to the strict methodological criteria that were applied in the process of selection of the research proposals. Even for the more specific and narrowly defined interventions selected for the program, some of the evaluation studies failed because the design could not be implemented. In the end, therefore, the contribution of the intervention studies to strategy development was modest.
The unique elements of the Dutch approach should not distract from the fact that the Dutch experience received important inputs from abroad. Its start is a late response to the British Black Report and is directly related to the efforts of the European Office of the World Health Organization to put health equity on national policy agendas [15]. During the program there were close contacts between members of the committee and researchers and policy-makers in other European countries, through the European Network for Interventions and Policies to Reduce Inequalities in Health [19], so that experiences in other countries could be taken into account. The report of the Independent Inquiry in Britain [20] acted as a rich source of ideas, while a recent Swedish report on tackling inequalities in health [21] strengthened the confidence in the usefulness of target setting for reducing inequalities in health.
The Dutch approach reflects the input of both researchers and policy-makers, although the balance between the two has oscillated over time. The first signals that health inequalities should be addressed came from researchers, but were picked up by policy-makers within the Ministry of Health in the mid-1980's who were then looking for opportunities to strengthen health policy (as opposed to health care policy) in the Netherlands. This small group of bureaucrats succeeded in launching and following through the first research program, but left the Ministry or changed posts before the program came to an end. Partly due to continuous personnel changes in the Ministry, the intensity of the exchanges between researchers and policy-makers gradually diminished during the second program. When the final report was published reactions from within the Ministry were rather cool, although the Minister, who had taken a personal interest in the matter, responded very favourably. At this stage, however, it seems that without a continuing "push" from the research-side the bureaucrats could easily loose interest altogether, particularly now that there are rapid changes of cabinet.
A major obstacle for a comprehensive package of policy measures seems to be the relatively weak position of the Ministry of Health as compared to other policy areas. It is obvious that a substantial reduction of health inequalities can be achieved only by involving other policy areas next to that of (preventive and curative) health care. This starting point seems to contrast with the ideas of the ministries in other policy areas, that seem to consider this issue as the responsibility of the Ministry of Health in particular. So far, the Ministry of Health does not seem to have a lot of success in convincing other policy areas of the importance of contributing to reducing inequalities in health.
The lack of success in mobilising other policy areas at the national level is probably partly related to the fact that the issue of inequalities is perceived as rather abstract by these other areas. This probably requires the issue of inequalities in health to be "re-phrased" for that specific policy area, in terms that fit within their ideas. Housing corporations for example do not consider themselves to be responsible for tackling health inequalities but they do feel responsibility for high quality living conditions, which then might automatically contribute to a better health status of people in lower socio-economic groups. Paradoxically, an approach in which the issue of inequalities in health is cut into small pieces, requires a steering. This forms the background of the plea of the committee for a steering group.
Remarkable progress has been made, not only in terms of knowledge production but also in terms of increased confidence among policy-makers and practitioners to take action to reduce inequalities in health. Many health agencies in the Netherlands are working to reduce socioeconomic inequalities in health. This is illustrated by the fact that the 'National Contract on Public Health', concluded in 2001 between many national and local agencies in the field of public health, has selected the reduction of socioeconomic inequalities in health as its first priority. Many local health agencies have already implemented some of the interventions discussed in this paper.
Acknowledgements
The research and development program was funded by the Ministry of Health, Welfare and Sports, through the Health Research and Development Council of the Netherlands.
The Program Committee was chaired by Prof. W. Albeda. Members (in addition to the authors of this paper) were: Prof. Dr H. Dupuis, Prof. Dr H.F.L. Garretsen, Prof. Dr P.J. van der Maas, Dr M. Mootz, and Dr R.W. Welschen.
This paper was written in the context of the project "Health Equity Research: Beyond the Sound of One Hand Clapping", of the AcademyHealth, funded in turn by the Rockefeller Foundation.
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| 15500696 | PMC529300 | CC BY | 2021-01-04 16:39:30 | no | Int J Equity Health. 2004 Oct 23; 3:11 | utf-8 | Int J Equity Health | 2,004 | 10.1186/1475-9276-3-11 | oa_comm |
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Int J Equity HealthInternational Journal for Equity in Health1475-9276BioMed Central London 1475-9276-3-91548259610.1186/1475-9276-3-9ResearchMothers' education but not fathers' education, household assets or land ownership is the best predictor of child health inequalities in rural Uganda Wamani Henry [email protected]är Thorkild [email protected]Åstrøm Anne Nordrehaug [email protected] James K [email protected] Stefan [email protected] Centre for International Health, University of Bergen, Armauer Hansen Building, N-5021 Bergen, Norway2 Ministry of Health, P.O Box 7272, Kampala, Uganda3 Department of Paediatrics and Child Health, Makerere University Medical School, P.O Box 7072, Kampala, Uganda4 Division of International Health (IHCAR), Karolinska Institute, Norrbacka, S-17176 Stockholm, Sweden2004 13 10 2004 3 9 9 6 4 2004 13 10 2004 Copyright © 2004 Wamani et al; licensee BioMed Central Ltd.2004Wamani et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Health and nutrition inequality is a result of a complex web of factors that include socio-economic inequalities. Various socio-economic indicators exist however some do not accurately predict inequalities in children. Others are not intervention feasible.
Objective
To examine the association of four socio-economic indicators namely: mothers' education, fathers' education, household asset index, and land ownership with growth stunting, which is used as a proxy for health and nutrition inequalities among infants and young children.
Methods
This was a cross-sectional survey conducted in the rural district of Hoima, Uganda. Two-stage cluster sampling design was used to obtain 720 child/mother pairs. Information on indicators of household socio-economic status and child anthropometry was gathered by administering a structured questionnaire to mothers in their home settings. Regression modelling was used to determine the association of socio-economic indicators with stunting.
Results
One hundred seventy two (25%) of the studied children were stunted, of which 105 (61%) were boys (p < 0.001). Bivariate analysis indicated a higher prevalence of stunting among children of: non-educated mothers compared to mothers educated above primary school (odds ratio (OR) 2.5, 95% confidence interval (CI) 1.4–4.4); non-educated fathers compared to fathers educated above secondary school (OR 1.7, 95% CI 0.8–3.5); households belonging in the "poorest" quintile for the asset index compared to the "least poor" quintile (OR 2.1, 95% CI 1.2–3.7); Land ownership exhibited no differentials with stunting. Simultaneously adjusting all socio-economic indicators in conditional regression analysis left mothers' education as the only independent predictor of stunting with children of non-educated mothers significantly more likely to be stunted compared to those of mothers educated above primary school (OR 2.1, 95% CI 1.1–3.9). More boys than girls were significantly stunted in poorer than wealthier socio-economic strata.
Conclusions
Of four socio-economic indicators, mothers' education is the best predictor for health and nutrition inequalities among infants and young children in rural Uganda. This suggests a need for appropriate formal education of the girl child aimed at promoting child health and nutrition. The finding that boys are adversely affected by poverty more than their female counterparts corroborates evidence from previous studies.
Child healthinequalitiesstuntingsocio-economictargetingmother educationUganda
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Background
Health outcomes are a result of a multi-layered conceptual framework with proximal and distal determinants [1]. Proximal (nearer in time and place to the terminal event) determinants such as feeding practices, injury and disease especially the infectious diseases are mediated through factors prevailing within households or the institutions serving children directly. At the distant level are factors operating through the socio-political and economic dimensions of the environment.
In recent decades, development agencies and governments have emphasized efficacious interventions to improve child survival and healthy development. However, most of these interventions have traditionally addressed proximal determinants with limited effort on distal determinants since many of them lie outside the health sector. Recent calls were made for interventions to address the more distal determinants of health outcomes [2,3]. To minimize inequities in child health and nutrition emphasis is made on correct identification and service delivery to the poor and most vulnerable in society.
To assess inequalities of child health and nutrition, the World Health Organisation recommends use of linear growth retardation (stunting) [4-6]. In the biomedical arena stunting is attributable to a wide range of prenatal and postnatal factors. These include: low birth weight [7-9], inadequate care and stimulation [10], and insufficient nutrition and recurrent infections [11]. Use of stunting avoids the measurement pitfalls like improper case definitions, time, and others usually associated with more traditional measures such as morbidity, mortality, and life expectancy. It also eludes problems associated with using monitory variables such as income or expenditure to compare health inequalities [12,13].
Stunting varies systematically with socio-economic status with the poorest most affected by it. This relationship is observed in a number of studies in the African region [14-18]. Unfortunately, proxies used to construct socio-economic indicator(s) vary. The relative household socio-economic position is sometimes developed by lumping indicators of socio-economic status into one global indicator, or derived from non-interventional feasible indicators. Additionally control for unmeasured effects of each indicator on others is often never done. This is detrimental to development of effective policy since choice of an indicator has interventional implications. This is especially critical in sub-Saharan Africa where evidence suggests that macro-economic growth alone is unlikely to significantly reduce stunting in medium terms [19]. Therefore efforts to address distal determinants of child health inequalities especially through the socio-economic pathway, requires that great care is taken in selecting indicators that are amenable to easy monitoring and feasible interventions [20].
The present study was conducted in a rural setting, in Uganda. It examines the independent influence of four different components of socio-economic status: mothers' education, fathers' education, household asset index, and land ownership on growth stunting, a proxy for health inequalities. Results of this study should provide policy makers and programme managers with useful information in child health and nutrition planning for rural areas.
Methods
Study area
The study was carried out in Hoima district, which is located in western Uganda and shares a border with the Democratic Republic of Congo along Lake Albert. The district has a population density of 80 per square kilometre with a total population of approximately 370,000, comprised of mainly peasant farmers of the Banyoro tribe [21]. The district has fertile soils and enjoys a bimodal rainfall pattern ranging from 800 – 1500 mm per annum enabling two harvests a year. Farming activities include cattle rearing, growing tobacco and coffee for cash; and maize, cassava, potatoes, beans and groundnuts for consumption.
Design and sampling
The present survey utilised a two stage probability proportional to size cluster design [22]. Data was collected with respect to infant- and young child feeding knowledge and practices, anthropometry and indicators for household socio-economic status. The sample was made large enough to estimate a prevalence rate of wasting similar to that assessed at the national level (4.0%), at 95% level of confidence [23], with 2% precision error and an assumed intra-cluster correlation coefficient of 0.11. The national census data [21] was used to estimate the population under-2 years of age in Hoima district (24,000). A sample size of 720 children was calculated based on the formula by Cochran [24], using the SampleXS software. At the first stage of sampling, a total of 72 villages were selected on probability proportional to population size basis. At the second stage, a total of 10 households were systematically sampled from each village selected at the first stage, providing a self-weighting sample with each child/mother pair in the district having an equal probability to be selected into the sample. A household was defined as a group of people living, cooking and eating together. One child under 2 years was enrolled per household after an informed verbal consent of the mother/caregiver. In case a household had two children in the target age group or twins one was selected randomly. Children with major handicap, disability or malformation were excluded from the sample. Problems encountered with sample selection included the existence of newly formed villages hitherto unrecorded in the census register. In one case an old village had been split; one of the "offspring" villages was selected randomly.
Data Collection
A structured questionnaire constructed in English and translated into Runyoro, the local language, was pilot tested and updated in survey similar settings and administered face-to-face to mothers/caregivers in their home settings. Trained non-medical university students gathered data in September and October 2002. To minimise bias, training of interviewers emphasized proper and random identification of respondents, eligible children, and questionnaire administration. A village leader followed data collectors through the village, and traditional village protocol was observed.
Information about the educational level of parents, household durable assets, materials of the dwelling structure and land ownership was collected as proxy makers of household socio-economic status. Child's age was obtained through birth certificates, health cards, or recall using a calendar of local events while taking precautions to minimize field errors [25]. Recumbent length measurements were taken with specially designed length boards that measured to the nearest 1 mm following standard recommendations [26]. The questionnaire was based on the demographic and health survey (DHS) questions [27] to avoid separate validity and reliability checks of the data collection instrument.
Measurements
The dependent variable (stunting) was constructed using child length, age and sex, and was defined as length less than minus 2 standard deviations (<-2 SD) from the median of the NCHS/WHO reference population [5].
The education level of fathers and mothers was assessed on scales ranging from (1) never went to school, to (7) college/university. Two categorical variables were constructed yielding four categories for mothers: (1) no formal education, (2) stopped in primary, (3) completed primary (minimum 7 years of school) and (4) stopped above primary. Five categories were yielded for fathers because they were more educated: (1) no formal education, (2) stopped in primary, (3) completed primary, (4) stopped in secondary, and (5) completed secondary 4 or above (minimum 11 years of school).
Household durable assets (cupboard, hurricane lamp, radio, bicycle, fuel, boat, telephone, refrigerator, motorcycle and car) were assessed as (1) available/ in working condition and (2) not available/ not in working condition. Four components of the dwelling structure were assessed including number of rooms, roof – (1) thatched, (2) corrugated iron sheets or tiles; floor – (1) mud, (2) cement; wall – (1) thatched, (2) mud and pole, (3) unburned bricks, (4) burnt bricks built with mud, (5) burnt bricks built with cement and (6) cement/concrete blocks. A household wealth (asset) index was constructed from 15 variables (household durable assets and dwelling structure) using principal components analysis [28]. The index scores were divided into quintiles (1) poorest, to (5) least poor.
Land is usually given special socio-economic status in many subsistence agricultural communities in Uganda [29]. In order to assess for its independent contribution to child well being land ownership was analysed independent of other household assets. It was assessed as a continuous variable using an open question "How much land does your family have for agriculture or any other activity?" A football pitch was used to estimate the size equivalent to one acre. A categorical variable was constructed in terms of; (1) no land at all, (2) one acre, (3) two acres, (4) three acres and (5) four or more acres.
Child age in months was assed as a continuous variable and a categorical variable was constructed yielding four categories: (1) 0–5 months, (2) 6–11 months, (3) 12–17 months, and (4) 18–23 months. Child sex was ordinal (1) male, (2) female.
Analysis
Data was entered in Epidata software [30], and was analysed in epi info (version 6.04d) and SPSS (version 12.0). Of 720 children recruited, 23 (3%) had missing data on anthropometric measurements or were extreme outliers and were dropped from the analysis of stunting. First, socio-economic indicators (mothers' and fathers' education, household wealth index and land ownership) were evaluated for the extent of shared variation by obtaining bivariate correlation coefficients (Spearman's rho). Second, unadjusted associations of stunting with socio-economic and demographic variables were examined by use of odds ratios. Third, backward conditional logistic regression was done simultaneously controlling for socio-economic and demographic factors. The pattern of stunting with sex across different socio-economic strata was evaluated with chi-square tests and bivariate logistic regressions. Mean age differences between boys and girls were assessed by the student t-test. Tests for trend across categories were performed by treating the categories as continuous variables in the logistic regression analyses. The clustering effect was not controlled for as the large number of primary sampling units (72) in the study minimizes it. The goodness-of-fit for regression models was assessed with Hosmer and Lemeshow test; with the null hypothesis that the model adequately fits the data (significant chi-square p-value implies that the model does not fit).
Ethics
Approval of the study was granted by Makerere University Faculty of Medicine Ethics and Research committee, the Uganda National Council for Science and Technology and the Regional Committee for Medical Research Ethics, West Norway (REK vest).
Results
Of the children studied 366 (51%) were females. The median age (inter-quartile range) was 10 (5–16) months for females and 11 (5–16) months for males respectively. The mean age difference between boys and girls was not statistically significant (p = 0.34). A total of 148 (21%) mothers and 59 (9%) fathers never attained any formal education (Table 1), while only 52 (7%) of mothers were educated up to secondary 4 or above. Ninety percent of mothers were married. One hundred seventy two (25%) of the children in the study were stunted out of which 105 (61%) were boys. The status of stunting ranged from 12% among children aged 0–5 months to 41% in children 18–23 months (Figure 1). There were 21 twins included in the study and 6 of them were stunted. Ninety percent of households owned any land. Median acreage was 2.0 and the mean was 3.6 acres.
Table 1 Frequency distribution, unadjusted and conditional multiple logistic regressions of stunting with socio-economic predictors (coefficients are expressed as odds ratios and p-values for the test of trend are indicated)
Variables Total Stuntedb Unadjusted Adjusted
na (%) n (%) OR 95%CI OR 95%CI
Child age in months
18–23 150 (22) 61 (41) 5.2 2.9–9.3*** 5.1 2.6–9.8***
12–17 189 (27) 56 (30) 3.2 1.8–5.7*** 3.0 1.6–5.8**
6–11 195 (28) 36 (19) 1.7 0.9–3.1 1.8 0.9–3.5
0–5 164 (23) 19 (12) 1.0 1.0
p-value for trend test <0.001 <0.001
Child sex is male 354 (49) 105 (31) 1.9 1.3–2.7*** 2.0 1.3–3.0**
Mother's education
None 148 (21) 45 (31) 2.5 1.4–4.4** 2.1 1.1–3.9*
Stopped in primary 282 (40) 80 (30) 2.3 1.4–3.9** 2.1 1.2–3.8*
Completed primary 131 (18) 22 (18) 1.2 0.6–2.2 1.0 0.5–2.0
Above primary 152 (21) 23 (15) 1.0 1.0
p-value for trend test <0.001 0.03
Father's education
None 59 (9) 17 (30) 1.7 0.8–3.5
Stopped in primary 179 (29) 47 (27) 1.5 0.9–2.6
Completed primary 151 (24) 36 (24) 1.3 0.8–2.3
Stopped in secondary 95 (15) 17 (18) 0.9 0.5–1.8
Secondary 4 or above 142 (23) 27 (20) 1.0
p-value for trend test 0.03
Household wealth index
1st quintile (Poorest) 139 (20) 38 (29) 2.1 1.2–3.7**
2nd quintile 140 (20) 36 (26) 1.7 0.9–2.9
3rd quintile 150 (21) 33 (24) 1.3 0.8–2.5
4th quintile 140 (20) 36 (26) 1.1 0.6–2.0
5th quintile (Least poor) 140 (20) 23 (17) 1.0
p-value for trend test 0.01
Land ownership
None 73 (10) 16 (22) 0.9 0.5 – 1.8
1 acre or less 179 (25) 39 (23) 0.9 0.6 – 1.6
2 acres 167 (24) 44 (28) 1.2 0.7 – 2.1
3 or 4 acres 152 (22) 37 (25) 1.0 0.6 – 1.8
5 acres or more 135 (19) 32 (24) 1.0
p-value for trend test 0.78
Hosmer-Lemeshow goodness-of-fit
chi-square p-value 0.90
*p < 0.05; **p < 0.01; ***p < 0.001
aTotals for some categories do not add up to 720 because of missing values
bBelow -2 SD height-for-age of the NCHS/WHO reference
Figure 1 Proportion of stunted children (<-2 SD of NCHS/WHO reference) among 720 children by sex and age group in western Uganda
Stunting distribution by socio-economic status
Unadjusted logistic regression analysis indicated that 3 socio-economic indicators (fathers' education, mothers' education and household wealth index) had a graded pattern with stunting across strata with the least educated or most poor being more likely to have stunted children. The corresponding p-values for test of trend was <0.001, 0.03 and 0.01 for mothers' education, fathers' education and household wealth index, respectively (Table 1). Land ownership however, showed no differentials with stunting across strata.
The extent of association between socio-economic indicators was examined. The correlations (Spearman's rho) between mothers' and fathers' education was 0.50; mothers' education and household wealth index = 0.37; mothers' education and land ownership = 0.12; fathers' education and household wealth index = 0.39; fathers' education and land ownership = 0.14; and household wealth index and land ownership = 0.18. Their colinearity was sufficiently low to allow them into the same model.
The four socio-economic indicators (mothers' education, fathers' education, household wealth index and land ownership), child age and sex were simultaneously entered in a backward conditional logistic regression. Only mothers' education, child age and sex appeared in the last step model. Children belonging to mother with no formal education or to mothers who stopped in primary school were significantly more likely to be stunted compared to their counterparts with mothers who were educated beyond primary school (OR = 2.1, Table 1). Being a boy (OR = 2.0) or older in age was also significantly associated with stunting.
Stunting distribution by sex and socio-economic status
Stunting differentials for both sexes within and across socio-economic indicators did not favour male children. There was a graded relationship of stunting among males unlike females with almost all socio-economic groups except land ownership (Table 2). More males were stunted in poorer socio-economic strata than their peers in better off strata. The trend was more marked with mothers' education (p < 0.001) and household wealth index (p = 0.01), less with fathers' education (p = 0.06) and none with land ownership (p = 0.64). The magnitude of the difference in stunting between males and females diminishes with improvement in socio-economic status. The socio-economically advantaged groups show no differences in stunting status between the two sexes. Compared to the proportion of stunting between boys and girls in the strata of mothers educated above primary, mothers with no formal education were significantly more likely to have stunted boys than girls (OR = 3.0).
Table 2 Comparison of stunting status by sex and socio-economic position with unadjusted odds ratios and 95% confidence intervals (CI) derived from bivariate logistic regression with girls in the comparison group (coefficients are expressed as odds ratios, p-values for test of trend and Pearson chi-square are also indicated)
Stunted children p-value Unadjusted odds ratio 95%CI
Males n(%) Females n(%)
Mother's education
None 33 (43) 12 (17) 0.01f 3.00 1.05–8.59*
Stopped in primary 44 (34) 36 (26) 0.18 1.33 0.53–3.38
Completed primary 15 (23) 7 (12) 0.16 2.34 0.69–7.87
Above primary 11 (17) 12 (14) 0.65 1.0
p-value for test of trend <0.001 0.14 0.11
Father's education
None 12 (40) 5 (19) 0.09f 1.65 0.45–6.03
Stopped in primary 30 (35) 17 (20) 0.04f 1.21 0.46–3.20
Completed primary 24 (31) 12 (17) 0.05f 1.37 0.49–3.87
Stopped in secondary 9 (21) 8 (16) 0.59 0.77 0.23–2.63
Ordinary level or above 16 (25) 11 (15) 0.14 1.0
p-value for test of trend 0.06 0.39 0.37
Index of household wealth
1st quintile (Poorest) 27 (40) 17 (25) 0.05f 2.17 0.81–5.81
2nd quintile 25 (34) 14 (21) 0.05f 2.44 0.88–6.73
3rd quintile 21 (35) 11 (15) 0.01f 2.60 0.90–7.56
4th quintile 20 (28) 10 (13) 0.02f 2.73 0.92–8.09
5th quintile (Least poor) 11 (15) 15 (22) 0.34 1.0
p-value for test of trend 0.01 0.34 0.28
Land ownership
None 9 (27) 7 (18) 0.42 0.77 0.23–2.61
1 acre 19 (25) 20 (22) 0.59 0.57 0.22–1.48
2 acres 28 (41) 16 (18) 0.01f 1.05 0.41–2.70
3 or 4 acres 26 (30) 11 (18) 0.07f 1.42 0.52–3.87
5 acres or more 20 (30) 12 (18) 0.08f 1.0
p-value for trend test 0.64 0.66 0.17
fFishers exact test; *p < 0.05
Discussion
This study examined how socio-economic indicators namely mothers' education, fathers' education, household asset index and land ownership relate with inequalities in child health and nutrition using growth stunting as the proxy for the inequalities. Findings showed that mothers' education was a robust predictor for inequalities of child health and nutrition. Secondly, boys were more affected by low socio-economic status than girls. Indirectly the study also indicates that it may be unsuitable to bunch together socio-economic indicators as they could have low covariance or could conceptually not be interchangeable and thus unable to serve as adequate proxies for one another.
While interpreting findings of this study one should consider the fact that some of the fixed factors known to influence linear growth such as birth weight [7,8] and maternal stature [9,31] were not controlled for in the analysis. Birth weight was dropped because of many missing data attributable to the fact that majority of mothers in Uganda give birth outside the health units, consequently missing information on birth weight. Unfortunately the study design excluded assessment of maternal stature. Additionally, in a cross-sectional study such as the present one, it is impossible to say anything about cause and effect relationships. Nonetheless, considering that our agenda was to examine socio-economic predictors for inequalities in health and nutrition, these limitations do not greatly trivialise the importance of the study.
The socio-economic predictor variables used in the study were assumed to represent distinct dimensions reflecting different domains of influences from the society. The relatively low colinearity between the indicators used supports this assumption. This favours discussions on choice of socio-economic indicators where researchers are urged to use a number of socio-economic indicators rather bunching them together as each could have its unique contribution [20,32].
Our results regarding the relationship of socio-economic status with stunting are similar to findings in other studies [14-18]. As one climbs up the socio-economic ladder, there is a remarkable drop in the rate of stunting observed. Interestingly despite taking care of some covariance that existed between different socio-economic indicators during the analysis, mothers' education emerged as the only independent socio-economic predictor for inequalities of child health and nutrition. This indicates that the association of fathers' education and household wealth index with stunting could be confounded by mothers' education.
This emphasizes the need for promoting the education of the girl child with a principal aim of improving child health, which is in line with recommendations by UNICEF [33]. However, for this to be achieved there is need for a rational approach that involves a greater strategic and collaborative relationship between different sectors of government and agencies.
Additionally, mothers' education could be used as a suitable indicator to guide effective targeting especially with regard to identifying the most vulnerable families in rural subsistence agricultural communities. This is in appreciation of the fact that achieving universal coverage for child-health interventions presently lies far beyond the capacity of many health systems in low-income countries [34,35]. It is also consistent with the recommendation that modern programming has to go beyond equitable targeting [2] in order hasten improvements in health outcomes.
It was surprising that land ownership and access, perceived by many in Uganda as the ultimate form of security or socio-economic status [29], showed no differentials with stunting. Obviously, this may be different in more densely populated parts of the globe. The fact that majority (90%) of the households had a piece of land could also reduce differences in nutritional status among children. However, assuming no extraneous confounding or misclassification biases these findings could imply that land ownership in rural subsistence agricultural set-ups is not critical for child health and nutrition or is at least not as vital as parents' education and household wealth index in child well being.
Concordant with findings in other studies [31,36], age correlated positively with stunting. However, disturbing results were observed with child sex. Among well-nourished children, sex differences are attributed to a normal pattern of dimorphism, with males tending to be taller and heavier than females. Finding of this study indicate that male children were more likely to be stunted than females. Surprisingly several studies report a similar pattern in Africa [8,16,23,37]. What was more disturbing however was that larger proportions of male children were stunted as one descends the socio-economic profile as compared to females. Stunting differentials with socio-economic status observed in this study could be solely attributed to boys. Unfortunately none of the literature cited disaggregate stunting with sex across socio-economic groups. There are also no documented beliefs, attitudes or practices that segregate against the boy child in Uganda. However, in evolutionary biology there is evidence of male vulnerability in response to environmental stress in early life [38].
Conclusions
This study highlights the importance of maternal education in child well being. From our data any increment in maternal education is likely to have a positive influence on child growth. Governments of low-income countries need to ensure that the girl child receives appropriate formal education. For effective targeting of families with children in greatest need or at highest risk of health and nutrition hazards, policy makers and programme managers could be guided by mothers' education. Findings that males appear to be more adversely affected by poverty than their female counterparts corroborate evidence from previous research.
Authors' contributions
All authors participated in the design of the study, the interpretation of findings and write-up of the manuscript. HW coordinated and supervised field data collection and performed the statistical analysis. All authors read and approved the manuscript.
Competing interests
The authors declare that they have no competing interests.
Acknowledgements
Financial support for this study was obtained from NORAD, the Norwegian government Quota Program and the NUFU collaborative project between the Department of Paediatrics and Child Health, Makerere University, Kampala, Uganda and the Centre for International Health, University of Bergen, Norway. Mothers and children who participated in the study are also gratefully acknowledged.
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| 15482596 | PMC529301 | CC BY | 2021-01-04 16:39:30 | no | Int J Equity Health. 2004 Oct 13; 3:9 | utf-8 | Int J Equity Health | 2,004 | 10.1186/1475-9276-3-9 | oa_comm |
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Environ HealthEnvironmental Health1476-069XBioMed Central London 1476-069X-3-101549622610.1186/1476-069X-3-10ResearchRisk factors for acute chemical releases with public health consequences: Hazardous Substances Emergency Events Surveillance in the U.S., 1996–2001 Ruckart Perri Z [email protected] Wendy A [email protected] Wendy E [email protected] Agency for Toxic Substances and Disease Registry, Division of Health Studies, Epidemiology and Surveillance Branch, 1600 Clifton Road, MS E-31, Atlanta, Georgia, USA2004 20 10 2004 3 10 10 7 6 2004 20 10 2004 Copyright © 2004 Ruckart et al; licensee BioMed Central Ltd.2004Ruckart et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Releases of hazardous materials can cause substantial morbidity and mortality. To reduce and prevent the public health consequences (victims or evacuations) from uncontrolled or illegally released hazardous substances, a more comprehensive analysis is needed to determine risk factors for hazardous materials incidents.
Methods
Hazardous Substances Emergency Events Surveillance (HSEES) data from 1996 through 2001 were analyzed using bivariate and multiple logistic regression. Fixed-facility and transportation-related events were analyzed separately.
Results
For fixed-facility events, 2,327 (8%) resulted in at least one victim and 2,844 (10%) involved ordered evacuations. For transportation-related events, 759 (8%) resulted in at least one victim, and 405 (4%) caused evacuation orders. Fire and/or explosion were the strongest risk factors for events involving either victims or evacuations. Stratified analysis of fixed-facility events involving victims showed a strong association for acid releases in the agriculture, forestry, and fisheries industry. Chlorine releases in fixed-facility events resulted in victims and evacuations in more industry categories than any other substance.
Conclusions
Outreach efforts should focus on preventing and preparing for fires and explosions, acid releases in the agricultural industry, and chlorine releases in fixed facilities.
==== Body
Background
Recent high-profile hazardous materials incidents, such as an explosion at a pharmaceutical supply plant in Kinston, North Carolina [1] and an explosion at a manufacturing plant that makes automotive insulation products in Corbin, Kentucky [2], highlight the need to determine risk factors for hazardous materials incidents to reduce the public health consequences from uncontrolled or illegally released hazardous substances. Previous analyses of risk factors for chemical releases have been restricted to one state, one chemical, or only a few years of data. An analysis of hazardous substance releases in Wisconsin found that ammonia releases occurred more frequently in the food processing, manufacturing, and agricultural industries; and ammonia releases were more likely than releases of all other chemicals to result in evacuation and injury [3]. Burgess et al. showed that chemical releases in Washington state inside buildings and releases involving three to five victims were more likely to require evacuation or sheltering in place [4]. An analysis of hazardous substance releases during 1990–1992 showed that ammonia, chlorine, and acids were frequently released and were more likely than other substances to result in injuries or evacuations [5].
A more comprehensive analysis is needed to determine which risk factors are associated with releases of hazardous substances that result in victims or evacuations. The Hazardous Substances Emergency Events Surveillance (HSEES) system, maintained by the Agency for Toxic Substances and Disease Registry (ATSDR), is an active, multistate surveillance system designed to collect and analyze information about acute releases of hazardous substances. Data elements captured in HSEES include time, date, and day of the week when the event occurred; event type (fixed-facility or transportation-related event); geographic information; factors contributing to the release; type of chemicals released; information about injured persons (victims); industry responsible for the event; and information about decontaminations and orders to evacuate. Data from HSEES were analyzed to identify potential risk factors associated with chemical releases involving victims or evacuations during 1996–2001.
Methods
Surveillance
HSEES events are defined as sudden, uncontrolled, or illegal releases of at least one hazardous substance that had to be removed, cleaned up, or neutralized according to federal, state, or local law. A substance is considered hazardous if it might reasonably be expected to cause adverse human health outcomes. Threatened releases are also included in HSEES, if the amount threatened to be released would have required removal, cleanup, or neutralization under federal, state, or local law and the threat led to an action to protect the public health (e.g., rerouting traffic, closing a road, or ordering an evacuation). Events involving only petroleum are excluded from HSEES.
The purpose of HSEES is to reduce morbidity and mortality associated with acute releases of hazardous substances. The goals of the system are to: 1) describe the distribution and characteristics of hazardous substances emergencies, 2) describe morbidity and mortality of employees, emergency responders, and the general public resulting from hazardous substance releases, 3) identify risk factors for morbidity and mortality, and 4) identify strategies that might reduce future morbidity and mortality resulting from the release of hazardous substances.
The pilot phase of the surveillance system was from January 1, 1990 through December 31, 1992. Data for 1996–2001, the most recent period for which complete data are available, were analyzed. Thirteen states participated in HSEES during the entire period analyzed: Alabama, Colorado, Iowa, Minnesota, Missouri, Mississippi, New York, North Carolina, Oregon, Rhode Island, Texas, Washington, and Wisconsin. An additional four states participated during portions of that period: Louisiana (2001), New Hampshire (1996), New Jersey (2000–2001), and Utah (2000–2001).
State health department personnel used a variety of sources (e.g., records and oral reports of state environmental agencies, police and fire departments, and hospitals) to collect information about the hazardous events. Before January 1, 2000, data were entered into a computerized data-entry system designed by ATSDR and were transmitted quarterly to ATSDR for quality control checks and analysis. Beginning on January 1, 2000, data were entered into a web-based application that enabled ATSDR to instantly access the data.
A standardized data-collection instrument was used to obtain information on each event. ATSDR provided the states with a training manual to ensure uniformity. A victim is defined as a person experiencing at least one documented adverse health effect (such as respiratory irritation or chemical burns) that probably resulted from the event and occurred within 24 hours after the release. The HSEES system does not identify the immediate cause of the adverse health effect other than the event itself. Official evacuations were recorded in the system if they were ordered by on-scene coordinators such as a fire or police chief, a member of a HazMat team, or some other type of official. Reasons for the evacuation are not captured.
Industry codes for the type of industry responsible for each HSEES event were assigned according to the 1990 Industrial Classification System of the United States Census Bureau [6]. The industry classification system consists of 243 codes. From this, 18 major industry categories were defined as follows: agriculture, forestry, and fisheries; construction; mining; manufacturing chemical and allied products; manufacturing petroleum and coal products; manufacturing (excluding chemical and allied products and petroleum and coal products); transportation; communications; utilities and sanitary services; wholesale trade; retail trade; finance, insurance and real estate; business and repair services; personal services (comprising industries such as private households, hotels/motels, dry cleaners, and beauty parlors); entertainment and recreation services; professional and related services (which includes hospitals and schools); public administration; and military. Individual chemicals that were released or threatened to be released were assigned to one of 11 substance categories (acids, ammonia, bases, chlorine, other inorganic substances, paints and dyes, pesticides, polychlorinated biphenyls, volatile organic compounds, mixtures of substances from different categories mixed before release, and other). Events that involved chemicals from more than one substance category were categorized as "multiple substances."
Analysis
Data were analyzed using SAS [7] logistic regression to identify potential risk factors associated with chemical releases involving victims or evacuations during 1996–2001. Events that involved only a threatened release were excluded from analyses that examined risk factors for events with victims. Fixed-facility and transportation-related events were analyzed separately. Potential risk factors included time of day (0000–0559, 0600–1159, 1200–1759, and 1800–2359), day of week (weekday vs. weekend), and season (December-February, March-May, June-August, and September-November) when the event occurred; type of general land use of the surrounding area in which the event occurred (industrial, commercial, residential, agricultural, other); release type; and substance category.
For each chemical reported, up to two entries for type of release could be selected from six choices (spill, air, fire, explosion, threatened release, other). To describe an event, release type was assigned in a hierarchic arrangement (fire and explosion, explosion, fire, air, spill, or threatened) according to all release types reported for an event. When an event had multiple release types, the highest rank was used to assign the release type. For example, an event that had a chemical released as both an air emission and a fire (or one chemical released by air and one chemical released by fire) would be classified as fire. Threatened releases were analyzed only for events with evacuations. The area of evacuation recorded if the evacuation zone was downwind/downstream of the release, a circular area, the building(s) or affected part of the building(s), both a circular area and downwind/downstream, or if no criteria were used.
For fixed-facility events, industry category and factor contributing to the release (improper mixing or filling; equipment failure; human error; system problem; beyond human control, including power failures and bad weather; illegal dumping or deliberate damage; other) were also included. Although up to two factors could have been selected for each event, we analyzed only the first factor selected, because only 13% of events had a second factor selected and specific combinations of factors were too sparse to provide meaningful analysis.
We examined the likelihood of an event resulting in a victim or evacuation for specific substance and industry categories separately. Substance categories were coded as yes/no; the referent group was all other substances. Similarly, industry categories were coded as yes/no; the referent group was all other industries. Because most transportation-related events were coded into the transportation industry (79%), the association between industry type and an event with a victim (or evacuation) was examined only for fixed-facility events. Mode of transportation, categorized as ground (i.e., truck), rail, water, air, and other, was also included in the analysis for transportation-related events.
To further specify risk factors, multivariate and stratified analyses were performed to examine whether certain combinations of variables significant as univariate predictors were associated with a greater likelihood of an event with a victim or evacuation. A multiple logistic regression model was constructed to examine the potential interactions among time of day, weekday, and season. For fixed-facility events, analysis stratified by industry category examined the association between specific substances and an event with a victim or evacuation within industries.
Results
During 1996–2001, participating state health departments reported 39,766 events to the HSEES system. The analysis of risk factors for events with at least one victim excluded 568 events that involved only a threatened release, and thus focused on 39,198 events (29,974 (76%) fixed-facility events and 9,224 (24%) transportation-related events). At least one victim was reported for 2,327 (8%) of the fixed-facility events with the number of victims per event ranging from 1 to 259 (mean = 4 and median = 1). Similarly, at least one victim was reported for 759 (8%) of the transportation events with the number of victims per event ranging from 1 to 65 (mean = 2 and median = 1).
The analysis of risk factors for evacuations focused on 39,622 events (30,190 (76%) fixed-facility events and 9,432 (24%) transportation-related events) because information about whether an evacuation was ordered was missing for 144 events. Evacuations were ordered for 2,961 (10%) fixed-facility events with the number of persons evacuated per event ranging from 0 (10 events) to 11,000 (mean = 112 and median = 20). For transportation events, 453 (5%) events involved an ordered evacuation; the number of persons evacuated per event ranged from 0 (3 events) to 8,700 (mean = 163 and median = 20). For fixed-facility events, the area of evacuation was primarily the building(s) or affected part of the building(s) (2,222 events, 75%), while for transportation events, the evacuation zone was primarily a circular area (199, 44%).
Events involving victims
Emergency chemical releases at fixed facilities involving victims were more likely to occur on weekdays than weekends (prevalence odds ratio [POR] = 1.16, 95% CI = 1.03–1.29) (Table 1). Furthermore, events at fixed facilities with victims were most likely to occur during 1200–1759 (POR = 1.90, 95% CI = 1.61–2.25) compared with the hours of 0000–0559. Transportation-related events were less likely to occur during specific hours or on weekdays. Seasonality was not associated with a higher likelihood of chemical releases with victims for either fixed-facility or transportation-related events.
Table 1 Univariate analyses for acute chemical releases involving victims, by type of event.
Variable Fixed Facilities Transportation
No. % with victims POR (95% CI) No. % with victims POR (95% CI)
Time of day
0000–0559 4117 4.3 referent 1205 9.8 referent
0600–1159 10104 7.4 1.78 (1.51–2.11) 3011 9.2 0.94 (0.75–1.18)
1200–1759 9289 7.9 1.90 (1.61–2.25) 2539 8.7 0.87 (0.69–1.11)
1800–2359 5069 7.4 1.78 (1.48–2.14) 1171 8.8 0.89 (0.67–1.17)
Day of week
Weekend 5765 7.0 referent 1226 7.0 referent
Weekday 24209 8.0 1.16 (1.03–1.29) 7998 8.4 1.22 (0.97–1.54)
Season
Dec.-Feb. 6643 7.3 referent 1631 8.3 referent
Mar.-May 7988 7.9 1.08 (0.96–1.23) 2812 7.4 0.88 (0.70–1.10)
Jun.-Aug. 8480 8.1 1.11 (0.99–1.26) 2794 8.9 1.08 (0.87–1.35)
Sep.-Nov. 6863 7.7 1.07 (0.94–1.21) 1987 8.5 1.02 (0.81–1.30)
Area*
Industrial 18936 5.6 0.59 (0.45–0.77) 2547 12.7 0.20 (0.15–0.27)
Commercial 5336 3.4 3.49 (2.67–4.57) 3385 2.9 0.46 (0.36–0.59)
Residential 2097 17.3 1.68 (1.53–1.85) 612 6.3 1.08 (0.98–1.19)
Agricultural 2253 22.1 1.70 (1.27–2.29) 1628 15.5 1.29 (1.02–1.64)
Other 1064 9.2 referent 903 15.9 referent
Release Type
Spill 11165 6.2 referent 8020 6.5 referent
Air 17280 7.1 1.16 (1.06–1.28) 945 18.3 3.25 (2.69–3.91)
Fire 878 22.8 4.47 (3.75–5.33) 134 35.8 8.08 (5.62–11.64)
Explosion 244 53.7 17.57 (13.51–22.86) 11 45.5 12.07 (3.67–39.68)
Fire and explosion 98 57.1 20.21 (13.45–30.38) 5 40.0 9.66 (1.61–57.91)
Substances†,‡
Acid 1832 14.8 2.20 (1.92–2.53) NA
Ammonia 2150 12.6 1.81 (1.58–2.07) 280 17.5 2.46 (1.79–3.38)
Chlorine 597 28.0 4.90 (4.07–5.89) 18 33.3 5.62 (2.10–15.01)
Other inorganics§ NA 968 10.7 1.40 (1.23–1.74)
Pesticides 810 14.8 2.13 (1.74–2.59) NA
Multiple substances¶ 1142 24.0 4.12 (3.57–4.75) 352 36.4 7.46 (5.92–9.41)
*Type of general land use of the surrounding area in which the event occurred.
†Only substance categories with a 95% CI greater than 1.0 are presented.
‡ Substance categories were coded as yes/no. The referent group is all other substances.
§Excludes acids, bases, ammonia, and chlorine.
¶Events with more than one hazardous substance released from different chemical categories.
Fixed-facility events with victims were most likely to occur in areas described as commercial, than in the referent group "other," while transportation-related events were more likely to occur in agricultural areas. Compared with spills, fire and explosion was the release type with the greatest likelihood of victims for fixed-facility events (POR = 20.21, 95% CI = 13.45–30.38). Releases of chlorine (POR = 4.90, 95% CI = 4.07–5.89) were most likely to result in fixed-facility events with victims compared with releases from all other chemical categories while releases of multiple substances from different categories (POR = 7.46, 95% CI = 5.92–9.41) were most likely to result in transportation events with victims.
Type of industry was examined as a potential risk factor for victims in fixed-facility events only. Industries classified as personal services were most likely to result in events with victims than were releases not involving these industries (POR = 8.24, 95% CI = 7.20–9.44) (Table 2). Factors identified as illegal dumping or deliberate damage were most likely to contribute to a fixed-facility event with at least one victim (POR = 15.93, 95% CI = 10.41–24.36) compared with "other" factors. Factors classified as "other" included maintenance, vehicular accident, fire, explosion, and factors that could not be classified into an existing category.
Table 2 Univariate analyses for acute chemical releases in fixed facilities, by events with victims and evacuations.
Variable Victims Evacuations
No. % with victims POR (95% CI) No. % with evac. POR (95% CI)
Industry*,†
Agriculture‡ 578 17.8 2.75 (2.21–3.42) NA
Business and repair services 265 23.0 3.76 (2.81–5.02) 268 15.7 1.74 (1.25–2.42)
Communications 33 21.2 3.33 (1.45–7.68) NA
Construction 319 15.4 2.26 (1.67–3.08) NA
Entertainment and recreation services 196 29.6 5.29 (3.88–7.21) 200 29.5 3.96 (2.91–5.38)
Finance, insurance & real Estate 108 37.0 7.36 (4.97–10.91) 107 41.1 6.59 (4.47–9.70)
Manufacturing§ 4594 9.6 1.39 (1.24–1.55) 4598 18.5 2.59 (2.38–2.83)
Personal services 1025 36.3 8.24 (7.20–9.44) 1064 23.7 3.03 (2.62–3.51)
Professional and related services 953 25.6 4.64 (3.99–5.41) 978 42.3 7.82 (6.85–8.94)
Public administration 273 22.7 3.70 (2.77–4.92) 277 22.4 2.72 (2.05–3.62)
Retail trade 451 33.0 6.45 (5.27–7.88) 456 36.6 5.65 (4.65–6.86)
Wholesale trade 1061 10.7 1.49 (1.22–1.82) NA
Factors
Beyond human control¶ 1076 2.2 0.58 (0.37–0.92) 1084 8.9 1.56 (1.19–2.05)
Equipment failure 15193 4.5 1.21 (0.97–1.51) 15186 7.1 1.23 (1.02–1.47)
Human error 5900 14.3 4.28 (3.43–5.35) 5930 15.6 2.97 (2.47–3.57)
Illegal dumping or deliberate damage 1152 26.7 15.93 (10.41–24.36) 1295 16.6 2.05 (1.59–2.65)
Improper mixing or filling 771 22.6 7.46 (5.69–9.77) 775 27.0 5.94 (4.71–7.49)
System problem 1838 0.9 0.23 (0.13–0.38) 1833 1.4 0.23 (.015–0.35)
Other 2393 3.8 referent 2425 5.9 referent
*Only industry categories with 95% CI greater than 1.0 are presented.
†Industry categories were coded as yes/no. The referent group is all other industries.
‡Includes forestry and fisheries.
§Excludes chemical and allied products and petroleum and coal products, which were analyzed as separate categories.
¶Includes power failures and bad weather.
Events involving evacuations
Emergency chemical releases at fixed facilities involving evacuations were more likely to occur on weekdays (POR = 1.26, 95% CI = 1.14–1.40) (Table 3). Furthermore, events at fixed facilities with evacuations were most likely to occur during 1800–2359 (POR = 1.69, 95% CI = 1.45–1.97) compared with the hours of 0000–0559. Fixed-facility events that resulted in evacuation orders were slightly more likely to occur during June through August than during December through February (POR = 1.12, 95% CI = 1.01–1.25). Transportation-related events were not more likely to occur during specific hours, on weekdays, or during a particular season.
Table 3 Univariate analyses for acute chemical releases involving evacuations, by type of event.
Variable Fixed Facilities Transportation
No. % with evac. POR (95% CI) No. % with evac. POR (95% CI)
Time of day
0000–0559 4130 6.5 referent 1237 4.9 referent
0600–1159 10188 10.4 1.67 (1.45–1.92) 3085 5.1 1.04 (0.77–1.41)
1200–1759 9363 10.2 1.64 (1.42–1.88) 2591 5.4 1.11 (0.82–1.51)
1800–2359 5100 10.5 1.69 (1.45–1.97) 1210 6.1 1.26 (0.89–1.78)
Day of week
Weekend 5790 5.0 referent 1262 7.7 referent
Weekday 24400 4.8 1.26 (1.14–1.40) 8170 8.8 0.95 (0.73–1.25)
Season
Dec.-Feb. 6683 9.2 referent 1682 5.4 referent
Mar.-May 8058 9.6 1.04 (0.93–1.17) 2860 4.6 0.86 (0.65–1.13)
Jun.-Aug. 8559 10.3 1.12 (1.01–1.25) 2849 4.7 0.87 (0.66–1.14)
Sep.-Nov 6890 10.1 1.10 (0.98–1.23) 2041 4.8 0.89 (0.67–1.20)
Area*
Industrial 18958 8.5 0.60 (0.48–0.75) 2579 5.7 0.61 (0.43–0.85)
Commercial 5438 5.2 3.11 (2.49–3.88) 3436 3.5 0.87 (0.63–1.19)
Residential 2158 22.3 1.50 (1.38–1.62) 637 5.0 1.12 (0.98–1.28)
Agricultural 2269 23.6 0.68 (0.51–0.89) 1681 7.9 0.86 (0.60–1.22)
Other 1089 5.9 referent 947 4.9 referent
Release Type
Spill 11129 6.9 referent 7999 3.1 referent
Air 17222 9.3 1.38 (1.27–1.51) 945 12.7 4.55 (3.61–5.72)
Fire 869 35.7 7.48 (6.40–8.75) 134 20.2 7.89 (5.08–12.25)
Explosion 242 36.8 7.85 (5.98–10.29) 10 20.0 7.81 (1.65–36.99)
Fire & explosion 96 46.9 11.90 (7.92–17.89) 4 25.0 10.42 (1.08–100.5)
Threatened 335 34.9 7.24 (5.72–9.17) 231 20.8 8.20 (5.82–11.54)
Substances†,‡
Acid 1850 9.6 1.46 (1.27–1.67) NA
Ammonia 2144 24.6 3.44 (3.09–3.83) 290 18.3 4.89 (3.57–6.69)
Chlorine 599 33.1 4.79 (4.03–5.71) 22 27.3 7.52 (2.93–19.31)
Multiple substances§ 1213 25.6 3.41 (2.98–3.90) 386 14.8 3.79 (2.81–5.10)
*Type of general land use of the surrounding area in which the event occurred.
†Only substance categories with a 95% CI greater than 1.0 are presented.
‡ Substance categories were coded as yes/no. The referent group is all other substances.
§Events with more than one hazardous substance released from different chemical categories.
Fixed-facility events with evacuations were most likely to occur in areas described as commercial compared with the referent group "other." Area type was not associated with ordered evacuations for transportation-related events. The release type fire and explosion was most likely to result in fixed-facility events with evacuations compared with spills (POR = 11.90, 95% CI = 7.92–17.89). Releases of chlorine were most likely to result in events with evacuations compared with releases from all other chemical categories for both fixed-facility and transportation-related events (POR = 4.79, 95% CI = 4.03–5.71 and POR = 7.52, 95% CI = 2.93–19.31, respectively).
We examined type of industry as a potential risk factor for evacuations in fixed-facility events only. Industries classified as professional services were most likely to result in events with evacuations compared with releases not involving these industries (POR = 7.82, 95% CI = 6.85–8.94) (Table 2). Factors identified as improper mixing or filling were most likely to contribute to releases with evacuations in fixed facilities (POR = 5.94, 95% CI = 4.71–7.49) compared with "other" factors.
Multivariate and stratified analyses of fixed-facility events
Results from the multiple regression analysis of temporal variables indicated an association of borderline significance between fixed-facility events with victims and the three-way interaction indicated by time of day (0600–1159), day of week (weekday), and season (March through May) (p = 0.06); no interaction among temporal variables was indicated for evacuations.
The likelihood of an event with at least one victim or evacuation in fixed facilities was examined for substances within industry category (Table 4). The strongest association was observed for acid releases in the agriculture, forestry, and fisheries industry for fixed-facility events involving victims (POR = 7.28, 95% CI = 2.02, 26.30). Releases of acids, chlorine, and multiple substances in the manufacturing industry had an elevated likelihood of both an event with at least one victim and an evacuation. There were fewer associations between substances and industry categories for events with evacuations compared with events with at least one victim.
Table 4 Multivariate analyses* for acute chemical releases in fixed facilities, by industry and substance category.
Industry Acids Ammonia Chlorine Pesticides Multiple Substances
POR (95% CI) POR (95% CI) POR (95% CI) POR (95% CI) POR (95% CI)
Victims
Agriculture† 7.28 (2.02–26.30) 2.65 (1.68–4.17)
Construction 5.92 (1.43–24.50)
Entertainment & recreation services 5.78 (2.80–11.94)
Manufacturing‡ 1.71 (1.27–2.29) 4.03 (2.69–6.05) 2.74 (1.90–3.96)
Personal services 3.21 (2.30–4.47)
Professional and related services 2.43 (1.49–3.99) 2.99 (1.23–7.27)
Public administration 3.99 (1.47–10.85)
Retail trade 3.37 (1.08–10.49)
Wholesale trade 4.19 (2.32–7.59) 2.35 (1.57–3.54)
Evacuations
Business & repair services 3.84 (1.55–9.48)
Entertainment & recreation services 3.60 (1.77–7.31)
Manufacturing‡ 4.31 (3.64–5.10) 3.32 (2.23–4.67) 2.64 (1.94–3.60)
Personal services 3.07 (1.72–5.45)
Retail trade 4.06 (1.23–13.38)
*Analysis stratified by industry category examined associations between specific substances and events with victims or evacuations within industries for categories that were significant in the univariate analyses.
†Includes forestry and fisheries.
‡Excludes chemical and allied products and petroleum and coal products, which were analyzed as separate categories.
Discussion
Almost 40,000 chemical release events were reported to HSEES during the 6-year period included in this analysis. Approximately 8% of the events resulted in almost 12,000 victims, and approximately 9% involved evacuation of more than 325,000 people. Because the public health consequences of chemical releases can be serious and affect large numbers of people, identifying the risk factors more likely to be associated with hazardous substance releases that result in victims or evacuations is important to target appropriate prevention activities.
For fixed-facility events, fire and explosion was the strongest single risk factor for events involving either victims or evacuations. Illegal dumping or deliberate damage also was strongly associated with fixed-facility events resulting in victims. For transportation-related events, explosion was the strongest single risk factor for events involving victims, and fire and explosion was the strongest for events involving evacuations. The observed associations between time of day (0600–2359) and weekday occurrence are likely to be influenced by production intensity, however information on production intensity is not available in HSEES to examine this relationship further. When temporal variables were modeled together, fixed-facility events with victims were more likely to occur on weekday mornings in the spring, which may coincide with the planting season. These results are consistent with a previous analysis that showed that acute hazardous substance releases with victims were more likely during the planting season in Midwestern states [8].
In the stratified analysis of substances within industry category, the strongest association was for acid releases in the agriculture, forestry, and fisheries industry for fixed-facility events involving victims. Previous analyses have shown that acute chemical releases are common in the agricultural industry, nitric acid and sulfuric acid are frequently released in agriculture events, and releases of acids result in significantly higher proportions of releases involving victims [5,8,9].
Chlorine releases in fixed facilities resulted in victims and evacuations in more industry categories than any other substance. This is consistent with an analysis that found chlorine releases had the greatest significant risk of having events with victims and were almost five times more likely than nonchlorine events to involve evacuations [10]. This is not surprising given that chlorine is a strong corrosive agent. Acute health effects of exposure to low levels of chlorine include sore throat, coughing, and eye and skin irritation; exposure to higher levels may cause severe burning of the eyes and skin, rapid breathing, narrowing of the bronchi, wheezing, bluish discoloration of the skin, and lung collapse [11]. Additionally, chlorine is one of the most frequently produced chemical substances in the United States, with more than 30 million tons produced annually [12]. Chlorine is widely used in a variety of processes, such as synthesizing other chemicals and making bleaches and disinfectants.
The HSEES system collected data in only 17 states during 1996–2001. Each state has different reporting requirements for the amount of hazardous substances released that has to be removed, cleaned up, or neutralized; and reporting of events to participating state health departments is not mandatory. Therefore, the total number of events, events with victims, and events with evacuations may be underestimated. However, HSEES is the only federal hazardous substances release database designed specifically to assess and record the public health consequences of hazardous substances emergency events.
The HSEES system captures more events than other federal reporting systems, such as the United States Environmental Protection Agency's (EPA) Risk Management Program (RMP) [13]. EPA's RMP requires all companies that use certain flammable and toxic substances to develop procedures for hazard assessment, prevention activities, and emergency response [14]. Because company-specific information (e.g., name and street address) is not available to HSEES at the federal level, RMP accident history data provides important insight regarding the hazardousness and regulatory practices associated with accidental releases of chemicals at United States manufacturing facilities [15].
These results indicate that prevention activities should focus on ways to reduce fire and/or explosion hazards in facilities that store, use, or manufacture hazardous substances such as distributing material safety data sheets to employees, keeping work areas dust free, and storing chemicals away from ignition sources. Previous analyses of HSEES data have shown that human error and equipment failure are the most frequent causes of acute chemical releases [10,16-18]; releases due to these causes may result in fires and explosions. Facilities should also have chemical inventories and risk management plans available for responders to enhance their response capabilities [19]. Additionally, security measures (e.g., surveillance cameras, chain-link fences, patrol guards, alarms) should be put in place to reduce deliberate chemical releases, such as theft [20]. Prevention activities also should target agricultural industries that use or store acids and industries that store, use, or manufacture chlorine.
Several state health departments participating in HSEES already have developed strategies aimed at reducing releases and injuries associated with chlorine. These activities include distribution of fact sheets to county emergency management agencies, fire departments, other first responders, and industries; and presentations to municipal water directors, engineers, and hotel/motel swimming pool owners and operators.
The predictors of public health consequences associated with acute chemical releases presented in this paper are broad categories of characteristics available in the national data. Future analyses of HSEES data may explore industry-specific root causes of events and risk of events with victims or evacuations for specific industries.
Conclusion
The results of this analysis should help guide prevention activities aimed at reducing emergency chemical releases and their associated victims and evacuations. Attention should focus on preventing fires and explosions, releases from illegal dumping and deliberate damage, and acid releases in the agricultural industry. Efforts should continue to educate industry, first responders and other users about the potential hazards of chlorine.
List of Abbreviations
ATSDR, Agency for Toxic Substances and Disease Registry
CI, confidence interval
EPA, Environmental Protection Agency
HSEES, Hazardous Substances Emergency Events Surveillance
POR, prevalence odds ratio
RMP, Risk Management Program
Competing Interests
The author(s) declare that they have no competing interests.
Authors' contributions
PZR led the writing of the manuscript and assisted with the statistical analysis. WAW led the statistical analysis and assisted with the writing of the manuscript. WEK conceived of the manuscript and participated in its writing and interpretation. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We extend our grateful appreciation to our partners in the participating state health departments, who, with diligence and dedication, researched and gathered much of the data for this publication. Without their assistance, ideas, and comments, this work would not have been possible.
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| 15496226 | PMC529302 | CC BY | 2021-01-04 16:36:31 | no | Environ Health. 2004 Oct 20; 3:10 | utf-8 | Environ Health | 2,004 | 10.1186/1476-069X-3-10 | oa_comm |
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Ann Clin Microbiol AntimicrobAnnals of Clinical Microbiology and Antimicrobials1476-0711BioMed Central London 1476-0711-3-211549623210.1186/1476-0711-3-21ResearchEarly detection of Pseudomonas aeruginosa – comparison of conventional versus molecular (PCR) detection directly from adult patients with cystic fibrosis (CF) Xu Jiru [email protected] John E [email protected] Philip G [email protected] B Cherie [email protected] J Stuart [email protected] Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Lisburn Road, Belfast, Northern Ireland, BT9 7AD, UK2 School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, Co. Londonderry, Northern Ireland, BT52 1SA, UK3 Northern Ireland Regional Adult Cystic Fibrosis Centre, Level 8, Belfast City Hospital, Lisburn Road, Belfast, Northern Ireland, BT9 7AB, UK4 Department of Respiratory Medicine, The Queen's University of Belfast, Belfast City Hospital, Lisburn Road, Belfast, Northern Ireland, BT9 7AB, UK2004 20 10 2004 3 21 21 29 7 2004 20 10 2004 Copyright © 2004 Xu et al; licensee BioMed Central Ltd.2004Xu et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Pseudomonas aeruginosa (PA) is the most important bacterial pathogen in patients with cystic fibrosis (CF) patients. Currently, routine bacteriological culture on selective/non- selective culture media is the cornerstone of microbiological detection. The aim of this study was to compare isolation rates of PA by conventional culture and molecular (PCR) detection directly from sputum.
Methods
Adult patients (n = 57) attending the regional adult CF centre in Northern Ireland, provided fresh sputum following airways clearance exercise. Following processing of the specimen with sputasol (1:1 vol), the specimen was examined for the presence of PA by plating onto a combination of culture media (Pseudomonas isolation agar, Blood agar & McConkey agar). In addition, from the same specimen, genomic bacterial DNA was extracted (1 ml) and was amplified employing two sequence-specific targets, namely (i) the outer membrane protein (oprL) gene locus and (ii) the exotoxin A (ETA) gene locus.
Results
By sputum culture, there were 30 patients positive for PA, whereas by molecular techniques, there were 35 positive patients. In 39 patients (22 PA +ve & 17 PA -ve), there was complete agreement between molecular and conventional detection and with both PCR gene loci. The oprL locus was more sensitive than the ETA locus, as the former was positive in 10 more patients and there were no patients where the ETA was positive and the oprL target negative. Where a PCR +ve/culture -ve result was recorded (10 patients), we followed these patients and recorded that 5 of these patients converted to being culture-positive at times ranging from 4–17 months later, with a mean lag time of 4.5 months.
Conclusions
This study indicates that molecular detection of PA in sputum employing the oprL gene target, is a useful technique in the early detection of PA, gaining on average 4.5 months over conventional culture. It now remains to be established whether aggressive antibiotic intervention at this earlier stage, based on PCR detection, has any significant benefits on clinical outcome.
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Introduction
Cystic fibrosis [CF] is the most commonly inherited fatal disease in persons originating from a white and European background, currently affecting approximately 30,000 adults and children in the US [1]. The defective gene carrying the mutation is carried in one in every 31 Americans [one in 28 Caucasians], equating to more than 10 million people being a symptomless carrier of the defective gene [1]. It is an autosomal recessive condition whereby two alleles carrying a polymorphism in the cystic fibrosis transmembrane conductance regulator [CFTR] gene phenotypically manifest the disease state through a variety of multiorgan problems, associated with a pharmacological disfunction to regulate sodium and chloride secretion across cell membranes. The most common complication of CF is the recurrence of chronic chest infections usually caused by bacterial pathogens [2]. CF patients continue to suffer from recurrent and chronic respiratory tract infections and most of their morbidity and mortality is due to such infections throughout their life [3]. These infections are usually dominated by Gram-negative organisms, especially by the pseudomonads, including Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia. However, with modern antibiotic management with improved antimicrobial agents, such as the aminoglycosides and carbapenems, CF patients have an improved survival, resulting in more adults in employment.
Previous studies have described alternative laboratory markers to conventional bacteriological culture for the detection of Pseudomonas aeruginosa in CF patients.[4,5] These markers have included serological testing of CF patients' sera for the presence of antibody to P. aeruginosa,[4] as well as molecular detection of DNA signature sequences of P. aeruginosa in CF patients [5]. Presently, the routine employment of molecular (PCR) detection of P. aeruginosa directly from the sputum of CF patients in the UK and Ireland is uncommon. Most clinical microbiology laboratories in the UK and Ireland, which support a CF centre, have developed standard operating procedures (SOPs) for the isolation of this organism from patients' sputum. Hence, it was the aim of this study to compare the conventional and molecular detection of P. aeruginosa from the sputum of adult patients attending the adult centre in Northern Ireland, as well as to estimate the lag time of conventional culture to detection, compared with molecular (PCR) detection.
Materials & Methods
Qualitative conventional detection of Pseudomonas aeruginosa from sputum
Duplicate sputa (1 ml minimum) specimens were collected from 57 adult patients with a well characterized history of CF in sterile (100 ml) plastic disposable containers. Sputum was collected immediately after a standardized session of physiotherapy and was stored at ambient temperature and was processed within 4 h from collection. Fresh sputum (1 ml min) was mixed with an equal mass (1:1) of Sputasol (Oxoid SR089A, Oxoid Ltd., Poole, England) and was incubated in a water bath at 37°C for 15 min, before further qualitative processing for the detection of Pseudomonas aeruginosa. Processed sputa (10 μ l) were inoculated and incubated, onto several selective media for the isolation of Pseudomonas aeruginosa, including:- Columbia Blood Agar (Oxoid CM0331) supplemented with 5% (v/v) defribinated horse blood, MacConkey Agar (Oxoid CM0007) and Pseudomonas Isolation Agar (PIA) (Oxoid CM0559 + SR0102). All media were incubated aerobically at 37°C for 48 h, unless otherwise stated. The PIA plates were incubated at room temperature for a further three days following initial 48 hrs incubation. In addition, all different phenotypes from the sputum of each patient were identified phenotypically employing a combination of conventional identification methods (e.g. oxidase), as well as the API Identification schemes (API 20NE, API 20E) (Biomérieux, Les Halles, France).
Molecular (PCR) detection of Pseudomonas aeruginosa from sputum
DNA extraction
All DNA isolation procedures were carried out in a Class II Biological Safety Cabinet (MicroFlow, England) in a room physically separated from that used to set up nucleic acid amplification reaction mixes and also from the "post-PCR" room in accordance with the Good Molecular Diagnostic Procedures (GMDP) guidelines of Millar et al[6], in order to minimise contamination and hence the possibility of false positive results. Bacterial genomic DNA was extracted directly from the patients' sputum, as well as from the reference strain Pseudomonas aeruginosa (Schroeter; Migula) ATCC 27853, by employment of the Roche High Purity PCR Template Preparation Kit (Roche, England), in accordance with the manufacturer's instructions. Extracted DNA was stored at -80°C prior to PCR amplification. For each batch of extractions, a negative extraction control containing all reagents minus sputum, was performed, as well as an extraction positive control with P. aeruginosa.
PCR amplification
All reaction mixes were set up in a PCR hood in a room separate from that used to extract DNA and the amplification and post-PCR room in order to minimise contamination. Initially PCR amplification conditions were optimised by separately varying magnesium chloride concentration, annealing temperature, primer concentration and DNA template concentration. Following optimisation, reaction mixes (100 μl) were set up as follows:-10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 200 μM (each) dATP, dCTP, dGTP and dTTP; 1.25 U of Taq DNA polymerase (Amplitaq; Perkin Elmer), 0.1 μM (each) of the each set of primers (exoA &oprL) (Table 1) and 4 μl of DNA template. The reaction mixtures following a "hot start" were subjected to the following empirically optimized thermal cycling parameters in a Perkin Elmer 2400 thermocycler: 96°C for 5 min followed by 40 cycles of 96°C for 1 min, 55°C for 1 min, 72°C for 1 min, followed by a final extension at 72°C for 10 min. Positive (P. aeruginosa ATCC 27853 DNA) and multiple negative (water) amplification controls were included in every set of PCR reactions. In addition, a broad-range/universal PCR was employed with each sputum to demonstrate successful extraction of bacterial DNA from the specimen, as well as lack of PCR inhibition, by employing the highly conserved 16S rDNA primers, PSL/PSR (Table 1), as previously described [7]. Any sputum which failed to amplify this locus was re-extracted and amplified until a positive signal was obtained.
Table 1 Oligonucletoides and optimised experimental PCR amplification conditions
Target Gene locus Primer 5'-----------------------3' Optimum MgCl2 concentration (mM) Annealing temperature Size of Amplicon (bp)
Eubacteria [7] 16S rRNA f: 5'-AGG ATT AGA TAC CCT GGT AGT CCA-3' 2.5 55°C 312
r: 5'-ACT TAA CCC AAC ATC TCA CGA CAC-3'
Pseudomonas aeruginosa [8] oprL1 f: 5'-ATG GAA ATG CTG AAA TTC GGC-3' 2.5 55°C 504
r: 5'-CTT CTT CAG CTC GAC GCG ACG-3'
Pseudomonas aeruginosa [9] exoA2 f: 5'-GAC AAC GCC CTC AGC ATC ACC AGC-3' 2.5 72°C 396
r: 5'-CGC TGG CCC ATT CGC TCC AGC GCT-3'
1, outer membrane lipoprotein; 2, exotoxin A.
Detection of amplicons
Following amplification, aliquots (10 μl) were removed from each reaction mixture and examined by electrophoresis (80 V, 45 min) in gels composed of 2% (w/v) agarose (Gibco, UK) in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 8.3), stained with ethidium bromide (5 μg/100 ml). Gels were visualised under UV illumination using a gel image analysis system (UVP Products, England) and all images archived as digital graphic files (*.bmp). Where a band was visualized at the correct expected size for either exoA or oprL loci, the specimen was considered positive for P. aeruginosa.
Results and Discussion
A comparison of the occurrence of P. aeruginosa in patients' sputum by conventional and molecular techniques is shown (Table 2). By culture, there were 30 patients positive for P. aeruginosa, whereas by molecular techniques, there were 35 positive patients. In 39 patients (22 P aeruginosa [PA] positive [+ve] & 17 P. aeruginosa [PA] negative [-ve]), there was complete agreement between molecular and conventional detection techniques. The oprL locus was more sensitive than the ETA locus, as the former was positive in 10 more patients and there were no patients where the ETA was positive and the oprL target negative, However, there were five patients who were culture positive but PCR -ve by both gene targets. Given that the universal 16S rDNA PCR was positive for these sputum specimens and that the appropriate molecular controls were working optimally, this may accounted for by potential phenotypic misidentification of P. aeruginosa, which has been recently described.[10] Alternatively, discrepant results (PCR+ve/culture-ve) could reflect true P. aeruginosa colonization with a false-negative culture result due to sample overgrowth by other bacteria or to the presence of non-cultivable organisms or auxotrophic mutations in the organism. Where a PCR +ve/culture -ve result was recorded (10 patients), we followed these patients and recorded that five of these patients converted to being culture-positive at times ranging from 4–17 months later, with a mean lag time of 4.5 months, whereas the remaining five patients remained negative for P. aeruginosa.
Table 2 Comparison of conventional culture with molecular detection of Pseudomonas aeruginosa from the sputum of adult CF patients
PCR (OprL) PCR (exotoxin A) Culture Frequency of patients (%)
+ + + 22 (38.6%)
+ - + 3 (5.3%)
- - + 5 (8.8%)
+ + - 3 (5.3%)
+ - - 7 (12.3%)
- - - 17 (29.7%)
In this study, two PCR assays were performed individually for the molecular detection of P. aeruginosa directly from the sputum of patients with CF. Two gene loci were targeted, as specific markers of P. aeruginosa, namely the exotoxin A (ETA) gene and the outer membrane lipoprotein (oprL) gene. ETA is produced by the majority of P. aeruginosa strains and can inhibit eucaryotic protein biosynthesis at the level of polypeptide chain elongation factor 2, similarly to diphtheria toxin.[9] OprL is an outer membrane lipoprotein which has been implicated in efflux transport systems, as well as affecting cell permeability.[8]
Pseudomonas aeruginosa is the most important bacterial pathogen in patients with CF [11], as demonstrated with high prevalence data in most of the National CF Registries. Chronic Pseudomonas colonisation of the major airways leading to delibating exacerbations of pulmonary infection, is the major cause of morbidity and mortality in patients with CF, hence it is important to be able to reliably detect P. aeruginosa from patients' sputum. Emerson et al. [12] recently published their findings of a US Cystic Fibrosis Foundation (CFF) registry-based study, which showed that infection related to P. aeruginosa was a major predictor of morbidity and mortality, whereby the 8-year risk of death parameter was 2.6 times higher in patients who had positive sputum cultures for this organism, as well as having a significantly lower percent predicted forced expiratory volume (FEV1). These workers suggested that early interventions may help decrease associated morbidity and mortality of young patients with CF.
More recently, Rosenfeld et al. [13] described the pathophysiology and risk factors for early P. aeruginosa infection in CF. These workers suggested that chronic lower airway infection with P. aeruginosa is associated with significant morbidity and mortality among CF patients [13]. However, they suggested that first acquisition of P. aeruginosa does not appear to cause an immediate and rapid decline in lung function, as early isolates are generally non-mucoid, antibiotic-sensitive and present at low densities, suggesting a possible "window of opportunity" for early intervention, as they describe [13]. This study also concluded that there are no controlled trials demonstrating clinical benefit in young children following early intervention, particularly for anti-pseudomonal therapy, as well as the long-term safety profile and optimal drug regimen.[13]
It is therefore important that primary diagnostic bacteriology laboratories have the ability to detect transient and early Pseudomonas colonization as early as possible, so that (i) aggressive antibiotic regimes may be considered,(ii). the patient is managed optimally, in an attempt to avoid early biofilm formation and chronic colonization with Pseudomonas and (iii) appropriate infection control precautions are considered. More recently, West et al. showed by a combination of serum IgG, IgA and IgM anti P. aeruginosa antibodies, in conjunction with these authors' Wisconsis Cystic Fibrosis Radiograph score, that P. aeruginosa infections occurred approximately 6–12 months before the organism was recovered from respiratory secretions.[4] In addition, this study demonstrated that mixing of young child with older chronically colonised children was associated with a significantly increased risk of P. aeruginosa acquisition.
Given that not all laboratories employ molecular detection methods for Pseudomonas aeruginosa, either from culture plates or patients' sputum, small numbers of Pseudomonas colonies (n = 1–2) may therefore be missed when present in the early stages of colonization preceding infection of the patient's airways, particularly where such single colonies are mixed alongside other phenotypically similar genera on the primary culture plate.[14] Pragmatic, practical and cost implications leave it impossible to qualitatively identify the total bacterial microflora present on non-selective primary plates from sputum. Therefore, any rapid molecular screening method should be encouraged to detect low copy numbers of organisms in the early stages of colonization/infection, where the main value of this diagnostic assay is in the rapid screening of patients with no or an intermittent history of Pseudomonas colonization. Although such assays are not generally available in most clinical diagnostic laboratories, these laboratories generally do have access to such technology at regional specialist microbiology centres and it may therefore be prudent to establish routine analysis of children's sputum, particularly from patients with no or an intermittent history by culture of Pseudomonas colonization, at annual review. Furthermore, it is very important to follow up any such patients, which demonstrate a PCR +ve/culture -ve finding from their sputum, in order to establish whether there is transient infection in patients, which never result in established colonization leading to chronic infection. Additionally, it is important to monitor such PCR +ve/culture -ve patients in terms of optimal antibiotic management and infection control.
Overall, our study demonstrated that molecular assays for the detection of P. aeruginosa were able to detect P. aeruginosa at an early stage, on average 4.5 months sooner than culture detection. However, it remains unclear what this "window of opportunity" potentially offers in terms of a decrease in P. aeruginosa-associated morbidity and mortality.
Authors' contributions
JEM, PGM and JSE jointly conceived the study and designed the experiments. JX executed and analyzed all practical aspects of the study, as well as analyzing the data. JEM executed certain molecular components of experimentation, analyzed the data and prepared the manuscript. BCM helped guide the molecular aspects of experimentation and was involved in drafting of the manuscript. PGM provided expert microbiological analysis and interpretation of data. JSE provided clinical expertise, interacted with the CF patients and was involved in interpretation of data, as well as critically reviewing the final manuscript. All authors read and approved the final manuscript.
Acknowledgements
JX was funded in association with a Teaching Company Scheme (TCS) award in co-operation with the BBSRC and PPL Therapeutics Ltd., Roslin, Edinburgh, Scotland.
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Rajan S Saiman L Pulmonary infections in patients with cystic fibrosis. Semin Respir Infect 2002 17 47 56 11891518 10.1053/srin.2002.31690
West SE Zeng L Lee BL Kosorok MR Laxova A Rock MJ Splaingard MJ Farrell PM Respiratory infections with Pseudomonas aeruginosa in children with cystic fibrosis: early detection by serology and assessment of risk factors. JAMA 2002 287 2958 2967 12052125 10.1001/jama.287.22.2958
Karpati F Jonasson J Polymerase chain reaction for the detection of Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Burkholderia cepacia in sputum of patients with cystic fibrosis. Mol Cell Probes 1996 10 397 403 9025076 10.1006/mcpr.1996.0055
Millar BC Xu J Moore JE Risk assessment models and contamination management: implications for broad-range ribosomal DNA PCR as a diagnostic tool in medical bacteriology. J Clin Microbiol 2002 40 1575 1580 11980924 10.1128/JCM.40.5.1575-1580.2002
Campbell PW Phillips JA Heidecker GJ Krishnamani MR Zahorchak R Stull TL Detection of Pseudomonas (Burkholderia) cepacia using PCR. Pediatr Pulmonol 1995 20 44 49 7478781
De Vos D Lim A JrPirnay JP Struelens M Vandenvelde C Duinslaeger L Vanderkelen A Cornelis P Direct detection and identification of Pseudomonas aeruginosa in clinical samples such as skin biopsy specimens and expectorations by multiplex PCR based on two outer membrane lipoprotein genes, oprI and oprL. J Clin Microbiol 1997 35 1295 1299 9163432
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Spilker T Coenye T Vandamme P LiPuma JJ PCR-based assay for differentiation of Pseudomonas aeruginosa from other Pseudomonas species recovered from cystic fibrosis patients. J Clin Microbiol 2004 42 2074 2079 15131172 10.1128/JCM.42.5.2074-2079.2004
Speert DP Molecular epidemiology of Pseudomonas aeruginosa. Front Biosci 2002 7 354 361
Emerson J Rosenfeld M McNamara S Ramsey B Gibson RL Pseudomonas aeruginosa and other predictors of mortality and morbidity in young children with cystic fibrosis. Pediatr Pulmonol 2002 34 91 100 12112774 10.1002/ppul.10127
Rosenfeld M Ramsey BW Gibson RL Pseudomonas acquisition in young patients with cystic fibrosis: pathophysiology, diagnosis, and management. Curr Opin Pulm Med 2003 9 492 497 14534401 10.1097/00063198-200311000-00008
Clarke L Moore JE Millar BC Garske L Xu J Heuzenroeder MW Crowe M Elborn JS Development of a diagnostic PCR assay that targets a heat-shock protein gene (gro ES) for detection of Pseudomonas spp. in cystic fibrosis patients. J Med Microbiol 2003 52 759 763 12909651 10.1099/jmm.0.05077-0
| 15496232 | PMC529303 | CC BY | 2021-01-04 16:38:17 | no | Ann Clin Microbiol Antimicrob. 2004 Oct 20; 3:21 | utf-8 | Ann Clin Microbiol Antimicrob | 2,004 | 10.1186/1476-0711-3-21 | oa_comm |
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Ann Clin Microbiol AntimicrobAnnals of Clinical Microbiology and Antimicrobials1476-0711BioMed Central London 1476-0711-3-221550068810.1186/1476-0711-3-22ResearchKilling Bugs at the Bedside: A prospective hospital survey of how frequently personal digital assistants provide expert recommendations in the treatment of infectious diseases Burdette Steven D [email protected] Thomas E [email protected] W Scott [email protected] Division of Infectious Disease, Department of Medicine, Wright State University School of Medicine, Dayton, Ohio 45409, USA2 Division of General Internal Medicine, Department of Medicine, Wright State University School of Medicine, Dayton, Ohio 45409, USA2004 22 10 2004 3 22 22 2 7 2004 22 10 2004 Copyright © 2004 Burdette et al; licensee BioMed Central Ltd.2004Burdette et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Personal Digital Assistants (PDAS) are rapidly becoming popular tools in the assistance of managing hospitalized patients, but little is known about how often expert recommendations are available for the treatment of infectious diseases in hospitalized patients.
Objective
To determine how often PDAs could provide expert recommendations for the management of infectious diseases in patients admitted to a general medicine teaching service.
Design
Prospective observational cohort study
Setting
Internal medicine resident teaching service at an urban hospital in Dayton, Ohio
Patients
212 patients (out of 883 patients screened) were identified with possible infectious etiologies as the cause for admission to the hospital.
Measurements
Patients were screened prospectively from July 2002 until October 2002 for infectious conditions as the cause of their admissions. 5 PDA programs were assessed in October 2002 to see if treatment recommendations were available for managing these patients. The programs were then reassessed in January 2004 to evaluate how the latest editions of the software would perform under the same context as the previous year.
Results
PDAs provided treatment recommendations in at least one of the programs for 100% of the patients admitted over the 4 month period in the 2004 evaluation. Each of the programs reviewed improved from 2002 to 2004, with five of the six programs offering treatment recommendations for over 90% of patients in the study.
Conclusion
Current PDA software provides expert recommendations for a great majority of general internal medicine patients presenting to the hospital with infectious conditions.
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PDAs (Personal Digital Assistants) are becoming widely used in medicine. A survey done by the America College of Physician predicted that 67% of physicians would be using PDAs by the end of 2002 [1]. These devices are used not only by the new generation of residents and physicians, but by all ages and all specialties [2,3]. PDA's are used not only as personal planners and contact lists but also for medical purposes such as patient billing and bedside medical references [4,5]. Physicians who have rapid and easy access to information are increasingly using that data when treating patients. This should ensure appropriate therapy and also reduce medical errors by ensuring proper dosing and treatment choices [6].
One medical specialty which has focused on PDA's application in medicine is infectious diseases [7]. There are numerous infectious disease programs which provide both expert recommendations regarding antibiotic choice as well as background information (epidemiology, diagnostic studies, source of pathogens, etc). We sought to determine the how often expert recommendations were available from the PDA infectious disease resources for the infectious conditions seen on our general internal medicine teaching service over a 4 month period of time. A secondary goal was to determine the change in the software over a 15 month period to provide expert recommendations.
Methods
Clinical Setting
Miami Valley Hospital is an 827 bed secondary and tertiary referral center, in Dayton, Ohio. It is an urban hospital that averages over three-thousand patient admissions per month. The internal medicine resident teaching service consists of two teams, each team with 2 senior residents and 2 interns who are supervised by an attending physician. There is a senior resident and intern on call in the hospital at all times. Patients were eligible to be admitted to resident's service if they were established within the Medical-Surgical Health Center of Miami Valley Hospital, if they were uninsured or if they had insurance but were without a local physician (a.k.a. private unattached patients). The general medicine teaching service averages approximately eight admissions per twenty-four hour period.
Patients and Problems
The patients were screened prospectively over a four month period (July 2002 through October 2002). The admitting senior resident prospectively recorded the chief complaint and initial differential diagnosis for each patient. One of us (SDB), as the chief resident during these months, gathered the data during morning report. Patients were considered appropriate for the study if they had a leading diagnosis or active alternatives that suggested an infectious disease at the time of admission. Cases selected were then assigned categories based on the "major clinical syndromes" from Mandell, Douglass and Bennett's Principles and Practice of Infectious Disease (PPID) (8) (Table 1). Syndromes were categorized by organ system and were considered only if they would commonly require hospitalization (for example, sinusitis was not evaluated).
Table 1 Appropriate inpatient clinical syndromes according to PPID (8) and the distribution of patients admitted to the general medicine teaching service from July through October of 2002.
Major Clinical Syndromes Patients
Fever
Fever of Unknown Origin (Neutropenia) 6 (2)
Upper Respiratory
Pharyngitis 2
Infections of head and neck 1
Pleuropulmonary
Acute Bronchitis 16
Chronic Bronchitis 20
Acute Pneumonia 31
Empyema 2
Chronic Pneumonia 1
Cystic Fibrosis 1
Urinary Tract 22
Sepsis Syndrome 3
Peritonitis and Other Intra-abdominal Infections 3
Cardiovascular
Endocarditis 2
Central Nervous System
Acute Meningitis 6
Encephalitis 1
Brain Abscess 1
Soft Tissue
Cellulitis and subcutaneous Tissue Infections 40
Lymphadenitis and Lymphangitis 2
Gastrointestinal
Inflammatory Enteritides 20
Abdominal Symptoms and Fever 7
Bone and Joint
Infectious Arthritis 1
Osteomyelitis 8
STD
Prostatitis, Epididymitis and Orchitis 1
Eye
Peri-ocular Infections 2
HIV
Pulmonary Manifestations in HIV 5
CNS Manifestations in HIV 1
Searching the PDA
A Sony Clie T615C (Palm OS) was the device used to access the software, but all programs evaluated were also available in the Pocket PC format (thus applicable to nearly 100% of PDAs in clinical use). Five PDA programs were initially searched to determine if expert recommendations were available. Software was chosen based on its availability to both the Palm OS and the Pocket PC and at the time of the initial software evaluation were the primary infectious disease programs available (since 2002 numerous other titles have been released to address infectious diseases). The PDA programs included: Sanford's Guide to Antimicrobial Therapy (SG) [9], John's Hopkins Antibiotic Guide (JHABx) [10], 5-Minute Infectious Diseases Consult ) (5MID) [11], 5-Minute Clinical Consult ) (5MCC) [12], and Pocket Medicine-Infectious Disease ) (PMID) [13]. The initial evaluation was performed with the most current software in October 2002 and reassessed in January 2004 with the latest versions of the available software. In addition, ePocrates ID ) (QID) [14] was also evaluated in January 2004 (but was not included in the October 2002 assessment as it was only available for the Palm OS at this junction).
A minimum of three attempts were used to locate conditions within each program. Searching was done using either the disease name or clinical problem that had led to the patient's admission. Synonyms were used when appropriate to increase the possibility of locating a condition within each program (for example: "urinary tract infection" was the first term searched and if no results available, then "pyelonephritis" was searched next and if still no results then "kidney infection" was entered into the database). Programs that were organized according to organ system (JHABx, QID only) were searched within the appropriate organ system, while programs that listed diagnoses alphabetically were searched accordingly. Expert recommendations were considered to be present and thus counted as a positive if the software had treatment recommendations present, whether related to antibiotic decision or "supportive care."
In order to determine the change in software capabilities over a 14 month period, the same patient data set was used to assess software programs in January of 2004. A comparison of the programs was then performed comparing the availability of expert recommendations.
Statistical Analysis
The data was collected by a single physician (SDB) and then validated independently by a second physician (TEH). The data was analyzed using descriptive statistics with simple frequencies and the confidence intervals were calculated according to standard formulas.
Results
Over the four month period, from July through October of 2002, there were 883 patients admitted to the resident service, of whom 212 had syndromes that were suspected on admission to be infectious in etiology (202 of which could be assigned to the pre-defined categories) (See Figure 1 and Table 1).
Figure 1 Results of patient screened for infectious etiologies over a 4 month period.
As shown in Table 2, treatment recommendations were available in the PDA in at least one program for one-hundred percent of the patients admitted during the software evaluation in January 2004. Expert recommendations were available in all six of the programs for 52% of the patients admitted. The Sanford Guide and ePocrates ID each offered expert recommendations regarding treatment for 100% of the patients, while John's Hopkins Antibiotic Guide, 5 Minute Clinical Consultant and 5 Minute Infectious Diseases both offered expert recommendations in over 95% of the patients. Pocket Medicine-Infectious Disease offered expert recommendations for the fewest number of patients.
Table 2 Number and percent of patients with infection- related clinical syndromes covered by the studied PDA programs as of January 2004.
Major Clinical Syndromes Patients SG QID JHABx 5MCC 5MID PMID Total % Rec
Fever 6 6 6 6 4 4 4 100
Upper Respiratory 3 3 3 3 3 3 3 100
Pleuropulmonary 71 71 71 71 71 69 31 100
Urinary Tract 22 22 22 22 22 22 0 100
Sepsis 3 3 3 3 3 3 0 100
Peritonitis 3 3 3 3 3 3 2 100
Cardiovascular 2 2 2 2 2 2 2 100
Central Nervous System 8 8 8 8 8 8 7 100
Soft Tissue 42 42 42 42 42 42 40 100
Gastrointestinal 27 27 27 27 27 27 7 100
Bone and Joint 9 9 9 9 9 9 8 100
STD 1 1 1 0 0 1 0 100
Eye 2 2 2 2 2 0 2 100
HIV 6 6 6 6 0 0 0 100
TOTALS 202 202 202 201 195 192 106 202
TOTAL % 100 100 99 97 95 52 100
95% Confidence Intervals 100% 100% 97.5% to 100% 94.5% to 99.5% 92% to 98% 45% to 59%
In regards to software changes between October 2002 and January 2004, each of the programs evaluated increased the number of patients for whom expert recommendations were available Table 3. The recommendations were not evaluated for changes in treatment over this time period, only the number of patients for whom recommendations were available was evaluated. The Sanford Guide had minimal improvements to make (initially offering expert recommendations for 96% of patients) while others improved significantly (JHABx improved from approximately 50% to over 96%). PMID, while offering treatment recommendations for the fewest number of patients, did show an improvement of 28% of the 14 month time period assessed. QID was assessed for the first time in 2004 when it was made available for both the Palm OS and Pocket PC and therefore no comparison data is available.
Table 3 Improvement in expert recommendations available in October 2002 as compared with January 2004.
Programs 2002 2004 Difference
SG 94% 100% +6%
QID NA 100% NA
JHABx 61% 99% +38%
5MCC 64% 97% +33%
5MID 58% 95% +37%
PMID 24% 52% +28%
Discussion
We were able to find expert recommendations regarding initial treatment of suspected infectious diseases on the PDA for 100% of patients admitted to the general medicine teaching service at Miami Valley Hospital over a 4 month period from July until October of 2002. As far as we know, this is the first ever prospective study of the breadth of coverage provided by PDA software for recommendations in infectious disease. Miller et al provide an excellent review and buyer's guide of many of the PDA software evaluated in this project and did sample the recommendations available for six selected infectious conditions [7].
This study should be interpreted in light of its potential limitations. First, we may have erred in our selection of which patients had infectious syndromes as the cause for admission. Patients' initial diagnoses might have been mistaken and the cause for admission was non-infectious. We have no information about how often such errors occurred. However, we attempted to assess the frequency of recommendations available for the initial diagnosis by the admitting physician rather than the discharge diagnosis.
A second potential limitation to our study is that searching errors occurred. Multiple steps were taken to limit the possibility that recommendations were credited that did not correctly match with the conditions sited or that recommendations were not found that did exist. Explicit criteria were used by an evaluator (SDB) familiar with both the PDA and the aforementioned software looking for management recommendations. This usually included antibiotic recommendations and occasionally recommendations regarding further evaluation. The possibility that we did not find recommendations which were indeed available seems unlikely because of the previously mentioned points as well as a systematic approach using multiple synonyms. Furthermore, the data was independently reviewed by a second physician and no inconsistencies were identified.
A third potential limitation is that of reproducibility. Since searching was done by an experienced user, searching errors would be more likely with less experienced user(s). Many of the programs require persistent use to learn the nuances and style of the programs and how to best access information. Therefore, in order for others to duplicate our results, users will need to develop some profiency with the software and be willing to perform searches using multiple synonyms.
Lastly, our study may have limits to its applicability. Our patients were admitted to an inpatient general internal medicine teaching service at an urban hospital in the United States. These data probably apply to similar inpatient services elsewhere within North America, but may not apply to either other specialty services (Pediatrics, Surgery, etc) or hospitals in other parts of the world.
While the limitations mentioned above apply to this study, they can also be expanded to include limitations in the software. No physician can access a software program for the first time and be expected to take full advantage of the recommendations available. It takes time and effort to maximize the clinical utility that is available on the PDA, which unfortunately, many clinicians do not accomplish. This paper was focused on treatment recommendations and did not evaluate the ability of the individual programs to assist with diagnosis. The diagnostic data and information other than treatment recommendations varies from program to program. This could be a topic for future research or may be available in other formats such as software reviews in PDA magazines.
This study demonstrates that these infectious disease programs have improved over time (table 3). The reasons for this are several. Many of the programs were newly released in 2002 and where advertised as a "works in progress." As time passed, they had time and resources to add more clinical data to their software (JHABx being the prime example) or they have released a more recent edition of the software (5MCC, 5MID, PMID). Secondly, the researcher (SB) had more time to become familiar with each program and therefore it is possible that part of the increased available recommendations is in part a "false positive" in that it was present in 2002 but not easily located (SG for example claims to have everything in the paperback version available in the PDA version, but accessing the data may be a challenge). This, as discussed previously, is an issue with software (often due to format or search engine) and increased experience with software definitely leads to increased clinical utility (both in regards to time and amount of information located). Table 3 also demonstrates the benefit of the "auto-update" feature that many of these programs possess. As new data is added to database, the device is able to access the central database and import these changes, thus making the software more dynamic rather than static. This allows the user the ability to use the latest recommendations, rather than material which may out of date. These features, allow with the compact size, mobility of the devices and the fact that multiple references may be available on a single device are just several examples of the benefit of using a PDA's in medicine.
Despite these potential limitations, our data suggests that PDAs provide expert recommendations for the majority of infectious clinical conditions encountered in practice by non-infectious disease general physicians. Since a majority of patients admitted to the hospital receive care from clinicians who are not specialists in infectious diseases, PDA's have the potential to have an impact on the quality of initial care by guiding not only the choice of antibiotics but also diagnostic testing and when to involve infectious disease specialists. Further research is needed to examine the efficiency of use; evidence supporting the recommendations, and the clinical impact of the use of these resources.
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Int J Health GeogrInternational Journal of Health Geographics1476-072XBioMed Central London 1476-072X-3-251550930110.1186/1476-072X-3-25ResearchDetecting spatiotemporal clusters of accidental poisoning mortality among Texas counties, U.S., 1980 – 2001 Nkhoma Ella T [email protected] Hsu Chiehwen [email protected] Victoria I [email protected] Ann Marie [email protected] Department of Epidemiology, University of North Carolina at Chapel Hill, CB#7435, 2106-B McGavran-Greenberg Hall, Chapel Hill, NC 27599–7435, USA2 Department of Public and Community Health, University of Maryland College Park, 2371 HHP Building, Valley Drive, College Park, Maryland, 20742, USA3 Department of Health Management and Policy, University of North Texas Health Science Center, 3500 Camp Bowie Blvd, Fort Worth, Texas, 76107, USA4 Department of Environmental and Occupational Health, University of North Texas Health Science Center, 3500 Camp Bowie Blvd, Fort Worth, Texas, 76107, USA2004 27 10 2004 3 25 25 21 7 2004 27 10 2004 Copyright © 2004 Nkhoma et al; licensee BioMed Central Ltd.2004Nkhoma et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Accidental poisoning is one of the leading causes of injury in the United States, second only to motor vehicle accidents. According to the Centers for Disease Control and Prevention, the rates of accidental poisoning mortality have been increasing in the past fourteen years nationally. In Texas, mortality rates from accidental poisoning have mirrored national trends, increasing linearly from 1981 to 2001. The purpose of this study was to determine if there are spatiotemporal clusters of accidental poisoning mortality among Texas counties, and if so, whether there are variations in clustering and risk according to gender and race/ethnicity. The Spatial Scan Statistic in combination with GIS software was used to identify potential clusters between 1980 and 2001 among Texas counties, and Poisson regression was used to evaluate risk differences.
Results
Several significant (p < 0.05) accidental poisoning mortality clusters were identified in different regions of Texas. The geographic and temporal persistence of clusters was found to vary by racial group, gender, and race/gender combinations, and most of the clusters persisted into the present decade. Poisson regression revealed significant differences in risk according to race and gender. The Black population was found to be at greatest risk of accidental poisoning mortality relative to other race/ethnic groups (Relative Risk (RR) = 1.25, 95% Confidence Interval (CI) = 1.24 – 1.27), and the male population was found to be at elevated risk (RR = 2.47, 95% CI = 2.45 – 2.50) when the female population was used as a reference.
Conclusion
The findings of the present study provide evidence for the existence of accidental poisoning mortality clusters in Texas, demonstrate the persistence of these clusters into the present decade, and show the spatiotemporal variations in risk and clustering of accidental poisoning deaths by gender and race/ethnicity. By quantifying disparities in accidental poisoning mortality by place, time and person, this study demonstrates the utility of the spatial scan statistic combined with GIS and regression methods in identifying priority areas for public health planning and resource allocation.
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Background
The accidental poisoning mortality rate has been increasing in the United States. In the 21-year period spanning 1981 to 2001, mortality rates due to accidental poisoning more than doubled from 2.0 per 100,000 in 1981 to 4.9 per 100,000 in 2001 [1]. The burden of accidental poisoning mortality in this period was more than four million years of potential life lost (YPLL) before age 65 [1]. Accidental poisoning mortality trends in Texas have mirrored national trends with a linear increase in rates from 1.5 per 100,000 in 1981 to 5.2 per 100,000 in 2001. During this period, 10,406 total deaths attributable to accidental poisoning have contributed to more than 250,000 YPLL before age 65 [1].
Accidental poisoning refers to the physiologic damage caused by inhalation, ingestion, or any other mode of exposure to various licit and illicit drugs, or chemicals such as those found in pesticides, household cleaning products and gases/vapors [2]. According to the US CDC classification standards, this category excludes poisonings that are associated with suicidal or homicidal intent. Several studies have examined geographic and temporal trends in accidental poisoning morbidity and mortality. Patterns of accidental poisoning have been shown to vary by gender, ethnicity, socioeconomic status, and region thus exhibiting non-random spatial or temporal distributions [3-5]. The study conducted by Hanson and Wieczorek [6] used the spatial scan statistic to identify spatial clusters of alcohol-related mortality. Altmayer et al. [7] reported significant disparities in premature mortality due to poisoning among certain geographic areas. An early study conducted in Ontario, Canada found spatial and temporal variation in accidental poisoning mortality rates, which also reported higher poisoning mortality in males than in females [8]. Kaufmann, Staes & Matte in their study on lead poisoning mortality reported elevated rates in less populated areas and different time trends in mortality by race and gender [9].
Many of the studies regarding poisoning mortality have examined exclusively excess mortality due to long-term exposure to specific agents and have not explicitly studied the possible persistence of mortality due to accidental poisoning across space and time. They often examined either spatial or temporal attributes of the burden of the disease. A statistic for examining spatial and temporal data concurrently is available through the use of SaTScan software. The objectives of this study are to detect the existence of spatial and temporal clusters of accidental poisoning mortality in Texas and to determine if there are variations with respect to race and gender among these clusters.
Results
From 1980 to 2001, there were 10,774 deaths attributable to accidental poisoning, resulting in an average annual age-adjusted mortality rate of 2.8 deaths per 100 000. From 1981 to 2001, a linear increase was observed in unintentional poisoning mortality rates among Texas counties (annual percent increase = 1.06, 95% CI: 1.05 – 1.06, p < 0.0001, see Figure 1). Age-adjusted mortality rates were observed to be higher in men than in women, and to be highest among the Black population (see Table 1).
Figure 1 Accidental poisoning mortality trends in Texas, 1981 – 2001.
Table 1 Characteristics of study population, Texas, 1980 – 2001
Population Average Total Population (%) Cumulative Deaths (%) Annual age-adjusted rate (per 100,000)
All 17,577,292 (100) 10,774 (100) 2.8
Male 8,677,605 (49.37) 7,593 (70.48) 4.0
Female 8,899,687 (50.63) 3,181 (29.60) 1.6
White 10,553,321 (60.04) 6,913 (64.16) 3.0
Male 5,187,210 (29.51) 4,661(43.26) 4.1
Female 5,366,111 (30.53) 2,252 (20.90) 1.9
Black 2,052,901 (11.68) 1,584 (14.70) 3.5
Male 991,066 (5.638) 1,063 (9.866) 4.9
Female 1,061,835 (6.041) 521 (4.836) 2.2
Hispanic 4,582,804 (26.07) 2,210 (20.51) 2.2
Male 2,305,930 (13.12) 1,820 (16.89) 3.6
Female 2,276,873 (12.95) 390 (3.620) 0.8
Other 388,266 (2.21) 67(0.62) 0.8
Male 193397 (1.10) 49 (0.45) 1.2
Female 194869 (1.11) 18 (0.17) 0.4
Poisson regression results
After adjusting for age, regression analysis revealed significant associations between gender and race/ethnicity with accidental poisoning mortality. Males were found to be at higher risk of death from accidental poisoning than females (see Table 2). Among the race/ethnicity categories, the highest effect estimate (rate ratio) was for the Black population. The White population exhibited the second highest risk estimate, and the populations in the "other" category exhibited the lowest risk of accidental poisoning mortality (see Table 2).
Table 2 Rate ratios (RR) for accidental poisoning mortality using Poisson regression adjusted for age, gender, and race/ethnicity.
Factor Rate Ratio 95% Confidence Interval p-value
Gender
Male 2.47 2.45 – 2.50 <0.0001
Female 1.00 (reference) --- ---
Race/Ethnicity
White 1.00 (reference) --- ---
Black 1.25 1.24 – 1.27 <0.0001
Hispanic 0.79 0.78 – 0.80 <0.0001
Other 0.24 0.22 – 0.25 <0.0001
Space-Time scan results
For the total population adjusting for age, gender and race/ethnicity, spatiotemporal scan analysis for accidental poisoning deaths among Texas counties revealed four significant clusters of high mortality rates (see Table 3). The most likely cluster was a "hotspot" cluster (i.e. relative risk > 2.0) [10] consisting of 16 counties located in the Gulf Coast and Southeast regions of Texas (see Figure 2). Another hotspot cluster was detected in the eastern portion of Texas and consisted of five counties. The primary cluster (i.e. most likely cluster) persisted for a shorter time period (four years) than the other clusters (see Table 3).
Figure 2 Statistically significant accidental poisoning mortality clusters among Texas counties, 1980 – 2001. See Table 3 for descriptive and effect information for each statistically significant cluster.
Table 3 Statistically significant spatiotemporal clusters of accidental poisoning mortality in Texas by gender and race/ethnicity
Cluster Time Period No. of Observed Cases Annual age-adjusted rate (per 100,000) Relative Risk p-value
All populations
Primary cluster 1998–2001 1095 5.7 2.056 0.001
Secondary cluster 1 1985–2001 637 6.8 2.448 0.001
Secondary cluster 2 1995–2001 1419 4.5 1.598 0.001
Secondary cluster 3 1995–2001 886 4.6 1.661 0.001
All Males
Primary cluster 1985–2001 575 10.9 2.744 0.001
Secondary cluster 1 1995–2001 960 6.8 1.711 0.001
Secondary cluster 2 1998–2001 685 7.4 1.863 0.001
Secondary cluster 3 1994–2001 704 6.4 1.620 0.001
All Females
Primary cluster 1997–2001 456 3.9 2.371 0.001
Secondary cluster 1 1996–2001 387 2.8 1.751 0.001
Secondary cluster 2 1997–2001 207 2.9 1.786 0.001
White Population
Primary cluster 1998–2001 785 7.4 2.480 0.001
Secondary cluster 1 1996–2001 886 5.2 1.743 0.001
Secondary cluster 2 1995–2001 598 4.8 1.601 0.001
White Males
Primary cluster 1998–2001 482 9.3 2.273 0.001
Secondary cluster 1 1995–2001 697 7.2 1.768 0.001
Secondary cluster 2 1994–2001 621 6.2 1.509 0.001
White Females
Primary cluster 1998–2001 300 5.6 2.940 0.001
Black Population
Primary cluster 1993–2001 345 6.3 1.800 0.001
Black Males
Primary cluster 1993–2001 236 9.1 1.867 0.001
Black Females
Primary cluster 1996–1998 2 2338.7 1048.700 0.033
Hispanic Population
Primary cluster 1985–2001 467 6.0 2.730 0.001
Secondary cluster 1 1994–2001 342 3.8 1.730 0.001
Hispanic Males
Primary cluster 1985–2001 410 11.2 3.130 0.001
Secondary cluster 1 1995–2001 239 6.0 1.663 0.001
Hispanic Females
Primary cluster 1994–2001 84 1.8 2.264 0.001
The spatiotemporal scan statistic also revealed four significant clusters for the male population (see Table 3). The primary cluster for the male population was a hotspot cluster consisting of 10 counties located in East Texas. The second most likely cluster consisted of nine counties in North Central Texas (see Figure 2). For the male population, the primary cluster persists from 1985 into the present decade. The primary cluster for the female population closely mirrors that of the total population in location and size and persists from 1997 into the present decade. The second most likely cluster for the female population encompasses a large area of frontier (i.e. sparsely populated) counties in the eastern half of Texas (see Figure 2).
There were no real hotspot clusters observed for the Black population (see Table 3). An extreme effect estimate or fluctuation was observed for the Black female population due to the small number of observed deaths in the numerator. The number of cases was two and the period of persistence extended over two years. The significant cluster for the Black population was observed in three highly populated counties in North Texas (see Figure 2). This cluster persists for eight years and into the present decade.
A hotspot cluster was observed for the total Hispanic population consisting of ten counties in eastern Texas and it persisted for 16 years and entered into the present decade (see Figure 2 and Table 3). Five of the counties in the total Hispanic population cluster included a hotspot cluster for Hispanic males persisting for the same period of time. For Hispanic females, a hotspot cluster was observed in Central Texas consisting of 45 counties. This cluster persisted over a seven-year period beginning in 1994 and entering into the present decade (see Figure 2).
The non-Hispanic White population exhibited a hotspot cluster in the Gulf Coast region of Texas, consisting of seventeen counties. The cluster persisted for three years and into the present decade (see Table 3). The same spatiotemporal cluster was observed for the non-Hispanic White male population, albeit with a slightly different effect estimate. The most likely cluster for the non-Hispanic White female population was also a hotspot cluster. This cluster is located in the same region as the total non-Hispanic White population and persists for the same time period but only includes fifteen counties (see Figure 2).
No significant clusters were detected for populations in the "Other" category. There were very few accidental poisoning deaths for this population and the population count in many of the counties was zero for this subgroup. This resulted in instability in rates and other estimates for this population group (see Table 1).
Spatial only and spatiotemporal scan adjusting for time non-parametrically results
To verify whether the detected clusters using "spatial-temporal" methods are truly geographic clusters or space-time clusters not explained by the time trend, we further conducted two analyses using 1) the purely spatial scan statistic and 2) the space-time scan statistic non-parametrically adjusting for time. The results of these additional analyses indicated that most clusters detected using the default method were in mostly identical regions detected in the earlier analysis, only with slightly lower relative risks (i.e., observed deaths/expected deaths). For instance for the "all population group", the detected primary cluster in West Texas with a relative risk of 2.4 remained a cluster, but reduced to relative risks of 2.2 and 2.1 using additional methods that adjusted for time trend. In addition, most of the detected clusters still persisted to the present decade after the adjustments of time trend. Similar results were observed for most clusters detected in 'Male", "Female", "Black", "Non-Hispanic Whites" and "Hispanic" subpopulations using additional analyses options (see Tables 4 and 5). The summary maps of these additional analyses are presented in Figures 3 and 4.
Table 4 Statistically significant spatial clusters of accidental poisoning mortality in Texas by gender and race/ethnicity
Cluster No. of Observed Cases Annual age-adjusted rate (per 100,000) Relative Risk p-value
All populations
Primary cluster 731 6 2.152 0.001
Secondary cluster 1 2408 3.6 1.285 0.001
Secondary cluster 2 520 3.7 1.326 0.001
Secondary cluster 3 148 4.1 1.468 0.011
Secondary Cluster 4 52 5 1.813 0.049
All Males
Primary cluster 624 9.7 2.433 0.001
Secondary cluster 1 1685 5 1.269 0.001
Secondary cluster 2 404 5.7 1.434 0.001
All Females
Primary cluster 962 2.1 1.278 0.001
White Population
Primary cluster 1725 4 1.351 0.001
Secondary cluster 1 290 4.8 1.622 0.001
Secondary cluster 2 114 4.6 1.539 0.015
White Males
Primary cluster 1030 5.7 1.399 0.001
Secondary cluster 1 217 7.3 1.782 0.001
Secondary cluster 2 269 5.8 1.414 0.001
White Females
Primary cluster 704 2.6 1.337 0.001
Black Population
Primary cluster 482 4.3 1.224 0.001
Black Males
Primary cluster 108 7.2 1.476 0.039
Black Females
Primary cluster 2 373.8 167.634 0.022
Hispanic Population
Primary cluster 500 5.3 2.459 0.001
Secondary cluster 1 120 3.9 1.788 0.001
Hispanic Males
Primary cluster 438 10 2.822 0.001
Secondary cluster 1 95 6 1.696 0.001
Hispanic Females
Primary cluster 128 1.2 1.524 0.001
Table 5 Statistically significant spatiotemporal clusters of accidental poisoning mortality in Texas by gender and race/ethnicity, adjusting for time nonparametrically
Cluster Time Period No. of Observed Cases Annual age-adjusted rate (per 100,000) Relative Risk p-value
All populations
Primary cluster 1984–2001 654 6.3 2.261 0.001
Secondary cluster 1 1983–2001 2257 3.6 1.306 0.001
Secondary cluster 2 1990–1999 331 4.4 1.590 0.001
Secondary cluster 3 1993–1999 1088 3.5 1.264 0.001
Secondary cluster 4 1992–1994 38 8.9 3.207 0.002
All Males
Primary cluster 1984–2001 588 10.0 2.521 0.001
Secondary cluster 1 1982–1993 797 5.9 1.475 0.001
Secondary cluster 2 1990–2001 337 6.4 1.605 0.001
Secondary cluster 3 1993–2001 1084 5.0 1.259 0.001
Secondary cluster 4 1992–1994 27 13.0 3.284 0.027
All Females
Primary cluster 1983–2001 916 2.1 1.320 0.001
White Population
Primary cluster 1983–2001 1390 4.3 1.439 0.001
Secondary cluster 1 1980–1997 220 5.5 1.832 0.001
Secondary cluster 2 1992–1994 32 11.0 3.679 0.001
Secondary cluster 3 1991–1999 207 4.6 1.536 0.003
Secondary cluster 4 1993–2001 544 3.7 1.246 0.019
White Males
Primary cluster 1983–2001 950 5.9 1.439 0.001
Secondary cluster 1 1980–1998 179 8.1 1.987 0.001
Secondary cluster 2 1993–2001 416 5.8 1.421 0.001
Secondary cluster 3 1990–1999 171 6.8 1.653 0.001
Secondary cluster 4 1992–1994 24 16.5 1.037 0.004
White Females
Primary cluster 1983–2001 638 2.7 1.409 0.001
Black Population
Primary cluster 1993–2001 122 6.2 1.758 0.002
Black Males
Primary cluster 1993–2001 236 7.2 1.477 0.001
Black Females
Primary cluster 1996–1998 2 2031.6 911.05 0.026
Hispanic Population
Primary cluster 1984–2001 477 5.6 2.592 0.001
Secondary cluster 1 1993–2001 368 3.2 1.483 0.001
Hispanic Males
Primary cluster 1983–2001 424 10.4 2.923 0.001
Secondary cluster 1 1989–2001 88 7.1 2.005 0.001
Hispanic Females
Primary cluster 1994–2001 84 1.5 1.920 0.002
Figure 3 Spatial accidental poisoning mortality clusters among Texas counties, 1980 – 2001. Figure includes statistically significant and non statistically significant clusters to facilitate comparison with Figure 1. See Table 4 for descriptive and effect information for each statistically significant cluster.
Figure 4 Accidental poisoning mortality clusters among Texas counties adjusting for time nonparametrically, 1980 – 2001. Figure includes statistically significant and nonstatistically significant clusters to facilitate comparison with Figure 1. See Table 5 for descriptive and effect information for each statistically significant cluster.
Discussion
The results from this study support the existence of spatiotemporal clusters of accidental poisoning mortality among Texas counties and show variations in these clusters by gender and race. This study also identifies several hotspot clusters. Moreover, the additional analyses (spatial only and adjusting for time non-parametrically) identify most clusters in almost identical regions but with slightly lower relative risks. This observation, along with the fact that both the results of the space-time scans with and without adjusting for time non-parametrically both demonstrate the temporal persistence of accidental poisoning mortality clusters into the present decade (at least 60% of the clusters persisted to the present decade using time-adjusting spatial scan), has provided supporting evidence that the clusters detected using the spatial-temporal method are geographic in nature, rather than an artefact of temporal trend.
The burden of excess accidental poisoning mortality was found to be highest in the Black population, followed by the non-Hispanic White population, and the least among the Hispanic population and populations of other race/ethnicity categories. Consistent with the literature, the male population also exhibited an elevated risk of accidental poisoning mortality when compared to the female population.
The results of this research presented both agreement and disagreement when compared other the results in which conventional techniques were used to identify geographic disparities by race/ethnicity, and gender. For instance, the results of this study were consistent with the literature in that the Black population was found to be at greatest risk of death from accidental poisoning [9,11]. In addition, the increased risk of accidental poisoning mortality observed in males when compared to females is consistent with what has been reported in other studies conducted in the United States [5,8,9,12]. On the other hand, the finding that the Hispanic population exhibited more than 20% less risk than the White population is unexpected. Other studies have found the Hispanic population to be either at increased risk or equal risk to the White population [5,11,13].
Although there are few studies in the literature that explicitly record cluster detection for accidental poisoning mortality, there are a variety of studies that have examined trends of accidental poisoning mortality in space and time [3-5,11,14]. Many of these studies have been conducted for specific toxic agents, e.g., alcohol, lead and pesticides but have not recorded the existence of spatial and temporal clustering of poisoning events and poisoning deaths.
There is also evidence in the literature to support the existence of temporal clustering of accidental poisoning mortality. In a study on organophosphate poisoning, Sahin, Sahin and Arabaci reported that deaths from accidental poisoning due to organophosphates were most frequent during certain months [4]. In a review of childhood poisoning, McGuigan reported temporal variations in symptomatic poisoning events over many years [14]. Sudakin, Horowitz, and Griffin used the space-time scan statistic to identify spatial and temporal clustering of accidental poisoning events due to pesticide exposures [15]. Therefore, the temporal clustering observed in the present study may be reflective of different types of poisoning agent exposures for each cluster.
The results of this study should be interpreted with several considerations. One, the process of cluster detection is necessarily ecological. The objective was to determine if there was an association between certain areas of Texas with excess accidental poisoning mortality. These results cannot be extrapolated to the level of the individual, i.e., one cannot interpret a relative risk above one as increased risk of accidental poisoning mortality for residents living in a given county. However, the risk estimate does provide valuable information about geographic disparity of accidental poisoning mortality.
Another consideration concerns the utilization of county-level data. The scan statistic may not have been sensitive to small areas of excess mortality that may have been detected at a higher geographic resolution. However, in Texas the county is the smallest geographic area for which there is routinely reported or available deaths and population estimate information. Also, the county is the political level at which health policy actions are instituted, since many of the local public health departments in Texas are county health departments.
A third consideration is that this study relied on county-level mortality data; and there may be variations in coding or reporting practices from county to county. Furthermore, in 1998 the International Classification of Diseases underwent its 10th revision, and there may be differences in mortality estimates based on the codes in the 9th and 10th revisions. This introduces the possibility of misclassification bias in the results. However, there is no evidence to indicate that the misclassification would be differential [2]. Hence, any misclassification would bias the effect estimates towards the null, thus underestimating the actual effect. Thus, the results of this study still provide useful information concerning health disparities at the county, regional and state level.
Conclusions
This study has demonstrated the existence of spatiotemporal clusters of accidental poisoning mortality in Texas. Additionally, this study has also shown variations in risk and clustering by gender and race/ethnicity. The results of this study have numerous implications. By quantifying accidental poisoning mortality disparities by geographic area, race/ethnicity, and gender, the results provide an evidence-base for health planning. The clusters identified in this study, specifically those that persist into the present decade, represent areas where health services, e.g., poison control centers and emergency rooms, may be deficient. Knowing the specific areas of excess mortality from accidental poisoning would help health policymakers to focus the scope of prevention programs and health care delivery, thus providing for efficient allocation of public health resources.
The findings from this study illustrate the use of the scan statistic and GIS in public health surveillance. Longitudinal data for accidental poisoning mortality was analyzed to identify hotspot clusters not only for the total population but also for subpopulations. Identifying geographic areas and specific populations, which are at an elevated risk of accidental poisoning mortality helps in the process of hypothesis generation for possible etiological mechanisms. For example, the most likely cluster for the total Texas population is in a heavily industrial area. The population in this area may have experienced excess mortality from other occupational exposure-related health outcomes (e.g. different types of cancers). One may further examine into the excess accidental poisoning mortality observed in this area in order to determine if it was due to occupational exposures.
The clusters identified in this study warrant further attention. Additional studies should be conducted to determine the causes behind the observed clustering, especially in the Gulf Coast region and the Central North Texas region where clustering was observed for the total Texas population and the two populations at greatest risk of accidental poisoning mortality, the Black population and the White population. More research is needed to assess different types of toxic exposures and their contribution to accidental poisoning mortality. Furthermore, the availability, locality and accessibility of health services such as poison control centers and health care facilities (hospitals and clinics) should be studied, giving special attention to those in non-urban counties.
Methods
Data sources and data processing
Three types of files were downloaded for the analysis. First, death files including the unintentional poisoning mortality data was obtained from Expert Health Data Programming Incorporated's Vitalweb (URL ). The data was extracted as counts by race, gender and age-group for ICD-9 codes 850 – 858 (unintentional poisoning by drugs) and 860–869 (accidental poisoning by solid, liquid, gas) for the years 1980 – 1998 and ICD-10 codes X40 – X49 (accidental poisoning by and exposure to noxious substances). The ICD-10 classification codes include exposure to drugs, gases and vapors, and other unspecified chemicals. Second, population (and estimates) files by year were obtained from the Texas State Data Center. Both deaths and population data included stratified information by age groups (16, representing the ages of 0–4 to 75 and above), gender (2, by male and female) and race (4, representing Blacks, Hispanics, Non-Hispanic Whites and Others). Third, county centroids information of latitude and longitude was downloaded from the 2000 U.S. Gazetteer portion of the US Census Bureau (Available online at ).
We performed the data processing by using an automated Microsoft Visual Basic Application (VBA) program to organize the downloaded county information of death counts, population at risk and geographic files (i.e. county centroids), before exporting them to SaTScan for analysis and for GIS mapping. The VBA program first imported, aligned, and then exported data tables to a SatScan compatible format. The program then invoked the SaTScan batch executable file (SaTScan version 4.0, freeware available from: URL: ) to perform scan analysis, and extracted cluster information from the SaTScan output file. Following this, the program linked the cluster dbase files with the county name file, and created a new file for mapping purposes. Using ArcGIS software (ESRI, ), we spatially joined the dbase file to a Texas county layer and produced maps displaying the poison mortality clusters of all populations and those in the populations stratified by gender, race/ethnicity, and gender-race/ethnicity combinations.
Poisson regression
The association between gender, race/ethnicity, or the combination of both and accidental poisoning mortality was explored using Poisson regression. Poisson regression was conducted on accidental poisoning deaths using age, gender, year and race as predictor variables and the natural logarithm of population size as the offset variable. Regression analysis was conducted using the GENMOD procedure from the SAS system for Windows version 8.2 (Cary, North Carolina: SAS Institute, Inc.1999–2001). To control for overdispersion, the Poisson model was used with the scaled deviance option. Estimates of the annual percent increase, and rate ratios for race and gender were calculated from the results of the analysis.
Spatial scan statistic
To detect potential spatiotemporal variations of poisoning mortality among Texas counties, this study utilized the Spatial Scan Statistic developed by Kulldorff [16,17] to detect clusters of unintentional poisoning mortality among Texas counties. This statistic has been used previously to detect clusters of breast cancer [18], and those specifically among Texas counties [19], prostate cancer [20], alcohol-related mortality [6], and diabetes prevalence [21] among others.
Many studies conducted on rare events such as unintentional poisoning deaths have aggregated the events to a higher geographic resolution, and have thus been unable to detect the existence of true clusters. The spatial scan statistic, however, is ideal for cluster detection of rare events since it includes the option of examining Poisson distributed data.
The space-time parameter of the spatial scan statistic places a cylindrical window on the coordinates grid for the locations studied and moves the center of the cylinder base over the grid so that the sets of geographic units covered by the window are constantly changing. The statistic assumes no predetermined cluster size and utilizes scan windows of various sizes (of the population at risk) with a maximum window size specified by the user. In the space-time scan, this spatial scan window serves as the base of a cylinder, with time acting as the height of the cylinder. The cylinder is continuously expanded and extended up to the maximum specifications set by the user. Whenever the cylindrical window finds a new death, SatScan calculates a likelihood function to test for elevated risk within the cylinder as compared with outside the cylinder. The likelihood function for any given cylindrical window (under the Poisson assumption) is proportional to:
(d/n)d([D - d] / [D - n])(D - d)I( ) (1)
where D is the total number of unintentional poisoning mortality deaths, d is the number of deaths within the space-time cylindrical window, and n is the expected number of cases after adjusting for any specified covariates. When SatScan is scanning for high rates, the indicator function I( ) is equal to 1 when the window has more cases than expected, and 0 if the observed cases are equal to or less than expected.
The present study utilized the retrospective space-time analysis for high rates using a Poisson model to calculate expected deaths due to unintentional poisoning in each county. The spatial scan window setting was set to a maximum cluster size of 25% of the study population, and the temporal scan window was set to a maximum cluster size of 90% of the study period. The covariates specified for this study were age group, gender, and race depending on the population being studied [16].
The "clusters" detected using the space-time scan statistic could be purely spatial, purely temporal or truly space-time clusters. To test whether these clusters are truly geographical in nature or are confounded by the temporal trend, i.e., whether there are any statistically significant geographical clusters or space-time clusters not explained by the time trend, we performed further analyses using (1) the purely spatial scan statistic and (2) the space-time scan statistic (non-parametrically) adjusting for time.
Authors' contributions
ETN was responsible for the design and implementation of the project and the final paper. ETN also contributed to data analysis. CEH contributed to database design, visual basic application programming and data analysis. CEH also assisted in the preparation and editing of the final paper. VIH contributed to the preparation and editing of the final paper, including reviewing the relevant literature. AMH was responsible for the layout and design of the illustrative maps and also assisted in preparing and editing the final paper. All authors read and approved the final manuscript.
Acknowledgements
The researchers extend their gratitude to the support provided by the Texas Department of Health through the project entitled: Survey of Physicians, Other Health Professionals and Providers for Emergency Response Purposes. Award No. 76376376302004-4 in the completion of this study.
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| 15509301 | PMC529305 | CC BY | 2021-01-04 16:39:01 | no | Int J Health Geogr. 2004 Oct 27; 3:25 | utf-8 | Int J Health Geogr | 2,004 | 10.1186/1476-072X-3-25 | oa_comm |
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Cardiovasc UltrasoundCardiovascular Ultrasound1476-7120BioMed Central London 1476-7120-2-221552211910.1186/1476-7120-2-22ResearchInfluence of oxygen tension on myocardial performance. Evaluation by tissue Doppler imaging Frøbert Ole [email protected] Jacob [email protected] Egon [email protected] Steen Hvitfeldt [email protected]øgaard Peter [email protected] Department of Cardiology, Center for Cardiovascular Research, Aalborg Hospital, Aarhus University Hospital, Denmark2 Institute of Pharmacology, University of Aarhus, Denmark3 Center for Model-based Medical Decision Support Systems, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark4 Skejby University Hospital, Aarhus, Denmark2004 2 11 2004 2 22 22 26 8 2004 2 11 2004 Copyright © 2004 Frøbert et al; licensee BioMed Central Ltd.2004Frøbert et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Low O2 tension dilates coronary arteries and high O2 tension is a coronary vasoconstrictor but reports on O2-dependent effects on ventricular performance diverge. Yet oxygen supplementation remains first line treatment in cardiovascular disease. We hypothesized that hypoxia improves and hyperoxia worsens myocardial performance.
Methods
Seven male volunteers (mean age 38 ± 3 years) were examined with echocardiography at respiratory equilibrium during: 1) normoxia (≈21% O2, 79% N2), 2) while inhaling a hypoxic gas mixture (≈11% O2, 89% N2), and 3) while inhaling 100% O2. Tissue Doppler recordings were acquired in the apical 4-chamber, 2-chamber, and long-axis views. Strain rate and tissue tracking displacement analyses were carried out in each segment of the 16-segment left ventricular model and in the basal, middle and apical portions of the right ventricle.
Results
Heart rate increased with hypoxia (68 ± 4 bpm at normoxia vs. 79 ± 5 bpm, P < 0.001) and decreased with hyperoxia (59 ± 5 bpm, P < 0.001 vs. normoxia). Hypoxia increased strain rate in four left ventricular segments and global systolic contraction amplitude was increased (normoxia: 9.76 ± 0.41 vs hypoxia: 10.87 ± 0.42, P < 0.001). Tissue tracking displacement was reduced in the right ventricular segments and tricuspid regurgitation increased with hypoxia (7.5 ± 1.9 mmHg vs. 33.5 ± 1.8 mmHg, P < 0.001). The TEI index and E/E' did not change with hypoxia. Hyperoxia reduced strain rate in 10 left ventricular segments, global systolic contraction amplitude was decreased (8.83 ± 0.38, P < 0.001 vs. normoxia) while right ventricular function was unchanged. The spectral and tissue Doppler TEI indexes were significantly increased but E/E' did not change with hyperoxia.
Conclusion
Hypoxia improves and hyperoxia worsens systolic myocardial performance in healthy male volunteers. Tissue Doppler measures of diastolic function are unaffected by hypoxia/hyperoxia which support that the changes in myocardial performance are secondary to changes in vascular tone. It remains to be settled whether oxygen therapy to patients with heart disease is a consistent rational treatment.
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Introduction
Low oxygen tension dilates coronary arteries and high oxygen tension is a coronary vasoconstrictor. Yet oxygen supplementation remains first line treatment in cardiovascular disease states such as myocardial infarction and pulmonary oedema.
Endothelium-dependent vasodilation is reduced in patients with ischemic heart disease [1] and such patients are believed to have a reduced ability to counteract the circulatory consequences of systemic (air line travel, high altitude stay) and regional (coronary artery stenosis) hypoxia. Spontaneous nocturnal hypoxia with desaturation for hours is a frequent phenomenon in patients with severe coronary artery disease [2]. Left atrial, left ventricular (LV), and right ventricular (RV) end-systolic diameter fall during simulated extreme altitude [3] and during moderate altitude exposure [4]. There is controversy concerning myocardial performance during hypoxia; improvement [4], no change [3,5,6]. and worsening [7] of left ventricular systolic function, have been described. One study looked at diastolic function expressed as E/A ratio and found a reduction with hypoxia [3].
Hyperoxia, a condition in which the total oxygen content of the body is increased above that normally existing at sea level, is associated with impairment of cardiac relaxation and increased left ventricular filling pressures in patients with and without congestive heart failure [8].
Tissue Doppler imaging (TDI) objectively derives measurements of contraction and relaxation velocities directly from the myocardium and thus yields information not previously accessible by echocardiography [9]. We used TDI to study myocardial performance in healthy volunteers and we hypothesized that hypoxia increases and hyperoxia reduces myocardial performance.
Material and Methods
Subjects
Seven healthy men, (age 25–46 (mean 38) years) completed the study. All subjects were normotensive, non-smokers, on no medication, had a normal left ventricular function by 2-D echocardiography, and had no family history of ischemic heart disease. The local ethical committee approved the study.
Ventilation system
We used a system consisting of a ventilator, a gas analyser with pulse oximeter, and a computer. Computer programs control the experimental procedure and continuously collect data from the ventilator and gas analyser [10].
Echocardiography
Echocardiography was performed from the apical acoustic window. Tissue Doppler recordings were acquired as digital loops in the apical 4-chamber, 2-chamber, and long-axis views [9]. To avoid aliasing, the settings of the ultrasound equipment and colour-coded area were adjusted to obtain the highest possible frame rate. We measured peak strain in the 16-segment left ventricular model and in 3 segments in the right. From the color-coded tissue tracking image, the motion amplitude toward the apex in systole was recorded in each segment. The global systolic contraction amplitude (GSCA) was calculated as the average shortening amplitude of all 16 segments. The peak E velocity was obtained by pulsed Doppler measurements of the mitral inflow at the tip of the mitral leaflets. We further assessed isovolumetric acceleration, TEI index (spectral and TDI), tricuspid regurgitation and pulmonary ejection time. The tissue E' velocity was obtained by Tissue Doppler at the lateral mitral annulus. E/E' has been proposed as a tool for assessing LV filling pressures that combines the influence of transmitral driving pressure and myocardial relaxation [11]. The TEI index (spectral or TDI), a combined measure of systolic and diastolic function, was assessed by Doppler time intervals from the mitral inflow and LV outflow tract or the time intervals were obtained by TDI at the lateral mitral annulus. The TEI index was calculated by a-b/b where the time interval "a" was measured from cessation to onset of mitral inflow and the time interval "b" was the duration of the LV outflow velocity profile. To minimize the variability of the measurements, all ECHO recordings were performed and analyzed in a blinded fashion by the same author (P.S.).
Study protocol
TDI was performed when the subjects had rested for 15 minutes breathing room air (SpO2 97.9 ± 0.1 %), after 5 minutes of respiratory equilibrium during hypoxia (SpO2 77.6 ± 1.2 %), and after 5 minutes of respiratory equilibrium during hyperoxia (SpO2 99.0 ± 0.2 %). Heart rate was continuously recorded on computer by means of the pulse oximeter. Blood pressure was measured once during each of the three respiratory steady state situations by an automatic blood pressure measuring device based on the oscillometric method.
Statistical analysis
All data are presented as mean ± SEM. Comparisons of the responses to changes in FiO2 (normoxia, hypoxia and hyperoxia) were made with a one-way repeated-measures analysis of variance. The Student-Newman-Keuls test was used post hoc to identify pairwise differences. Differences were considered statistically significant when P < 0.05.
Results
Respiratory parameters and hemodynamics
FiO2 and FeO2 decreased with hypoxia and increased with 100% oxygen breathing (table 1). FeCO2 and tidal volume were unchanged in all three test situations, reflecting that the subjects were in respiratory steady state. There was a small, but statistically significant decrease in respiratory rate from normoxia to hypoxia, which might reflect that the subjects were more accustomed to the test situation at this stage.
There was a significant increase in heart rate with hypoxia and a decrease with hyperoxia (figure 1). Neither systolic (126 ± 6, 123 ± 8, 122 ± 12 mmHg, respectively, P = ns) nor diastolic blood pressure (80 ± 5, 78 ± 8, 81 ± 6 mmHg, respectively, P = ns) changed significantly with test situation.
Figure 1 Bar graph depicting heart rate during normoxia, hypoxia and hyperoxia. ** P < 0.01 vs. normoxia.
TDI and spectral echocardiography, hypoxia
A significant increase in strain rate was found in 4 segments (figure 2). GSCA increased with hypoxia (9.76 ± 0.41 vs. 10.87 ± 0.42, P < 0.001, figure 3). Tissue tracking displacement was reduced in all three right ventricular segments (figure 4) and systolic tricuspid regurgitation increased with hypoxia (figure 5). The TEI index (spectral or TDI) and E/E' did not change with hypoxia.
Figure 2 Comparison of peak systolic strain in the 16-segment left ventricular model illustrating the effects of hypoxia and hyperoxia. A, Anterior; B, basal; D, distal; I, inferior; L, lateral; M, mid; P, posterior; S, septal. * P < 0.05, ** P < 0.01 vs. normoxia.
Figure 3 Tissue tracking score index on the basis of the 16-segment left ventricular model illustrating the effects of hypoxia and hyperoxia.
Figure 4 Comparison of peak systolic strain in the right ventricle model illustrating the effects of hypoxia and hyperoxia. * P < 0.05.
Figure 5 Bar graph depicting systolic tricuspid regurgitation during normoxia, hypoxia and hyperoxia. ** P < 0.01 vs. normoxia.
TDI and spectral echocardiography, hyperoxia
Hyperoxia worsened left ventricular function. Strain rate was reduced in 10 segments (figure 2) with preponderance in the lateral and anterior segments. GSCA was reduced (8.83 ± 0.38, P < 0.001 vs. normoxia). Tissue tracking displacement did not change in the right ventricular segments (figure 4) and systolic tricuspid regurgitation was unchanged compared with normoxia (figure 5)). The TDI TEI index was significantly increased with hyperoxia (0.32 ± 0.04 vs. 0.45 ± 0.05, P < 0.001) and the spectral TEI index showed similar changes. E/E' did not change with hyperoxia.
Discussion
The main findings of the present study of healthy volunteers are: 1) hypoxia increased strain rate and tissue tracking displacement. 2) Hypoxia increased tricuspid regurgitation and reduced right ventricular tissue tracking displacement. 3) Hyperoxia reduced strain rate and tissue tracking displacement and increased the TEI index. The novelty of our findings, when compared to the literature, is the demonstration that longitudinal myocardial function, and thus the function of the subendocardium, is sensitive to moderate changes in inspired oxygen.
Patients with ischemic heart disease and heart failure frequently encounter hypoxia but the consequences of hypoxia on left ventricular function remain a matter of controversy, in part because of differences in methodology and the measured parameters. In early studies using roentgenkymograms [12] and dye injection [13] hypoxia was shown to increase cardiac output at rest despite reduced [13] or unchanged [12] stroke volume. The authors explained the increase in cardiac output by increased heart rate [12,13] Myocardial blood flow in the left and right ventricles increased at high altitude in dogs studied with radioactive microspheres [14] and in healthy controls using positron emission tomography [15]. In a study using M-mode echocardiography at high altitude [4] percent fractional shortening and velocity of circumferential fiber shortening remained normal while LV isovolumetric contraction time shortened. Echocardiography was also employed in a simulated ascent of Mount Everest [3] and an insignificant increase in fractional shortening and ejection fraction was found. It is generally accepted that right-sided pressures increase with hypoxia [3,14] We speculate that the improvement in tissue tracking displacement in our study reflects the systemic vasodilation which is another consequence of hypoxia [16]. On the other hand, RV-systolic function decreased which is likely to be correlated to the increased systolic pulmonary pressure reflecting the increased pulmonary vascular resistance during hypoxia.
Hemoglobin saturation in healthy persons increase very little from breathing room air to breathing 100% oxygen. Nevertheless, profound cardiovascular effects were found. This is probably because of an anticipated increase in oxygen dissolved into plasma from 0.32% to 2.09% [17]. Hyperoxia is a possible product of oxygen therapy when administered to patients with heart disease during acute illness. Hyperoxia reduces cardiac output as documented with roentgenkymograms [12], dye injection [18,19]., echocardiography [20] heart catheterisation [8] and indirectly by measurement of isometric systolic tension by means of strain gauge in dogs [21]. Reduced cardiac output with hyperoxia has even been demonstrated in patients with myocardial infarction using dye injection [22]. Some of the reduction in cardiac output may be explained by the observation that hyperoxia reduces heart rate as seen in our study and previously [19,23-25] Reduced heart rate is, however, not an entirely consistent finding [8,20,22,26] but in these four studies this could be because of high sympathetic tone (stay in a hyperbaric chamber [20], acute myocardial infarction [22], open heart surgery [26], heart catheterisation [8]). We found that heart rate increased with hypoxia and decreased with hyperoxia. This might have affected our measures of LV systolic and diastolic function but we consider this unlikely on the basis of our previous studies [11,27] demonstrating that TEI index, strain rate and the tissue tracking was unrelated to heart rate. During hypoxia as well as hyperoxia no change in LV filling pressure was noted as the E/E' ratio was unchanged. The individual parameters in the E/E' ratio are known to be influenced by heart rate but as a ratio it seems independent of heart rate and load conditions [11,27]. Therefore, we did not perform any adjustments for heart rate in the evaluation of LV diastolic function.
Because of the finding of no change [19] or a discrete rise [18,22] in blood pressure, a reduction in cardiac output results in an increased systemic vascular resistance during hyperoxia [18,19,22] Regardless of the fact that sympathetic tone may be affected by hyperoxia, even after complete sympathetic blockade myocardial contractile force remains reduced [26]. In the present study both tissue tracking displacement and strain rate worsened during hyperoxia. It seems plausible that this deterioration could be explained by systemic vasoconstriction [21] increasing afterload.
In conclusion, hypoxia improves and hyperoxia worsens systolic myocardial performance in healthy male volunteers. TDI measures of diastolic function are unaffected by hypoxia/hyperoxia which support that the changes in myocardial performance are secondary to changes in vascular tone. It remains to be settled whether oxygen therapy to patients with heart disease is a rational treatment that may sometimes be harmful or whether supplemental oxygen consistently results in an overall gain in delivered oxygen.
Acknowledgements
The study was supported by The Danish Heart Foundation (no 03-1-2-12-22050), the John and Birthe Meyer Foundation, Karen Elise Jensens Fond, the Novo Nordisk Foundation, and the Danish Medical Research Council.
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Mak S Azevedo ER Liu PP Newton GE Effect of hyperoxia on left ventricular function and filling pressures in patients with and without congestive heart failure Chest 2001 120 467 473 11502645 10.1378/chest.120.2.467
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Poulsen SH Nielsen JC Andersen HR The influence of heart rate on the Doppler-derived myocardial performance index J Am Soc Echocardiogr 2000 13 379 384 10804435 10.1067/mje.2000.104061
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| 15522119 | PMC529306 | CC BY | 2021-01-04 16:38:29 | no | Cardiovasc Ultrasound. 2004 Nov 2; 2:22 | utf-8 | Cardiovasc Ultrasound | 2,004 | 10.1186/1476-7120-2-22 | oa_comm |
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Health Qual Life OutcomesHealth and Quality of Life Outcomes1477-7525BioMed Central London 1477-7525-2-591550070010.1186/1477-7525-2-59ResearchPredicting gender differences as latent variables: summed scores, and individual item responses: a methods case study Pietrobon Ricardo [email protected] Marcus [email protected] Ulrich [email protected] Laurence D [email protected] Danny O [email protected] Timothy [email protected] Division of Orthopaedic Surgery, Center for Excellence in Surgical Outcomes, Duke University Medical Center, Box 3094, Durham, NC 27710, USA2 Department of Health and Physical Education, Center for Excellence in Surgical Outcomes, Duke University Medical Center, Durham, NC, USA3 Center for Excellence in Surgical Outcomes, University Hospital Basel, Department of General Surgery and Surgical Research, Basel, Switzerland4 Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA5 Department of Internal Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA2004 25 10 2004 2 59 59 10 5 2004 25 10 2004 Copyright © 2004 Pietrobon et al; licensee BioMed Central Ltd.2004Pietrobon et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Modeling latent variables such as physical disability is challenging since its measurement is performed through proxies. This poses significant methodological challenges. The objective of this article is to present three different methods to predict latent variables based on classical summed scores, individual item responses, and latent variable models.
Methods
This is a review of the literature and data analysis using "layers of information". Data was collected from the North Carolina Back Pain Project, using a modified version of the Roland Questionnaire.
Results
The three models are compared in relation to their goals and underlying concepts, previous clinical applications, data requirements, statistical theory, and practical applications. Initial linear regression models demonstrated a difference in disability between genders of 1.32 points (95% CI 0.65, 2.00) on a scale from 0–23. Subsequent item analysis found contradictory results across items, with no clear pattern. Finally, IRT models demonstrated three items were demonstrated to present differential item functioning. After these items were removed, the difference between genders was reduced to 0.78 points (95% CI, -0.99, 1.23). These results were shown to be robust with re-sampling methods.
Conclusions
Purported differences in the levels of a latent variable should be tested using different models to verify whether these differences are real or simply distorted by model assumptions.
Statistical ModelsOutcome AssessmentPsychometricsGenderLow-back painDisability Evaluation
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Background
Clinical researchers frequently use statistical models in an attempt to model outcomes that are not directly measured, also known as latent variables. Examples of such latent variables include mental health, quality of life, and physical disability. Although groups of items (questions) known as outcome scales can be assumed to measure latent variables, it is methodologically challenging to aggregate item responses into scores that accurately and reliably represent the latent variable.
The aim of this study is to point that the choice of models with biased assumptions can lead to different conclusions regarding the associations between latent variables and predictors. Three alternative methods are presented: Prediction of latent variables measured as summed scores using linear regression models, prediction of individual item responses using logistic regression models and propensity scores to control for differences in item responses, and prediction of latent variables using Item Response Theory models with covariates. Since all three methods are statistically sophisticated, they will be described using the technique of "layers of information", and used to evaluate the purported association between gender and disability. Specifically, we will test whether this association can be explained by different reporting patterns.
Methods
Method of layers of information
The method of "layers of information" was designed to explain complex statistical methods to audiences with a variety of previous quantitative backgrounds. Each layer is associated with a progressive level of complexity; thus, ensuring that readers with different needs can understand the technique to a level that will enable them to at least understand the statistical method of a clinical study (first layer) and ultimately to apply the statistical method to a new research study (last layer). In the current study, we have used five layers of information: (1) General description, (2) Examples of previous clinical applications, (3) Data requirements, (4) Statistical Theory, and (5) Analysis and Reporting.
Layer 1 – General description
A latent construct is a concept not directly measured, but that can be estimated through proxy measures. Physical disability is an example since its level is frequently inferred from responses given to a series of items in an outcomes scale measuring patients' ability to perform activities of daily living. Because latent variables cannot be directly measured and predicted, several statistical techniques were devised to approach this problem (Figure 1).
Figure 1 Graphical description of three models to predict a latent construct 1a. Prediction based on summed scores 1b. Prediction based on regression on individual items 1c. Prediction based on latent variables
1. Outcome prediction based on summed scores
The most common approach is to simply add patients' responses to each item; thus, creating a summed score. Summed scores are then used to determine significant predictors in a regression model (Figure 1a).
Two assumptions underlie this strategy. First, we assume that the contribution of each item to the latent variable is known. For example, in a disability scale where patients are questioned about their ability to "raise a glass of water" and to "raise a 40-pound bag", researchers assume that they know the exact amount of disability associated with each of the activities stated by these items. In a scale that does not discriminate between the level of disability associated with each item, the assumption would be that answers to each of these items would represent the same amount of disability, when, in fact, they may not.
The second assumption when using summed scores is that each item measures the latent construct without any interference from extraneous factors. For example, it is assumed that two individuals with the same neck disability level, but different educational levels would have similar answer patterns for an item such as "I feel pain in my neck after reading for more than two hours". In this example this assumption might not be true since individuals with different educational levels may have different levels of exposure to a two-hour reading session and consequently have a different perception of the disability caused by such activity. Therefore, in spite of having the same disability level, they would probably provide different answers to the same item. This phenomenon is known as Differential Item Functioning, previously known as item bias.
2. Outcome prediction based on responses to individual items
A second approach is to use answers from each item and then determine how each predictor is associated with individual item responses (Figure 1b). Although apparently simple, this model no longer measures the association of each predictor with the latent variable of interest since individual items, and not the latent construct, is part of the model. In addition, if different items have contradictory levels and directions of association with each predictor, making inferences about the construct may be difficult or impossible.
3. Outcome prediction based on latent variables
The last and most recent approach is to use statistical models that will concomitantly determine the latent construct level and its association with the predictor of interest (Figure 1c). The main advantage of this method is that the assumptions made for summed scores are no longer necessary while, in contrast with the prediction based on individual items, a latent variable is still assumed. The main underlying assumptions of IRT models are that the association between item responses and the latent variable obeys a constant pattern across items, usually an S-shaped pattern, and that patterns of item-response are not influenced by any factor extraneous to the latent variable. Additional requirements include more powerful computers to execute the computations as well as larger sample sizes.
Layer 2 – Examples of previous clinical applications
Outcome prediction based on summed scores
In a study designed to predict factors associated with post-treatment disability after lower-extremity soft tissue sarcoma, Davis [1] calculated summed scores from scales measuring impairment [2,3], physical disability [4], and quality of life [5]. Although the authors did not report whether the four scales complied with the assumptions of a linear regression model described in our first layer, they found that large tumor size, bone resection, motor nerve sacrifice, and complications were associated with poor outcomes.
Outcome prediction based on responses to individual items
In a study evaluating the prediction of visual disability based on individual objective measures of visual impairment, Bandeen-Roche [6] regressed individual items of Activities of Daily Vision scale [7] and then compared their results to the prediction based on summed scores. These authors found that whereas most vision covariates were similarly associated with different item responses, visual acuity was much more strongly associated with two activities ("difficulty reading signs at night and during the day", and "watching television") than with others ("descending steps in either type of light"). In addition, male gender and a greater number of comorbid conditions were also preferentially associated with difficulty watching television.
Although these models bring new insights into the association between individual physical activities and their respective predictors, they cannot clarify whether these were true predictors or whether they simply presented different reporting patterns.
Prediction based on latent variables
To our knowledge, although multiple previous clinical research projects have used IRT for the determination of scale scores [8], no previous clinical articles have used IRT models with concomitant predictors. Potential clinical applications are any situations where the researcher is attempting to predict a latent construct based on a group of variables [8], but where a possibility of different reporting patterns or items with an association with different levels of the latent construct are present.
Layer 3 – Data requirements
Outcomes
First, a latent construct has to be measured through a set of proxy variables. These indicators may have responses in various formats, including dichotomous (yes/no), ordinal (e.g., a little, moderate, a lot), or nominal (alternatives without a rank). IRT models assume that the latent construct is continuous and, in most cases, unidimensional, meaning that one single latent construct is assumed.
Predictors
Predictors can be continuous or categorical variables.
Sample size
Previous studies have estimated that, for logistic regression models, one should have at least 10 events per predicting variable [9], while for multiple linear regression models this number reduces to four (Freedman 1989). For IRT, some studies have estimated that models can be estimated with as few as 250 respondents, although 500 would be ideal in most scenarios [11]. This number may vary; however, depending on the response heterogeneity to the items in the original sample. As general rule, more heterogeneous responses usually require smaller sample sizes.
Layer 4 – Statistical theory
Outcome prediction based on summed scores
Differences in summed scores according to a set of predictor or covariates can be described using linear regression. In these models, the summed score is represented by y using a linear combination of predictor variables xj, where j represents several predicting variables 1, 2, ..., p. It is assumed that no missing values are present for every observation. The fitted values, or predicted summed scores, are then the sum of coefficients βj multiplying each of the xj plus an intercept β0, although the later may be absent in some models. This model can be represented by:
y - β0 + β1 x1 +...+ βp xp
Ordinary least-squares models estimate the coefficients to minimize the squared sum of residuals. If the response and predictors corresponding to the ith of n observations are yi, xi1 ,..., xip, then the fitting criterion chooses the βj to minimize:
The standard statistical theory of linear models makes the first formula more explicit by writing the model for the ith observation as:
This model makes the following assumptions: The ci are independently and identically distributed; the ci have mean zero and finite variance σ2; the ci have a normal distribution.
Outcome prediction based on responses to individual items
Individual responses to dichotomous items can be predicted by generalized linear models using a binomial distribution and, most commonly, a logit link function that will bound the probability of an answer to be between 0 (answer = no) and 1 (answer = yes). The logit link can be expressed by:
where π is the probability of a positive answer andx is a vector with item responses . To linearize the function, the dichotomous response for each item can be algebraically transformed to:
Notice that, in contrast to linear models, the logistic model does not have an error term since it models the probability of an event directly that will determine the variability of the binary outcome. Logistic models are estimated by maximum likelihood, which is a method to estimate regression coefficients that will maximize the likelihood of obtaining the data ( p(0|x), where 0 is the latent construct.
One of the problems with the prediction based on individual items is that items do not individually represent the latent construct. Therefore, if one is to predict individual answers, it would be interesting to at least account (adjust) for responses of the same patient to other items. This adjustment can be accomplished by propensity scores [12], which reduce all remaining items to a single composite variable that appropriately summarizes their responses. Compared to the multiple adjustment performed in logistic regression models, propensity scores have the advantage of making the adjustment more transparent.
It is important to notice that although the covariates are used as predictors for the item-response, it is still impossible to infer whether this association was distorted by an association between item responses and extraneous variables rather than the association between item responses and the latent trait.
Outcome prediction based on latent variables
Although multiple models have been described for the regression of latent variables on predictors [6], we will concentrate on IRT. IRT assumes that the response of patients to individual items can be modeled with a two-level logistic regression where the log odds of patient i providing a positive answer to an item j is represented by:
Where βj represents the difficulty of item j and ui represents the trait level associated with subject i. This equation holds true in the simplest IRT model known as Rasch or one-parameter logistic (1PL). Other models – two-parameter logistic, ordinal logistic – among others – are used according to the types of response alternatives presented by each item.
Adding one additional parameter λ to represent the extent to which item j can discriminate between subjects of different trait levels, we obtain:
Finally, if we add a predictor to this equation we will have
where γ is the regression coefficient for predictor x. This model allows several advantages over the two models previously described in this layer, including the absence of assumptions from summed scores as well as the summarization of all items into a single latent variable. The most frequent assumptions in IRT models are that a single construct is measured and that observations are independent, conditional on the latent variable. Different IRT models will have different assumptions about the extent to which assumptions of summed scores can be relaxed. For example, 1-Parameter. Logistic Regression models assume that each item measures the latent trait with equivalent strength.
One important practical aspect, when making use of IRT models with predictors, is to check quadrature point approximation used in the random-effects estimator. As a rule of thumb, if the coefficients do not change by more than a relative difference of 0.01%, then the choice of quadrature points does not significantly affect the outcome and the results may be confidently interpreted. Two aspects of random-effects models have the potential to make the quadrature approximation inaccurate: large group sizes and large correlations within groups [16].
Layer 5 – Analysis and reporting
Data analysis
To illustrate a practical application of the previously described models, we will use data from a cohort study of patients with low-back pain to evaluate the gender-disability association. Specifically, we will evaluate whether female patients either have more severe disability or simply whether they are more likely to give positive answers to some items while having equivalent physical disability levels.
Several studies have found that, compared to men, women are usually associated with higher initial disability and pain scores after low-back pain episodes [14,15]. However, it is usually unnoticed that these studies do not directly measure disability, a latent construct, but rather measure patients' responses to items that are hypothesized to measure disability. In other words, the hypothesis is that the instrument accurately measures the construct, although the instrument is rarely re-evaluated by the time of measurement. In support of this important caveat is that previous studies have found that women have different responses to the stress caused by low-back pain when compared to men [16]. Therefore, the question of whether women really present with higher disability levels, simply have a different response to items measuring disability or both have higher disability and have a different response is open.
A description of the cohort used for this analysis is presented in detail elsewhere [17]. Briefly, the cohort contains data on 1,633 patients with low-back pain answering 23 dichotomous items from the Roland Questionnaire modified by Patrick [18,19]. The item content for this scale is presented in Table 1. The outcome of interest is physical disability represented by items of the modified Roland Questionnaire, and the main effect is gender. The association between these variables is adjusted for several potential confounders, including marital status (married, other), presence of workman's compensation (yes/no), and presence of private insurance (yes/no). All analyses were performed using Stata 8.0 for Linux (Stata Corporation, College Station, TX). Because Item Response Theory model with predictors are very computer intensive and individual models may take over 24 hours to run in personal computers, a special arrangement of the operating system was instituted to obtain maximal performance. These changes included establishing maximal priority (renice set to -20 to the Stata process, and running in a Linux "bigmem" kernel 4.20 with random allocation memory of 4 gigabytes). Additional measures to increase computational speed included data collapsing, frequency weights, and matrices with previous beta coefficients used as priors.
Table 1 Item content for the modified Roland Questionnaire
1. I stay home most of the time because of my back problem or leg pain (sciatica)
2. I change position frequently to try and get my back or leg comfortable
3. I walk more slowly than usual because of my back problem or leg pain (sciatica)
4. Because of my back problem, I am not doing any of the jobs I usually do around the house
5. Because of my back problem, I use handrail to get upstairs
6. Because of my back problem, I have to hold onto something to get out of an easy chair (comfortable padded chair)
7. I get dressed more slowly than usual because of my back problem or leg pain (sciatica)
8. I only stand for short periods of time because of my back problem or leg pain (sciatica)
9. Because of my back problem, I try not to bend or kneel down
10. I find it difficult to get out of a chair because of my back problem or leg pain (sciatica)
11. I have trouble putting on my socks (or stockings) because of the pain in my back or leg
12. I find it difficult to turn over in bed because of my back problem or leg pain
13. I sleep less well because of my back problem
14. I avoid heavy jobs around the house because of my back problem
15. Because of my back problem, I am more irritable and bad tempered with people than usual
16. Because of my back problem, I go upstairs more slowly than usual
17. I stay in bed most of the time because of my back or leg pain (sciatica)
18. I keep rubbing or holding areas of my body that hurt or are uncomfortable
19. My back or leg is painful almost all the time
20. I only walk short distances because of my back problem
21. Because of my back problem, my sexual activity is decreased
22. Because of my back problem, I am doing less of the daily work around the house than I would usually do
23. I often express concern to other people what might be happening to my health
Data preparation
Briefly, our sample is composed by 1,633 individuals with a diagnosis of low-back pain. Most patients are females (52.3%), married (69.9%), white (83.0), and with medical insurance (68.3%).
For linear and logistic regression models the data were placed in wide format, with individual variables representing patient responses to each item. For IRT models the data were presented in long compressed format (Figure 2).
Figure 2 Sequence of Stata commands for the execution of the three sets of model
Prediction based on summed scores
When comparing the crude association between summed scores and gender, it was found that female patients had scores that were on average 1.46 (95% CI 0.73, 2.08) points higher than their male counterparts in a 0–23 scale. This association was further tested in a linear regression model (Figure 1a) controlling for gender, insurance status (including workman's compensation), marital status, and income. The full model demonstrated that, adjusted to potential confounders, women report on average 1.32 (95% CI 0.65, 2.00) more points in the modified Roland scale than men. After backwards deletion, none of the previous potential confounders were proven to be substantial confounders using a cut point of 10% change the original point estimate.
Since the distribution of summed scores of the modified Roland Questionnaire was not normal, we used regression diagnostics using plots to determine that the relationship between predicted and observed values did not display any violations of the regression assumptions. This was confirmed by a Ramsey regression specification error test (RESET) for omitted variables (p = 0.7371) although the Breusch-Pagan / Cook-Weisberg test demonstrated a trend towards heteroscedacity (p = 0.0777).
In order to further verify the robustness of this association, an ordinal logistic regression model was used with cut-points at 0–7 (low summed score), 8–15 (medium summed score), and 16–23 (high summed score). This model was considered to adequately comply with the proportionality assumption (p = 0.776). Results for the ordinal regression model demonstrated that the predicted probability of a male having low, intermediate, and high scores were progressively decreasing: 0.38, 0.33, and 0.28, respectively. This pattern was in contrast with women, where the probabilities were ascending: 0.32, 0.33, and 0.35, respectively.
In summary, all results from models using summed scores point to a significant association between female gender and high disability scores. It is unclear; however, whether this association can be explained by high disability levels or simply different report patterns between men and women.
Prediction based on responses to individual items
As a next step, the association between individual item responses and gender was evaluated using logistic regression models stratified by propensity scores adjusting for responses to other items (Figure 1b). Propensity scores were determined by running logistic regression models that evaluated the probability of a positive response to an item adjusted for all remaining items and covariates except gender. These scores were then used to classify all observations into five different propensity score percentiles. The distribution of each of the covariates was found to be balanced among all four groups, indicating that the propensity scores were effective in "randomizing" the groups (Alcouffe 1999).
The analysis across propensity strata demonstrated contradictory results, with male patients being significantly associated with positive responses to items 4 ("Because of my back problem, I am not doing any of the jobs I usually do around the house") and 8 ("I only stand for short periods of time because of my back problem or leg pain (sciatica)"), while female patients were significantly associated with positive responses on items 7 ("I get dressed more slowly than usual because of my back problem or leg pain (sciatica)"), 15 ("Because of my back problem, I am more irritable and bad tempered with people than usual"), 17 ("I stay in bed most of the time because of my back or leg pain (sciatica)"), and 19 ("My back or leg is painful almost all the time"). No single item was consistently associated with gender across all propensity score strata.
A new model was then built adjusting for scores pooled across strata. The results demonstrated that most items were not associated with either gender, items 4 ("Because of my back problem, I am not doing any of the jobs I usually do around the house") and 8 ("I only stand for short periods of time because of my back problem or leg pain (sciatica)") being positively associated with male gender while items 7 ("I get dressed more slowly than usual because of my back problem or leg pain (sciatica)") and 15 ("Because of my back problem, I am more irritable and bad tempered with people than usual") being associated with female gender (Figure 3).
Figure 3 Odds ratio of having a positive response to an item* *ORs above one represent a positive association between a positive item response with being a male
Since logistic regression models do not control for the latent variable one cannot test whether the association between gender and individual item responses is related to an association with disability or simply caused by women being more likely to provide a positive response to a certain item in spite of having the same degree of disability.
Prediction based on latent variables
Finally, IRT models (Figure 1c) were used to determine the association between gender and IRT scores. First, a crude association between male and IRT scores was calculated based on all 23 items. This model demonstrated that female gender continued to be significantly associated with higher disability (coefficient 0.34, log likelihood test p < 0.001). Notice that this value is presented in a new scale that can no longer be compared to the previous scores obtained from the modified Roland scale with a range from 0 to 23.
To test the hypothesis that some items might present different reporting patterns, we tested for interaction terms between each item and gender. Our results demonstrated that items 7 ("I get dressed more slowly than usual because of my back problem or leg pain (sciatica)", Figure 4a), 14 ("I avoid heavy jobs around the house because of my back problem", Figure 4b), and 17 ("I stay in bed most of the time because of my back or leg pain (sciatica)", Figure 4c) presented significant interactions with gender. An interaction with gender indicates that the item response is affected by gender; thus, demonstrating different reporting patterns. Although interpretations of item content are speculative, items 7 and 14 may indicate that male and female patients interpret these questions as a different type and level of activity, respectively, while item 17 may be associated with differential behaviors in relation to disability across genders.
Figure 4 Item characteristic curves for items demonstrating differential item functioning 4a. Item 7 4b. Item 14 4c. Item 17
A new IRT model was then calculated, but now excluding all items with differential reporting patterns. The difference in disability reporting between men and women was reduced (coefficient 0.04, log likelihood p = 0.08), indicating that gender was no longer significantly associated with disability. In fact, when the same items were excluded from the summed score, a multiple linear regression model demonstrated that the difference between female and male patients had been reduced to 0.78 points (95% CI, -0.99, 1.23) on the original 0–23 scale (p = 0.06), a reduction of 53.4% compared to the original difference.
Bootstrapping methods were used in the linear regression model to verify whether the association was robust after multiple sampling procedures had been applied to the models. The results demonstrated a variation of only 13.2%; thus, indicating that these results are robust provided that the sample is representative of the study population.
In conclusion, one could infer that although women still have slightly more disability than men, much of the previously reported differences using the modified Roland were inflated by the presence of items with different reporting patterns in scales measuring disability.
Conclusions
We used three different regression models to investigate the association between gender and disability. Although summed models demonstrated a significant association between gender and disability, these models did not allow us to test whether this purported difference was related to the latent construct disability or to items presenting with differential item functioning. Analysis of the association within individual items demonstrated inconsistent associations with gender, with some items presenting a strong positive association with male gender while others had a positive association with female gender. Since these associations were made with the item response rather than the latent variable, it was impossible to verify whether these were valid representations of the construct of interest, associations with disability, or simply the effects of differential item functioning. Last, we examined the association between gender and disability measured as a latent variable. After removing items with differential item functioning, the association with gender was lessened and no longer significant. Therefore, we concluded that although a small difference between genders in relation to the disability associated with low back pain does exist, much of it is caused by differential item functioning than a true association with the disability construct.
In summary, we advocate that the measurement of the association between latent variables and covariates be systematically performed using a combination of regression models to ensure that observed associations are not distorted by differential item functioning.
Authors' contributions
RP: design, analysis, manuscript writing; MT: design, analysis, manuscript revision; UG: design, analysis, manuscript revision; LDH: design, manuscript revision; DOJ: design, manuscript revision; TC: data collection, design, manuscript revision.
Acknowledgements
Sources of funding: partially funded by the Orthopaedic Trauma Association.
We would like to thank Mrs. Suzana S. M. Albano for editorial support.
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| 15500700 | PMC529307 | CC BY | 2021-01-04 16:38:12 | no | Health Qual Life Outcomes. 2004 Oct 25; 2:59 | utf-8 | Health Qual Life Outcomes | 2,004 | 10.1186/1477-7525-2-59 | oa_comm |
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Int Semin Surg OncolInternational seminars in surgical oncology : ISSO1477-7800BioMed Central London 1477-7800-1-101553325110.1186/1477-7800-1-10ResearchThe role of thallium-201 and pentavalent dimercaptosuccinic acid for staging cartilaginous tumours Choong Peter FM [email protected] Toshiyuki [email protected] John [email protected] Stephen [email protected] Rodney [email protected] Department of Orthopaedics, University of Melbourne, St. Vincent's Hospital, Melbourne, Australia2 Department of Medical Imaging, St. Vincent's Hospital, Melbourne, Australia3 Department of Pathology, St. Vincent's Hospital, Melbourne, Australia4 Department of Medical Imaging, Peter MacCallum Cancer Institute, Melbourne, Australia5 Division of Surgical Oncology, Peter MacCallum Cancer Institute, Melbourne, Australia2004 8 11 2004 1 10 10 18 8 2004 8 11 2004 Copyright © 2004 Choong et al; licensee BioMed Central Ltd.2004Choong et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Introduction
Heterogeneity of cartilage tumours may confound accurate diagnosis and grading resulting in under and over treatment. Improved preoperative assessment of malignancy and grade would be invaluable for developing a rational plan for treatment. We examined correlations between nuclear tracer avidity and malignancy grade in cartilage tumours.
Methods
Between 1996 and 2000, 92 consecutive patients with cartilaginous tumours (50 benign, 42 non-metastatic malignant) underwent nuclear scanning. Thallium-201 (TL-201) and pentavalent dimercaptosuccinic acid (DMSAV) were used as nuclear isotopes. Scanning with these agents was performed on separate days 48 hours apart. Static and SPECT images were obtained at 30 m and 4 h after injection of nuclear tracer. Pathology review was undertaken blinded to the results of the nuclear scans and correlations between histologic results and trace uptake at 4 hours examined.
Results
25 patients with negative DMSAV had benign tumours. 15/17 tumours with positive TL-201 had malignant tumours. 11/13 patients with both positive DMSAV and TL-201 scans had intermediate or high grade tumours and 4 of these developed metastases. We have developed an algorithm for the management of patients with tumours that aims to avoid over treatment of low grade tumours and under treatment of high grade tumours.
Conclusion
Functional nuclear scanning with TL-201 and DMSAV complements other imaging modalities in the management of cartilaginous tumours.
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Background
Traditionally, the determination of malignancy and its grade in cartilage tumours has been from the combination of history, radiographic features with or without computed tomography and histologic examination [1-3]. More recently, magnetic resonance imaging has also been employed [4-6]. However, cartilage tumours are recognized for their histologic heterogeneity [7-9] and bizarre physical features, such that reliance on anatomic imaging and accuracy of biopsy alone for interpretation of state of malignancy or benignity may be misleading. While the recognition of high grade malignancy is not difficult, the differentiation between benign and low grade (grade I) tumours can present a diagnostic dilemma [10,11]. Such a dilemma may lead inadvertently to under-or over-treatment of cartilage tumours.
Functional nuclear scans employ isotopes that become substrates for various cellular metabolic cycles and therefore, are useful for determining the metabolic activity in these tissues [12]. Given that malignant tumours are more metabolically active than benign tumours, and that there is a relationship between grade of malignancy and metabolic activity, functional nuclear scans may help to differentiate between cartilage tumours of varying metabolic activity, which in turn, may shed some light on their state of malignancy.
We now report our experiences with 2 radio-siotopes, Thallium-201 (Tl-201) and pentavalent dimercaptosuccinic acid (DMSAV) for determining the metabolic activity of cartilage tumours, and their value in differentiating between malignant and benign cartilage tumours in 92 consecutive cartilage tumours. We describe their role in the development of our current regimen for treating cartilage tumours.
Methods
Patients
Between January 1997 and December 2000, 92 consecutive patients were referred to our institution for investigation and management of suspected chondral tumours. There were 50 females and 42 males with a median age of 45 years (range 16–87 years).
Tumours were located in the humerus (17), chest wall (3), femur (27), hand (3), knee (3), vertebra (1), scapula (7), tibia (16), pelvis (9), foot (4), radius (2). All patients had previously unoperated tumours and no patient presented with metastases.
Investigations
All patients were examined with plain radiographs, computed tomography and magnetic resonance imaging. From 1997 onward, TL-201 and/or DMSAV scans were also performed on patients with suspected chondral tumours.
Tl-201 and DMSAV scintiigraphy were conducted at two centers (SVH, PMCI) with similar imaging protocols. All studies were conducted using a gamma camera. A pre-determined dose of radioisotope was administered intravenously and scintigraphic images obtained at 30 minutes (early phase) and 3–4 hours (late phase) after injection in all cases. Early phase static images were acquired over the area of interest and late phase images consisted of whole body imaging and SPECT over the area under investigation. A low energy high resolution, parallel-hole collimator was used and image acquired in a 128 × 128 matrix for 5 minutes. A simple grading system was devised for late phase isotope uptake with NO UPTAKE indicating no tumour uptake greater than background activity and INCREASED UPTAKE indicating definite activity greater than the background level. The background level referred to is that of the tissue within which the tumour arose, that is, bone. We selected the late phase results for correlation with the results of histological examination of the surgical specimens because early uptake at 30 minutes may also represent peritumoural inflammation or vascularity, which may confound the interpretation of results. In contrast, late phase uptake represented true tumour uptake of isotope.
Tumours
The pathologist (JS) is the designated lead pathologist on the Victorian Bone Tumour Registry. For the purpose of this study, assessments of surgical tissue and histologic re-evaluation were conducted without prior knowledge of the results of the functional nuclear scans. There were 50 benign tumours and these consisted of enchondromata (29), osteochondromata (17), chondroblastoma (3), and chondromyxoid fibroma (1). There were 42 malignant tumours and these consisted of 38 central chondrosarcoma and 4 dedifferentiated chondrosarcoma. Of these malignant tumours, 22 were graded as grade I, 12 as grade II and 8 as grade III according to.
Treatment
All tumours in this study were treated with excision. In those tumours that were considered to be benign or grade 1 malignancy as based on history, examination, plain radiography, magnetic resonance imaging and functional scanning, were treated by careful intralesional curettage, burring with a high speed dental burr, pulsatile lavage, chemical cautery with phenol and then the defect filled with polymethylmethacrylate cement. In those tumours that were considered to be clearly malignant and interpreted as grade 2 and higher or if the functional nuclear scans showed significant uptake, wide resection was employed.
Because of the histologic heterogeneity of cartilage tumours, the majority of tumours were not biopsied prior to definitive surgery. Biopsy was considered if there was doubt about the diagnosis, or if the anticipated treatment was potentially far greater than may have been required. If biopsy was preferred but could not be safely performed preoperatively, for example, a periacetabular tumour, frozen section drill biopsy would be conducted as part of the initial surgical approach to the tumour.
Follow-up
No patients were lost to follow-up. Patients with malignant tumours were reviewed every 3 months for the first 2 years and 6 monthly after that for a further 2 years with a plan for yearly review for the following 4 years. Computed tomography of the chest was performed every 6 months, and plain radiographs obtained of the operated area at each visit.
The median follow-up was 3.7(0–6) years. At last review, 85 patients were alive without disease, 2 were alive with pulmonary metastases. 3 patients had died of metastatic disease, one patient died from intraoperative complications and 1 patient died from a pulmonary embolus 1 month after surgery. There were no local recurrences.
Results
Thallium scans
Eighty seven of ninety two patients underwent thallium scanning. Of these, 17 patients had increased uptake on the delayed scans, and 70 patients had no uptake on the delayed scans.
DMSA(V) scans
Eighty three of ninety two patients underwent DMSA(V) scanning. Of these, fifty eight patients had increased uptake on the delayed scans and twenty five patients had no uptake on the delayed scans.
Combined thallium and DMSA(V) scans
Seventy eight of ninety two patients had both thallium and DMSA(V) scanning. There was no uptake with Thallium and DMSA(V) scanning in twenty patients. Thallium scanning was negative and DMSA(V) was positive in forty five patients. There was uptake on both Thallium and DMSA(V) scanning in 13 patients. There was no case where there was thallium uptake without DMSA(V) uptake.
Correlation between functional scanning and state of benignity /malignancy of tumours
a. Thallium scans Of the 50 benign tumours, 45 underwent thallium scanning. Forty-three tumours had negative scans and 2 had positive scans. Of the 42 malignant tumours, 27 had negative thallium scans and 15 had positive scans.
b. DMSA(V) scans Of the 50 benign tumours, 47 had DMSA(V) scans. Twenty five of these tumours had negative scans and 22 had positive scans. Of the 42 malignant tumours, 36 had DMSA(V) scans. All 36 of these were positive. No malignant tumour was DMSA(V) negative.
c. Combined thallium and DMSA(V) scans There 78 tumours that were scanned with both thallium and DMSA(V). Of the 42 benign tumours, 2 had positivity forboth thallium and DMSA(V), 20 had positivity only for DMSA(V) and 20 had no uptake on either scan.
Of the 36 malignant tumours that were scanned with both isotopes, 11 showed positivity for both thallium and DMSA(V), while 25 tumours showed positivity for DMSA(V) only. No malignant tumour was negative to thallium scanning.
Correlation between functional scanning and tumour grade in 42 choindrosarcomas
a. Thallium scans Of the 42 chondrosarcomas, 7 grade II and 8 grade III tumours had positive thallium uptake. No grade I tumour had thallium uptake. Twenty two grade I tumours, and 5 grade II tumours showed no uptake.
b. DMSA(V) scans Thirty six of the 42 chondrosarcomas had DMSA(V) scans. All were positive (20 grade I, 11 grade II, 5 grade III). No chondrosarcoma was DMSA(V) negative.
Correlation between functional scanning and metastasis
No patient with a negative thallium scan developed metastasis. Six of seventeen patients with positive thallium scans developed metastases. In contrast, four of fifty eight patients with positive DMSA(V) scans developed metastases. In patients where both scans were performed, four of thirteen patients developed metastases.
Discussion
Anatomic imaging such as computed tomography [13] and magnetic resonance imaging [4-6,14] provide excellent morphologic delineation and localisation of bone tumours. These tests are invaluable in the surgical planning for patients with musculoskeletal tumours However, these tests do not always give an indication of the biologic behaviour of the tumour, particularly if there are subtleties between benign and low grade malignant states [15]. Cartilaginous tumours of bone are characterized by radiologic and histologic heterogeneity [7,8] that may give rise to diagnostic dilemas. Benign tumours such as enchondromas and osteochondromas may appear large and bizarre giving an impression of biologic aggressiveness, particularly those in the hand, while some chondrosarcomas such as a clear cell chondrosarcoma may be small, well defined, slow growing and apart from osteolysis have no other hallmark of malignancy [16-18] such as cortical destruction or soft tissue extension to express its malignant phenotype. One of the major difficulties in orthopaedic oncology is the differentiation between enchondroma and grade 1 chondrosarcoma [19].
Conventional technetium monodiphosphonate skeletal scans are employed to identify uni-or multifocal disease when bone tumours are suspected [12]. For positivity, this scanning modality relies on the interactions between tumour and host bone that incite an osteoblastic response by adjacent bone and does not necessarily imply malignancy. For example, enchondroma and chondrosarcoma frequently demonstrate a similar uptake of nuclear tracer, without differentiating between benignity and malignanc.
Reliance on preoperative histologic diagnosis for determining the nature of a cartilage lesion can also be difficult. Biopsy of a suspicious cartilage tumour is most helpful when clear malignancy is demonstrated because a benign finding does not necessarily exclude malignancy. The histologic heterogeneity of cartilage tumours, however, is well recognized and unless the biopsy accurately targets the most malignant part of the tumour, there is a risk that the histologic diagnosis may under-report the state of malignancy if this should exist.
Functional nuclear scans reflect the metabolic activity of tumours and may provide important information regarding their biologic behaviour. In this regard, hypermetabolic tumour tissue is likely to be more active than surrounding normal tissue and this difference may be valuable for distinguishing between benign and malignant tumours. Similarly, when dealing with grades of malignancy, a higher level of metabolic activity may be expected from high grade tumours in comparison to lower grade counterparts. Since the late 1970s, thallium (Tl-201) scanning has been used extensively as a safe method to assess ventricular function in patients with myocardial ischaemia and the results have been interpreted as reflecting relative levels of myocardial metabolic activity. As Tl-201 is a potassium analogue, its uptake into cells depends on the sodium potassium ATPase dependent pump. Tl-201 is a readily available, cyclotron produced radionuclide that decays by electron capture with a half-life of approximately 73 hours. The liver, spleen, kidneys, myocardium, thyroid, choroids plexus of the lateral ventricles and testis normally demonstrate avidity for Tl-201, with very minimal uptake in healing surgical wounds. Tl-201 is thought to accumulate less well within connective tissue, which contains inflammatory cells and almost undetectable in necrotic tissue. Localization of Tl-201 within tumours appears to be influenced by tumour vascularity, tumour cellularity, the metabolic rate of the tumour and the histological type of tumour. Thallium uptake studies [20-23] in patients with bone tumours have been used to predict response to preoperative chemotherapy by correlating the histological degree of tumour necrosis to changes in Tl-201 uptake. The correlation between thallium uptake and tumour metabolic activity, thus makes it a good candidate tracer to assess malignancy in chondromatous tumours. 99Tcm-Dimercaptosuccinic acid (DMSA) is another readily available isotope, which was first described as an isotopic agent for investigating the renal parenchyma in a variety of disease entities [24-26]. Subsequently, the pentavalent form of 99Tcm-dimercaptosuccinic acid (99Tcm-(V)DMSA) developed a recognised role for imaging medullary thyroid carcinoma, [27-30] and this role was further investigated in relation to assessing bone metastases and other neoplastic conditions such as multiple myeloma, osteosarcoma and chondrosarcoma. [31-33]; [28,34]. Of note, uptake of DMSA(V) is reported to be absent in vertebral collapse and osteoarthritis [35].
Our study has demonstrated that the combined use of Tl-201 and DMSA(V) scanning may be correlated with the benignity, malignancy and grade of cartilaginous tumours. For a tumour that is hetereogeneous not only on radiologic but also pathologic appearance, the use of functional nuclear scanning may help to differentiate particularly between the low grade and benign lesions. Of note, no malignant lesion had a negative DMSA(V) scan, and only 2 out of 45 benign tumours showed thallium uptake in our study.
While this modality of investigation does not replace the value of combining elements from the clinical presentation, plain radiographic changes and pathology, functional nuclear scanning may increase the confidence of diagnosing a low grade or benign tumour, and the availability of information preoperatively may also be helpful for tumours that are deeply situated and difficult to biopsy. With regard to biopsy, localized thallium or DMSA(V) positivity may be useful for guiding tissue sampling from the most metabolically active site, thus enhancing the procurement of potentially the most aggressive/malignant part of the tumour [12].
Although, the median duration of follow up in this study was limited to 3.7 years, the high proportion of metastases in patients whose sarcomas were thallium positive may suggest a role for thallium scanning as a prognostic indicator for metastasis in chondrosarcoma. Interestingly, the combination of DMSA(V) with thallium scanning did not improve the prognostic value for metastases. This may be attributed to the fact that the spread of DMSA(V) positivity amongst the tumours was greater than thallium positivity, whereas the latter was positive only in patients with grade II and III chondrosarcomas, which are the ones that mainly metastasise.
We cannot explain why 2 out of 45 benign tumours were thallium positive. The specimens were reviewed by a panel of pathologists expert in bone pathology and none conferred a diagnosis of sarcoma. In both of these cases, DMSA(V) positivity was also observed. It is important to note that these tumours occurred in the hand and foot, although not part of the syndrome of enchondromatosis. It is appreciated that small bone enchondromata may behave locally in a very aggressive manner without any cytologic evidence of malignancy [19]. However, small bone chondrosarcoma are also know to be fatal and careful inspection of imaging and pathology is required to ensure distinction between benign and malignant lesions which may behave similarly [36,37]. No conclusion can be drawn about the biologic significance of our finding of 2 benign small bone tumours with thallium uptake, in view of the very small numbers of these in our study. One patient had a small toe amputation and the other had curretage, chemical cautery and cementation of a phalangeal lesion. Neither have represented with local or systemic recurrence of disease.
As a result of this study, we now undertake the following sequence of scanning and treatment (Figure 1). All patients with a suspected chondral tumour would undergo a DMSA(V) scan. If this is negative, no further scanning is performed and the tumour is regarded as benign because our study had indicated that no malignant tumour had a negative DMSA(V) scan. These tumours would then be treated on their individual merits. If there is uptake on the delayed DMSA(V) scans our study had indicated that half of these are likely to be malignant therefore, patients would undergo thallium scanning. If there is no uptake, then based upon our study, the result would be interpreted as benign cartilage tumour or grade 1 chondrosarcoma. Patients may then undergo intralesional curettage with chemical cautery and cementation or wide excision. If there is uptake on the thallium scan, our study demonstrated that there is a very high likelihood that these tumours would be grade II or III and a wide excision would be recommended.
Figure 1 Algorithm for the use of DMSA(V) and thallium scanning for cartilaginous tumours. Thal : Thallium-201; DMSA(V) : Pentavalent dimercaptosuccinic acid +ve : positive uptake; -ve : no uptake.
Surgical treatment for cartilage lesions vary widely [38]. Intralesional curettage together with local adjuvant treatment is often recommended for benign tumours but the management of chondrosarcoma follows the same principles as espoused for all sarcomas [39,40]. Wide resection with a cuff of normal tissue radially and axially around the tumour is encouraged. The importance of good margins is highlighted by the predilection for local recurrence of incompletely excised chondrosarcoma [41-43], and also because of the risk of chondrosarcomas recurring at a higher maliginancy grade with its associated increased risk of metastasis [44]. Inaccurate diagnosis and or grading may thus result in under- or over treatment of cartilage neoplasms that may later manifest as troublesome local recurrence or unnecessary loss of function from ablative surgery.
The management of grade 1 tumours remains controversial. Some authors have recommended intralesional curretage, adjuvant treatment and cementation for these lesions [45,46]. In a series of 40 enchondromata and low grade chondrosarcomas, Bauer et al. observed a local recurrence of 0.09 over 10 years using this modality of treatment. In all cases, local control was subsequently achieved by repeating the earlier surgery on the recurrence. Neither recurrence occurred as a higher grade of tumour. In contrast, some have recommended wide excision of grade I lesions and so called "borderline" chondrosarcoma for fear of tumour recurrence at a higher grade, which would portend toward a poorer outcome [47,48]. The relationship between thallium positivity and higher grades of chondrosarcoma in our study suggests that it would be important to review in a prospective manner the outcome of surgery based upon our algorithm of preoperative scanning.
Conclusion
There has been rising interest in studying non-invasive techniques of imaging cartilage tumours to try and determine their biologic aggressiveness prior to definitive surgery. The advantages of functional nuclear scanning with DMSA(V) and thallium are that they are easy to perform in the majority of nuclear medicine departments, the isotopes are easily available and the costs are not prohibitive. We have found that the information derived from our study complements other imaging modalities and apart from improving our understanding of cartilaginous tumours, has also assisted us in developing a strategy for their treatment.
Competing interests
The author(s) declare that they have no competing interests.
List of abbreviations
TL-201 – Thallium -201
DMSA(V) – Pentavalent dimercaptosuccinic acid
Authors contribution
Prof. Peter Choong Surgeon, carried out surgery, participated in clinical and diagnostic practice. Wrote and prepared manuscript
Dr. Toshikunisada Orthopaedic oncology fellow, collected diagnostic data
Dr. Stephen Schlicht Nuclear physician, Participated in manuscript preparation, and conduct of diagnostic tests
Dr. Rodney Hicks Nuclear physician, Participated in manuscript preparation and conduct of diagnostic tests
Dr. John Slavin Pathologists, participated in blinded review of patients histology
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| 15533251 | PMC529308 | CC BY | 2021-01-04 16:38:35 | no | Int Semin Surg Oncol. 2004 Nov 8; 1:10 | utf-8 | Int Semin Surg Oncol | 2,004 | 10.1186/1477-7800-1-10 | oa_comm |
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RetrovirologyRetrovirology1742-4690BioMed Central London 1742-4690-1-361551627010.1186/1742-4690-1-36Short ReportSIVdrl detection in captive mandrills: are mandrills infected with a third strain of simian immunodeficiency virus? van der Kuyl Antoinette C [email protected] den Burg Remco [email protected] Mark J [email protected] Rob A [email protected] Albert DME [email protected] Ben [email protected] Department of Human Retrovirology, Academic Medical Centre, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands2 Artis, Plantage Kerklaan 38–40, 1018 CZ Amsterdam, The Netherlands3 Department of Virology, Erasmus Medical Centre, P.O Box 1738, 3000 DR Rotterdam, The Netherlands2004 1 11 2004 1 36 36 30 9 2004 1 11 2004 Copyright © 2004 van der Kuyl et al; licensee BioMed Central Ltd.2004van der Kuyl et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
A pol-fragment of simian immunodeficiency virus (SIV) that is highly related to SIVdrl-pol from drill monkeys (Mandrillus leucophaeus) was detected in two mandrills (Mandrillus sphinx) from Amsterdam Zoo. These captivity-born mandrills had never been in contact with drill monkeys, and were unlikely to be hybrids. Their mitochondrial haplotype suggested that they descended from founder animals in Cameroon or northern Gabon, close to the habitat of the drill. SIVdrl has once before been found in a wild-caught mandrill from the same region, indicating that mandrills are naturally infected with a SIVdrl-like virus. This suggests that mandrills are the first primate species to be infected with three strains of SIV: SIVmnd1, SIVmnd2, and SIVdrl.
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Findings
To date over 30 strains of simian immunodeficiency virus (SIV) have been isolated from African primate species and sequenced [1]. Mandrills (Mandrillus sphinx) are quite exceptional among African monkeys in that they harbour two distinct SIV strains, designated SIVmnd1 and SIVmnd2, with a separate geographic distribution [2,3] (see also Figure 1A). SIV infections are mostly non-pathogenic in their natural hosts. SIVmnd1, despite high virus levels in chronically infected mandrills, has only a small effect on the T-cell counts, and primary infection does not induce clinical symptoms [4,5]. However, two cases of immunodeficiency were reported in mandrills after long-term (>18 years) SIV infection [6]. In 2003, a 20-year old male captive mandrill (mandrill CAS) housed at Artis Zoo (Amsterdam, The Netherlands), suffering from heart failure and poor general condition, was found to be positive for serum SIV antibodies. Inspection of the other three mandrills of his group, a ten-year old female (mandrill REB) and their offspring (mandrills RAF, 3 years old and HAB, 2 months old), showed that the female and one of the offspring (HAB) were also SIV antibody-positive.
Figure 1 A) Geographic distribution of the genus Mandrillus, based upon mitochondrial haplotypes (adapted from [10]), and the SIV strains they harbour. B) Phylogenetic tree generated with Kimura-2-parameter distances and the NJ option of the MEGA package , from 267 nt of the mitochondrial cytochrome b gene of the four captive mandrills and reference sequences from GenBank (accession numbers are indicated). C) Phylogenetic tree generated with the NJ option of MEGA, based upon Kimura-2-parameter distances of SIVpol nucleotide fragments from captive mandrills CAS and REB, and reference sequences for SIVdrl, SIVmnd1, and SIVmnd2, respectively. Numbers shown are bootstrap confidence levels (BCL). D) SIV-pol amino acid consensus sequence from captive mandrills compared with homologous sequences from SIVdrl and SIVmnd2, respectively. The translated SIV-Artis sequence is the consensus sequence of 14 PCR clones derived from two animals. The YMDD motif within the catalytic core of the RT enzyme is underlined.
Both EDTA-plasma and PBMC (isolated with the OptiPrep system (Nycomed, Oslo, Norway)) was obtained from the animals for further analysis. Our goal was to investigate whether the monkeys were virus carriers, and which strain of SIV they harboured. To detect both SIVmnd1 and SIVmnd2, we designed two nested primers sets based on published pol gene sequences that amplify an RT fragment of 282 nucleotides (Table 1). Nucleic acids were isolated from PBMC by a procedure using silica and guanidium thiocyanate [7]. cDNA was synthesized with the 3'primer and AMV-RT (Roche Diagnostics, Penzberg, Germany). PCR amplifications were performed using the following protocol: denaturation for 5 min at 95°C and amplification for 35 cycles (first PCR) or 25 cycles (second PCR) of 1 min at 95°C, 1 min at 55°C, and 2 min at 72°C, followed by an extension of 10 min at 72°C. Products were cloned with the TOPO TA cloning kit (Invitrogen, San Diego, Calif.). Sequencing of at least four clones per sample was done with the Bigdye Terminator Cycle Sequencing kit and an ABI 377 automated sequencer (both from ABI, Foster City, Calif.), using M13 forward and M13 reverse primers. For species identification, fragments of the mitochondrial 12S and cytochrome B genes were amplified [8], and sequenced.
Table 1 PCR primers used to amplify Mandrillus SIV-pol
Primer Sequence ('5→'3) Description Fragment size
SIVmnd1A AGATATAGGGGATGCCTATT 5' first primer A-B = 356 nt
SIVmnd1B TCTTCCACTTATCTGGGTGT 3' first primer
SIVmnd1C AGATTATAGACCCTATACTGC 5'second primer C-D* = 282 nt
SIVmnd1D CATCCAATGAAAGGGAGGTTC 3' second primer
SIVmnd2A GGACATAGGGGATGCCTATT 5' first primer A-B = 356 nt
SIVmnd2B CTGTCCATTTCTTTGGGTGC 3' first primer
SIVmnd2C GGACTTTAGAAAGTACACTGC 5'second primer C-D* = 282 nt
SIVmnd2D CATCCACTCAAAGGGAGGTTC 3' second primer
SIVdrlA GGATGTAGGTGATGCCTATT 5' first primer A-B = 356 nt
SIVdrlB CTGTCCACTTCTTTGGATGC 3' first primer
SIVdrlC = SIVmnd2C 5'second primer C-D* = 282 nt
SIVdrlD CATCCATTCATAAGGAGGATTG 3' second primer
* corresponding to nucleotides 2503–2784 of SIVdrl (acc. no. AY159321)
Mitochondrial 12S and cytochrome B sequences were identical in all four animals and confirmed that the monkeys were M. sphinx [9,10]. In addition, the cytochrome B sequences were indistinguishable from the recently described northern mandrill haplotype (Figure 1B) [10], suggesting that the captive animals descended from founders originating from a locale north of the Ogooué River (see Figure 1A). SIV-pol fragments could be amplified from PBMC of both adult mandrills CAS and REB with the primer set specific for SIVmnd2, but not with SIVmnd1 specific primers. However, analysis of the cloned fragments showed that these were 96–97% identical to SIVdrl-1FAO (GenBank acc. no. AY159321) isolated from a drill monkey (Mandrillus leucophaeus), with a lower sequence identity to SIVmnd2 (± 85% to GenBank acc. no. AF367411), and to SIVmnd1 (<64% to GenBank acc. no. M27470). Although SIVmnd2 and SIVdrl are more closely related to each other than the two SIVmnd strains, SIVdrl has several mismatches with the PCR primers designed for detection of SIVmnd2. This could explain why one seropositive animal was tested as PCR-negative. Therefore, we designed a new primer set that amplifies the same gene fragment based on the SIVdrl sequence (Table 1). Reanalysis of the mandrill PBMC samples with this drill-specific primer set again resulted in only two positive samples from the two adult animals: mandrills CAS and REB. Sequence analysis confirmed the high similarity to SIVdrl-1FAO (97%), and a lower similarity to SIVmnd2 (87%). SIV pol fragments from both mandrills were 98–99% similar to each other. Clones obtained from a single animal with the two primer sets were not identical to each other (98–99% identity), suggesting that each PCR amplified a subset of the virus population (Figure 1C).
Mandrills presently inhabit Cameroon, Gabon, and the southwestern part of the Republic of Congo. Two mitochondrial haplotypes are described in this species, separated by the Ogooué River in Gabon [10]. Interestingly, the distribution of mandrill SIV strains follows approximately the same geographic distribution, with SIVmnd1 being present in the southern part of the mandrill range, and SIVmnd2 in the northern part. Drill monkeys are found in Nigeria and Cameroon separated from the mandrill territory by the Sanaga River (Figure 1A). Mandrills and drills are currently believed to be non-sympatric, but it is not unlikely that the situation was different in the past.
SIVmnd2 and SIVdrl are closely related, and both are equidistant from SIVmnd1. SIVmnd2 is found in northern mandrills, which are closest to the current drill habitat. A wild-caught mandrill from south Cameroon was found to harbour a SIVdrl virus strain [3,11], suggestive of cross-species transmission [11]. Multiple cross-species transmissions are now believed to obscure the evolution and distribution of SIV strains in African primate species [1]. SIV cross-species transmissions are ongoing, and African green monkey strains have recently been detected in patas monkeys and baboons [1,12,13], species that are found in close proximity to each other.
All four mandrills examined here were born in captivity. Male CAS was born in 1983 at the now closed Wassenaar Zoo (The Netherlands), and moved to Artis Zoo in 1986. The female, REB, was born in Budapest Zoo (Hungary), and moved to Artis Zoo when she was 5 years old. Their offspring, RAF and HAB, were both born in Amsterdam. Exposure to drills during their lifetime is unlikely as none of the zoos kept drills (M. leucophaeus) at any time. Drills are rare in European zoos, and only the zoos of Nikolaev (Ukraine) and Saarbruecken (Germany) reported keeping both drills and mandrills in a 1992 survey [14]. So, it is improbable that the Artis mandrills acquired SIVdrl from a recent contact with captive drills. Another way of acquiring a drill SIV strain could be if one of the monkeys was actually a hybrid between a drill and a mandrill. Hybridisation between different species of Cercopithecinae is possible, and offspring is sometimes fertile depending upon the exact species. The genus Mandrillus cannot hybridise in the wild, as the habitats of the two species do not overlap, but it does so in captivity. The morphologic differences between female drills and mandrills are less obvious than those between males and are mainly noticeable in the colouration of the muzzle and the size of the animal. A single hybrid M. sphinx × M. leucophaeus has been reported from Vienna Zoo, Austria, in a 1992 survey [14], and two hybrid M. leucophaeus × M. sphinx were described from a Wildlife Rescue Centre in Cameroon [15]. Mitochondrial sequencing as performed in this study cannot alone be used to resolve hybridisation, as it only characterises the mother lineage. The male mandrill is, however, unlikely to be a hybrid as its description fits exactly that of a male mandrill. The female was also listed in the Artis Zoo database records as a non-hybrid. She was registered with the European studbook programme (ESB) for mandrills supervised by the Budapest Zoo, Hungary, and was born from registered mandrill parents.
Because SIVdrl was also found in a wild-caught Cameroonian mandrill [3,11], it is plausible that a SIVdrl-like virus is naturally present in mandrills, making them the only primate species that is naturally infected with three strains of SIV. The presence of SIVdrl in one of the two adult captive mandrills occurred probably through transmission from a wild ancestor, and this animal probably infected the other mandrill once joined in Artis Zoo. Sexual and mother-to-child transmission of SIV in mandrills have been reported to be rare [16,17]. Here, in offspring born to a SIV-positive mother SIV could not be detected. The persistence of maternal antibodies could explain why the 2-month old young tested seropositive, although there is a possibility of the animal having a viral copy number below the detection limit of the PCR assay. Transmission of SIV between mandrills and drills could have taken place either by biting or sexual contacts. SIVmnd1 is mainly transmitted between males during aggressive contacts [17], and might also be transmitted when fighting off other receptive primates species. Sexual transmission could have occurred during interbreeding. Each of these possibilities requires an overlap of habitats, which could have existed in the past.
If we assume that SIVdrl(Artis) left Africa at least ten years ago (when the female was born in captivity), its genome conservation is remarkable: only 2 conserved amino acid differences separate the consensus pol-sequence from the SIVdrl reference sequence (Figure 1D). However, to gain a deeper insight into the characteristics and evolution of this virus strain, a full-length sequence, or at least additional sequences of the gag or env regions, would be required. Only then could it be determined whether the virus carried by the captive mandrills is really SIVdrl or a novel recombinant virus. Several recombinant SIVs have been described in naturally infected primate species (see: [1]). Unfortunately, further sequence analysis is difficult due to shortage of material as the monkeys were euthanized soon after the SIV antibody test results became known.
Competing interests
The author(s) declare that they have no competing interests.
Authors'contributions
ACvdK designed the study, analysed the sequences, and drafted the manuscript. RvdB carried out the PCR assays and performed the cloning and sequencing. MJH did the medical examinations and collected the blood samples. RAG carried out the SIV antibody assays. ADMEO and BB conceived of the study, and participated in its coordination.
Acknowledgements
The authors thank Wim van Est for generating Figure 1A.
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Takehisa J Harada Y Ndembi N Mboudjeka I Taniguchi Y Ngansop C Kuate S Zekeng L Ibuki K Shimada T Bikandou B Yamaguchi-Kabata Y Miura T Ikeda M Ichimura H Kaptue L Hayami M Natural infection of wild-born mandrills (Mandrillus sphinx) with two different types of simian immunodeficiency virus AIDS Res Hum Retroviruses 2001 17 1143 1154 11522184 10.1089/088922201316912754
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| 15516270 | PMC529309 | CC BY | 2021-01-04 16:36:37 | no | Retrovirology. 2004 Nov 1; 1:36 | utf-8 | Retrovirology | 2,004 | 10.1186/1742-4690-1-36 | oa_comm |
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J Transl MedJournal of Translational Medicine1479-5876BioMed Central London 1479-5876-2-331548557310.1186/1479-5876-2-33ResearchThe effect of chemotherapy combined with recombination mutant human tumor necrosis factor on advanced cancer Liu Xing [email protected] Xiang-fu [email protected] Zhi-weng [email protected] Huishan [email protected] Xinyuan [email protected] Changmin [email protected] Chuan [email protected] Guoxiang [email protected] Department of Oncology, Union Hospital of Fujian Medical University, Fujian, Fuzhou, 350001, P. R. China2004 14 10 2004 2 33 33 27 5 2004 14 10 2004 Copyright © 2004 Liu et al; licensee BioMed Central Ltd.2004Liu et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Past studies suggested that tumor necrosis factor (TNF) assisted anti-tumor treatment and intensified the sensitivity of chemotherapy. However its clinical application has been curbed because of its low purity, high dosage, and strong toxicity. This research, through perspective random clinical control experiment, observed the therapeutic effect of the treatment of late malignant tumor through the injection of recombinant mutant human tumor necrosis factor (rmhTNF) combined with general chemotherapy and its adverse reactions.
Methods
105 patients with advanced malignant tumor were randomly divided into trial group, 69 patients, and control group, 36 patients. Injection of rmhTNF 4 × 106u/m2 was given to the trial group, from the 1st to 7th days, the 11th to 17th days combined with chemotherapy course. The chemotherapy plan was as follows: CAP for patients with the NSCLC; FAM for patients with gastric cancer; FC for patients with colorectal cancer. One treatment cycle lasted for 21 days and two cycles were scheduled. The control group was given only the same chemotherapy as the trial group.
Results
In the trial group there was 1 CR case and 12 PR cases, and the response rate is 13/69 (18.84%); in the control group 1 PR case, the response rate 1/36 (2.78%). The response rate of the trial group was significantly higher than that of the control group (P = 0.022). The response rate for NSCLC in the trial group was 8/17 (47.06%), and 1/6 (16.67%) in the control group. The response rates for gastric cancer and colorectal cancer in the trial groups also were higher than those of the control groups. After the treatment the KPS is 89.00 ± 9.92 in the trial group, and 84.17 ± 8.84 in the control group, with a significant difference between the two groups (P = 0.028). The adverse reactions of rmhTNF injection included: pain in the injection area, chill, hardening and swelling and redness in the injection area, fever, ostealgia and myosalgia, and cold-like symptoms. All these adverse reactions were mild and bearable.
Conclusions
The administration of rmhTNF injection in combination with general chemotherapy is an effective and secure means in treating advanced malignant tumor.
TumorTumor necrosis factorBiological therapyChemotherapyComplex therapy
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Tumor necrosis factor (TNF) is a polypeptide produced by monocytic macrophages and T-lymphocytes stimulated by endotoxin[1]. It has been well shown in vivo or in vitro that TNF assists anti-tumor treatment and intensified the sensitivity of chemotherapy to many different kinds of tumor cells [2-5]. However its clinical application has been curbed because of its low purity, high dosage, and strong toxicity. Via the cooperation between the Forth Military Medical University and Shanghai Celstar Bio-pharmaceutical Holding Co. Ltd, an injection of recombinant mutant human tumor necrosis factor (rmhTNF) which is a genetic engineering TNF of a high activity and low toxicity, has been produced by rebuilding natural TNF with protein engineering technics. Phase I clinical trial (tolerance test) showed that the patients had good tolerance. and the toxicity of rmhTNF was slight As a participant of the Phase II and Phase III clinical study of rmhTNF, we observed the therapeutic effect of the treatment of late malignant tumor through the injection of recombinant mutant human tumor necrosis factor combined with general chemotherapy and its toxicity in a multi-center, random clinical control Phase II and Phase III clinical study of rmhTNF during October 2000 ~ May 2002. The results are reported as below.
1. Materials and Methods
1.1 Patients
105 patients from our department with advanced malignant tumors diagnosed via pathologic or cytological examinations were randomly collected, of which 23 were non-small cell lung cancer, 50 gastric cancers and 32 were colorectal cancers. Moreover, 79 of them were males and 26 of them were females. The range of their ages was 25~70, with the median age 52 years old. All of them were during their later phases of either recrudescent or metastatic cancers, had no sugary indexes and were taking conservative medicine treatment. KPS scoring for them before treatment were ≥ 60, neutrophil ≥ 2.0 × 109/L, PLT ≥ 100 × 109/L and Hb ≥ 90 g/L. They had almost normal functions of the heart, liver and kidney and at least had one evaluable tumor focus. They had not taken any other anti-tumor treatment one month before the experiment and they had a prospective survival time > 3 months. All of the patients were well informed and written consents were signed. Then they were randomly divided into two groups, i.e. the trial group of 69 patients and the control group of 36 patients, the conditions of the two groups were comparable (P >0.05, see Table 1). In the individual group of three tumors, the stage of the disease is of no significant difference (P >0.05, see Table 1).
Table 1 The General Data of the Trial and Control Group
Item Trial group (n = 69) Control group (n = 36) P
Sex 0.362
Male 50 29
Female 19 7
Median age (year) 52 51 0.437
Type of tumors 0.638*
NSCLC 17 6
Gastric cancer 32 18
Colorectal cancer 20 12
KPS () 85.31 ± 6.07 86.69 ± 7.81 0.334
Clinical stage 0.630
III 11 8
IV 58 28
NSCLC III 5 3 0.621*
IV 12 3
Gastric cancer III 4 3 0.684
IV 28 15
Colorectal cancer III 2 2 0.620*
IV 18 10
*The p value is calculated by Fisher exact chi-square because n < 30 or some cells have expected count less than 1.
1.2 Source of drugs
Injection of rmhTNF which was in powder form of 500 × 105u/tube (number 00061) was made and provided by the Forth Military Medical University and was stored at 4°C in refrigerator.
1.3 Treatment protocol
For the trial group, injection of rmhTNF 4 × 106u/m2 was given from the 1st to 7th days, the 11th to 17th days combined with chemotherapy course. The chemotherapy plan was as follows: CAP (CTX 750 mg/m2, d1, ADM 40 mg m2, d1, DDP 30 mg m2, d1-d3) for patients with the non-small cell lung cancer; FAM (5-FU500 mg/ m2, d1-d5, ADM 40 mg/ m2, d1, MMC 6 mg/ m2, d1) for patients with gastric cancer; FC (5-FU 500 mg/ m2, d1-d5, CF 100 mg/ m2, d1-d5) for patients with colorectal cancer. One treatment cycle lasted for 21 days and two cycles were scheduled. For the control group, only the same chemotherapy as the trial group was given. Signs, symptoms and adverse reactions were carefully observed during the treatment. Weekly examinations of blood routine were performed before and after treatment, while liver and kidney functions, urine routine, EEG, liver ultrasonic and chest X-ray examinations were performed before and after every treatment cycle. CT examinations of the evaluable tumor focuses were performed one time before treatment, when treatments were finished and after 4 weeks of the finish.
1.4 Evaluation of response and toxicity
1.4.1 Evaluation of response
Complete response (CR) is that, the disappearance of all lesions and no appearance of new disease for at least 4 weeks. Partial response (PR) is defined as a reduction by at least 50% in the sum of the products of the two longest diameters of all lesions maintained for at least 4 weeks with no appearance of new disease. Minimal response (MR) is different from PR with a reduction by at least 25%, but not more than 50%. Stable disease (SD) is a less than 25% reduction or less than 25% increase in the sum of the products of the two perpendicular diameters of all measured lesion with no appearance of new disease. Progression disease (PD) is that, an increase greater than 25% over the size present at entry into the study or, for patients who respond, the size at time of maximum regression, or the appearance of new areas of malignant disease. CR+PR were rated as response rate.
1.4.2 Toxicity
The toxicity was followed the WHO acute and sub-acute toxic rating
2. Results
2.1 Response to treatment
After two treatment cycles, the trial group had 1 CR case, 12 PR cases, 11 SD cases and 18 PD cases, and the response rate was 13/69(18.84%). The control group, on the other hand, had 1 PR case, 4 MR cases, 19 SD cases and 12 PD cases, and had a response rate 1/36 (2.78%). The response rate of the trial group was significantly higher than that of the control group (P = 0.022, see Table 2).
Table 2 The Response Rate in the Trial and Control Group after Two Treatment Cycles
Group n CR PR MR SD PD Response rate P
Trial 69 1 12 11 27 18 13/69 (18.84%) 0.022
Control 36 0 1 4 19 12 1/36 (2.78%)
The response rate for NSCLC of the trial group was 8/17 (47.06%), higher than that of the control group 1/6 (16.67%) but without statistic significance (P = 0.208). The response rates for gastric cancer was 12.50% (4/32), while for the controls were 0.00% (0/18) without statistic significance (P = 0.283). The response rates for colorectal cancer was 5.00% (1/20), while no response case in the controls group (0/12), but there was still no statistic significance (P = 1.000, see Table 3).
Table 3 The Response Rate in the Trial and Control Group for Different Type of Tumors
Group n CR PR MR SD PD Response rate p*
NSCLC Trial 17 1 7 3 4 2 47.06% (8/17) 0.208
Control 6 0 1 1 3 1 16.67% (1/6)
Gastric cancer Trial 32 0 4 6 13 9 12.50% (4/32) 0.283
Control 18 0 0 2 9 7 0.00 (0/18)
Colorectal cancer Trial 20 0 1 2 10 7 5.00% (1/20) 1.000
Control 12 0 0 1 7 4 0.00 (0/12)
*The p value is calculated by Fisher exact chi-square because n < 30 or some cells have expected count less than 1.
2.2 Life quality
Before the treatment there was no significant difference of the general status (KPS) between the two groups (P > 0.05). After the treatment the KPS was 89.00 ± 9.92 in the trial group, and 84.17 ± 8.84 in the control group, with a statistic significant difference (p = 0.028, see Table 4).
Table 4 Before and after Treatment, KPS Value in the Trial and Control Group
Group Trial (n = 69) Control (n = 36) P
Before treatment () 85.31 ± 6.07 86.69 ± 7.81 0.334
After treatment () 89.00 ± 9.92 84.17 ± 8.84 0.028
2.3 Toxicity
Fever, cold-like symptoms, ostealgia and myosalgia, chill, pain in the injection area, hardening and swelling and redness in the injection area were much more happened in the trail group compared with the control group (P < 0.01 = , while there were no significant differences between the two groups on the frequencies of anemia, leukopenia, thrombocytopia and nausea / vomiting (P > 0.05). No abnormalities correlated with drugs were found in liver or kidney functions, urine routine, EEG and blood pressure (see Table 5).
Table 5 Toxicity in the Trial and Control Group
Toxicity Trial (n = 69) Control (n = 36) P*
0 I II III IV 0 I II III IV
Anemia 37 16 8 7 1 21 9 4 1 1 0.665
Leukopenia 28 23 9 9 0 17 13 4 2 0 0.570
Thrombocytopia 59 5 2 2 1 31 3 1 1 0 0.557
Nausea / Vomiting 32 16 19 2 0 18 4 11 2 1 0.476
Fever 52 12 5 0 0 36 0 0 0 0 0.000
Eruption 67 2 0 0 0 36 0 0 0 0 0.274
Cold-like symptoms 47 19 3 0 0 36 0 0 0 0 0.000
Ostealgia / Myosalgia 46 21 2 0 0 36 0 0 0 0 0.000
Chill 38 31 0 0 0 36 0 0 0 0 0.000
Pain in injection area 13 41 15 0 0 36 0 0 0 0 0.000
Hardening, swelling and redness in injection area 42 17 10 0 0 36 0 0 0 0 0.000
*The p value is calculated by Fisher exact chi-square because n<30 or some cells have expected count less than 1.
3. Case report
A 54-year-old man hospitalized at August 27, 2001 with complains of "left chest pain accompanied by cough and hard breath for half a month". Physical examination after hospitalization showed: enlarged lymph node of 3 × 3 cm above the right clavicle, hard and immobile. Chest CT on September 27, 2001 (see Figure 1) showed: conglomeration of a size of 5.5 × 4.2 cm at the left lower hilus pulmonis, large amount of accumulation of fluid in the left thoracic cavity, enlarged lymph nodes in the mediastinum. Biopsy of the lymph node above the right clavicle showed: transferred adenocarcinoma. Cancer cells were found in the fluid in the thoracic cavity after centrifugation. The diagnosis was "Adenocarcinoma on the left lower lung, stage T4N3M0IIIb". Chemotherapy of protocol CAP + rmhT NF injection (i.m.) was given from October 4 to November 14, 2001. Two weeks later, a clear relief of hard breath and cough was found. After two periods of therapy (November 16, 2001), physical examination showed shrinkage of lymph node of 0.5 × 0.5 cm above the right clavicle, and Chest CT (see Figure 2) showed: clear shrinkage of conglomeration of 3.0 × 2.5 cm at the left lower hilus pulmonis, small amount of accumulation of fluid in the left thoracic cavity. A callback of CT one month later showed: conglomeration was of the size of 4.0 × 2.8 cm. The curative effect was confirmed as "PR".
Figure 1 Chest CT before treatment (27-Sep-2001) show that conglomeration of a size of 5.5 × 4.2 cm at the left lower hilus pulmonis, large amount of accumulation of fluid in the left thoracic cavity, enlarged lymph nodes in the mediastinum.
Figure 2 Chest CT after treatment (16-Nov-2001) show that clear shrinkage of conglomeration of 3.0 × 2.5 cm at the left lower hilus pulmonis, small amount of accumulation of fluid in the left thoracic cavity.
4. Discussion
Tumor necrosis factor (TNF) is a polypeptide produced by monocytic macrophages and T-lymphocytes stimulated by endotoxin. It can act as modulator to immunity and induces anti-tumor effects in hosts. It also has direct cytotoxic effects and inhibitory effects on cellular growth. It can kill the tumor cell without notable toxic effect on the normal cells [1]. Studies have shown that the anti-tumor mechanism of the tumor necrosis factor includes 1) killing the tumor cell directly [3]; 2 inducing the apoptosis of tumor cells [3]; 3) reversing more drug resistance of tumor cell and improving the sensitiveness of chemotherapy [3]; 4) destroying the blood supply of tumor tissue [4]; 5) increasing the killing effects of immune-effect cells on the tumor cells [3]. However, its clinical application has been curbed because of its low purity, high dosage, and strong toxicity. Studies have also shown that, a higher anti-tumor effect and lower toxicity were got by modified some structure of TNF. Nakamura [6] prepared a novel recombinant tumor necrosis factor-α (TNF) mutant (mutant 471), in which 7 N-terminal amino-acids were deleted and Pro8Ser9Asp10 was replaced by Arg-Lys-Arg, and compared its biological activity with that of wild-type recombinant TNF. Mutant 471 had a 7-fold higher anti-tumor activity against murine L-M cells in vitro, and a higher binding activity to TNF receptors on L-M cells, than wild-type TNF. Kamijo[7] reported that TNF(C-Phe), in which the C-terminal leucine of TNF molecule was replaced by phenylalanine, was 20-times as potent in induction of differentiation of human myelogenous leukemia cells (U-937 cells) as the parent TNF(N-Met). The rmhTNF has been obtained successfully from hTNF-α by gene engineering technology which was mutated by deleting 7 amino acids at the N-terminus, replacing Pro8Ser9Asp10 by Arg-Lys-Arg, and substituting Leu157 with Phe [8]. Phrase I clinical trial indicated that, all of 32 patients who were randomly grouped into six groups were treated with different dose: 2.5 × 105u/m2, 5 × 105u/m2, 1 × 106u/m2, 2 × 106u/m2, 3 × 106u/m2, 4 × 106u/m2, were well tolerable.
The present study has shown that shortly after the combined chemotherapy with rmhTNF, 24/69 (34.78%) of the focuses were more or less absorbed or subsidized, of which there were 1 CR case and 12 PR cases, with the response rate was 13/69 (18.84%), while in the control group only 5/36 (13.89%) cases had reduced focuses with only 1 PR case and the response rate 1/36 (2.78%). The response rate of the trial group was significantly higher than that of the control group (P = 0.022). Analysis of the therapeutic effects in different kind of diseases showed that the response rate for NSCLC of the trial group was 8/17 (47.06%), higher than that of the control group 1/6 (16.67%), which also means that TNFα has suppressive effects on lung adenocarcinoma[9]. The reason why there was no statistic significance (P > 0.05) between the two groups may be due to too small samples in this investigation. The response rates for gastric cancer and the colorectal cancer were both quite low, i.e. 4/32 (12.50%) and 1/20 (5.00%), respectively, while for the controls were 0/18 and 0/12, respectively (0.00%) and without statistic significance (P > 0.05). Such a result may be mainly due to the fact that most of the patients had taken combined chemotherapy before and that samples in both groups were quite small. It was thought [2] that the anti-tumor activity of TNF may co-activate some chemotherapy drugs. Results of the present study showed that the combined therapy of TNF and chemotherapy may be still effective on previously chemotherapy drug resistant tumors, which also means that TNF has co-activating effects on chemotherapy drugs. Moreover, after two treatment cycles, scoring for the general status (KPS) was 89.00 ± 9.92 in the trial group and 84.17 ± 8.84 in the control group, with a statistic significant difference (P = 0.028). It shows that the combined rmhTNF chemotherapy may benefit the quality of the patients' life.
It has already been reported[2-5,10,11] that the toxicity of TNF in phase I or II clinical trials are mainly chill, fever, local redness, swelling and pain, hypotension, nausea, vomiting, myalgia, fatigue and diarrhea, while pulmonary hemorrhage and severe hepatic dysfunction also have been observed[12]. But there are no reports on the toxicity of rmhTNF. The present study showed that some of the patients had pain in the injection area, chill and hardening, swelling and redness in the injection area of which the incidences were 81.2%(56/69), 44.9%(31/69) and 39.1% (27/69), respectively, but of a low degree and the patients had good tolerance. The symptoms of fever, ostealgia and myosalgia and cold-like symptoms were more in the trail group than that in the control group, all of which were of grade I or II. They mainly happened when the drugs were given at the 3rd or 5th times, and can be relieved after taking 25 mg metacen, and can disappear automatically after the finish of the therapy. There were no significant differences between the two groups on the number of cases which showed marrow suppression or nausea / vomiting, and on the degree of those symptoms. There were no cases which showed rmhTNF-correlated abnormal liver or kidney functions, urine routine, EEG and blood pressure.
In summary, combined therapy of rmhTNF and chemotherapy has co-activating and sensitivity improving effects on the treatment of advanced malignant tumors, and may increase the recent responsive effectiveness with an improvement of the general status and quality of patients' lives. The main adverse reactions of the local injection of rmhTNF are pain in the local injection area, chill, hardening and swelling and redness in the injection area, fever, ostealgia and myosalgia and cold-like symptoms, all of which were of light to moderate degree and are tolerable.
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| 15485573 | PMC529310 | CC BY | 2021-01-04 16:39:24 | no | J Transl Med. 2004 Oct 14; 2:33 | utf-8 | J Transl Med | 2,004 | 10.1186/1479-5876-2-33 | oa_comm |
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J NeuroinflammationJournal of Neuroinflammation1742-2094BioMed Central London 1742-2094-1-181547947410.1186/1742-2094-1-18ReviewUsing animal models to determine the significance of complement activation in Alzheimer's disease Loeffler David A [email protected] Department of Neurology, William Beaumont Hospital Research Institute, Royal Oak, MI 48073, USA2004 12 10 2004 1 18 18 5 8 2004 12 10 2004 Copyright © 2004 Loeffler; licensee BioMed Central Ltd.2004Loeffler; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Complement inflammation is a major inflammatory mechanism whose function is to promote the removal of microorganisms and the processing of immune complexes. Numerous studies have provided evidence for an increase in this process in areas of pathology in the Alzheimer's disease (AD) brain. Because complement activation proteins have been demonstrated in vitro to exert both neuroprotective and neurotoxic effects, the significance of this process in the development and progression of AD is unclear. Studies in animal models of AD, in which brain complement activation can be experimentally altered, should be of value for clarifying this issue. However, surprisingly little is known about complement activation in the transgenic animal models that are popular for studying this disorder. An optimal animal model for studying the significance of complement activation on Alzheimer's – related neuropathology should have complete complement activation associated with senile plaques, neurofibrillary tangles (if present), and dystrophic neurites. Other desirable features include both classical and alternative pathway activation, increased neuronal synthesis of native complement proteins, and evidence for an increase in complement activation prior to the development of extensive pathology. In order to determine the suitability of different animal models for studying the role of complement activation in AD, the extent of complement activation and its association with neuropathology in these models must be understood.
Alzheimer's diseaseanimal modelscomplement activationtransgenic mice
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Background
Alzheimer's disease and complement activation
A variety of inflammatory processes are increased in regions of pathology in the Alzheimer's disease (AD) brain [1-4]. There is a reciprocal relationship between this local inflammation and senile plaques (SPs) and neurofibrillary tangles (NFTs); both SPs and NFTs, as well as damaged neurons and neurites, stimulate inflammatory responses [5], and inflammatory processes exert multiple effects, some of which promote neuropathology [6-8]. Numerous retrospective studies have shown that long-term administration of nonsteroidal anti-inflammatory drugs (NSAIDs) to individuals with arthritis significantly reduces the risk for these individuals for developing AD [9]. These findings, together with the demonstration of elevated glial cell activation [10-12], complement activation [13-15], and increased acute phase reactant production [16-19] at sites of pathology in the AD brain, support the hypothesis that local inflammation may contribute to the development of this disorder [20]. Although a short-term trial of AD patients with the NSAID indomethacin suggested protection from cognitive decline [21], subsequent trials with other anti-inflammatory drugs have found no evidence for slowing of the dementing process [22-25]. These findings underscore the current perception of CNS inflammation as a "double edged sword" [26,27], with neuroprotective roles for some inflammatory components and neurotoxic effects for others [28-30].
The significance of complement activation, a major inflammatory mechanism, in AD is particularly problematic. The complement system is composed of more than 30 plasma and membrane-associated proteins which function as an inflammatory cascade. Complement activation promotes the removal of microorganisms and the processing of immune complexes. The liver is the main source of these proteins in peripheral blood, but they are also synthesized in other organs including the brain [31]. Protein fragments generated during activation of the system enzymatically cleave the next protein in the sequence, generating a variety of "activation proteins" with diverse activities (Table 1). Three complement pathways, the classical, alternative, and lectin-mediated cascades, have been identified (Fig. 1). Full activation results in the generation of C5b-9, the "membrane attack complex" (MAC), which penetrates the surface membrane of susceptible cells on which it is deposited and may result in cell death if present in sufficient concentration. The presence of early complement activation proteins [32-37] and of the MAC [38-42] has been demonstrated by immunocytochemical staining in the AD brain. Subsequent studies found that complement activation increases Aβ aggregation [43,44] and potentiates its neurotoxicity [45], attracts microglia [46,47], promotes microglial and macrophage secretion of inflammatory cytokines [48,49], and induces neuronal injury, and sometimes neuronal death, via the MAC [50]. These findings suggested that complement activation might contribute to the neurodegenerative process in AD. However, recent studies have also revealed neuroprotective functions for some complement activation proteins, including in vitro protection against excitotoxicity [51,52] and Aβ-induced neurotoxicity [53], as well as anti-apoptotic effects [54,55]. Further, C1q, the first complement protein to be deposited on cell membranes during activation of the classical complement sequence, may facilitate the clearance of Aβ by microglia [56], although this is controversial [57]. Understanding the role of complement activation in AD is of clinical relevance because some complement-inhibiting drugs are available, and others are being developed (see reviews by Sahu and Lambris [58], and Morgan and Harris [59]). Conditions for which these agents are currently being investigated include stroke [60], organ transplantation [61], glomerulonephritis [62], ischemic cardiomyopathy [63], and hereditary angioedema [64]. Modulation of CNS complement activation in experimental animal models of AD, both by treatment with complement-inhibiting drugs and by generation of AD-type pathology in complement-deficient animals, should be useful for obtaining a greater understanding of the role of this process in the development of AD-type pathology. Unfortunately, knowledge of the extent of complement activation in animal models is lacking. This paper will review (a) criteria for an optimal animal model to study this issue, (b) present knowledge about complement activation in animal models of AD, and (c) additional animal models which offer alternatives for addressing this question.
Table 1 Biological activities of complement activation proteins, with relevance to AD.
Name Biological activity
C1q Enhances Aβ aggregation [43,44]; may facilitate Aβ clearance [56]; enhances Aβ-induced cytokine secretion by microglia [49]
C3a Anaphylatoxin (increases capillary permeability) [155] ; protects neurons vs. excitotoxicity [52]
C3b Immune adherence and opsonization [89] (may facilitate Aβ clearance by phagocytic microglia)
C4a Anaphylatoxin (weak) [156]
C5a Anaphylatoxin; protects neurons vs. excitotoxicity [51]; chemotaxic attraction of microglia [46,47]; inhibits apoptosis 54; increases cytokine release from Aβ-primed monocytes [48]
C5b-9 Neurotoxicity [50]; sublytic concentrations may have both pro- and anti- inflammatory activities [157]
Figure 1 Schematic diagram of classical, alternative, and lectin complement activation pathways. There is evidence for activation of the classical and alternative pathways in the AD brain. (Adapted from Sahu and Lambris, 2000 [58]).
Criteria for an optimal animal model for studying AD-related complement activation
While animal models of human disease generally have similar pathological findings to the human disorders, distinct differences remain. These models may be appropriate for studying some aspects of a disease process, while less suitable for others. To determine the significance of complement activation in the development of AD-type pathology, for example, some animal models may be of value primarily for investigating the relationship between early complement activation and SP and NFT formation, whereas others may be more relevant for studying the role of the MAC in neuronal loss.
1. Complete activation of complement
Investigators at the Academic Hospital Free University in Amsterdam first reported the presence of early activation proteins in the classical complement cascade in the AD brain [32-34,36,37]. The MAC was not detected. However, further studies by other laboratories convincingly demonstrated the MAC, by a variety of techniques, in AD specimens [38-42]. The Dutch group has more recently reported detection of the MAC in brain specimens from subjects with dementia with Lewy bodies who met CERAD neuropathological criteria for AD [65]. The MAC has similarly been reported in SPs from subjects with Down's syndrome [66] and with familial British dementia [67], disorders in which typical AD-type neuropathology is present. An optimal animal model for studying AD-related complement activation should therefore have complete complement activation.
2. Association of complement activation proteins with neuropathology
Complement proteins are detectable on or closely associated with SPs, NFTs, and dystrophic neurites in the AD brain. These findings are in agreement with in vitro studies indicating that Aβ and tau protein, the major components in SPs and NFTs, can fully activate human complement [42,68-71]. Although the above studies suggested that complement is activated principally by the aggregated forms of Aβ and tau, soluble, non-fibrillar Aβ may also be capable of activating complement [72]. In contrast to the robust staining of complement proteins in mature plaques, immunoreactivity to these proteins in diffuse plaques has generally been below the level of detection, though it has been reported in some studies [36,73,74]. Complement activation in the AD brain is increased primarily in regions containing extensive pathology (e.g., the hippocampus and cortex), and whether early complement components are also present in the diffuse plaques that develop in the AD cerebellum is controversial [74,75]. The above findings suggest that complement activation in an optimal animal model of AD should be associated with SPs and, in those models in which neurofibrillary pathology occurs, with NFTs.
3. Initiation of complement activation early in development of pathology
How the increased complement activation in AD relates to the development of SPs and NFTs, and to neuronal loss, is unclear. Immunocytochemical staining for complement activation proteins in the aged normal human brain is generally faint, and may be below the level of detection [42,69,73]; of relevance is a recent report describing extensive neuron-associated C1q reactivity in a cognitively normal subject with neuropathological findings limited to diffuse cortical plaques [76]. Elderly "high pathology controls," lacking dementia but with increased numbers of entorhinal NFTs and neocortical Aβ deposits, have a slight increase in the percentage of C5b-9-immunoreactive plaques in comparison with aged normal subjects, though this percentage is far lower than in the AD brain [39]. A recent study in our laboratory [77] used enzyme-linked immunosorbent assay (ELISA) to measure the concentrations of two early complement activation proteins, C4d and iC3b, in brain specimens from AD and normal subjects. ELISA is more sensitive than immunocytochemical staining, though it provides no information regarding the cellular association of complement immunoreactivity. Increased concentrations of these early complement activation proteins were present in some aged normal specimens. These reports suggest that early complement activation may increase prior to the development of plaques and NFTs. Similar findings are desirable in an optimal animal model for studying AD-related complement activation.
4. Increased CNS production of native complement proteins
Both mRNA expression and protein synthesis of native complement proteins are increased in the AD brain [78-80]. (Note: the distinction between detection of native complement proteins, vs. detection of complement activation proteins, has frequently been blurred. In some studies in which immunoreactivity to complement activation proteins (C3c, C4c, C4d) has been reported, the antisera used were also capable of detecting the respective native complement proteins (C3 or C4) [40,80]. Only when antisera are used whose immunoreactivity is limited to activation-specific neo-epitopes can complement activation be confirmed. The paucity of antisera which can detect complement activation proteins in experimental animal models is a significant obstacle to determining the extent of complement activation in these models.) In addition to neurons, complement proteins are synthesized by other cells in the CNS including microglia, astrocytes, oligodendrocytes, and endothelial cells [31]. The biological effects of these activation proteins are mediated by numerous regulatory proteins including CD59, clusterin, vitronectin, C1-inhibitor, C4-binding protein, decay-activating factor, and Factor H, which inhibit different steps in the complement cascade. All of these regulatory proteins are produced in the human brain, but less is known about their CNS synthesis in other species [31]. The status of some of these regulatory proteins in AD is unclear; for example, there are conflicting reports regarding the up-regulation of C1-inhibitor [81,82] and CD59 [41,82,83]. Thus, while an optimal animal model for studying AD-related complement activation should have up-regulated CNS synthesis of complement proteins, the alterations that should be present in complement regulatory proteins are less clear.
5. Alternative as well as classical complement activation
Complement activation in the AD brain was initially thought to be limited to the classical pathway, but recent reports have also indicated increased concentrations of the alternative activation factors Bb and Ba, and Factor H, a regulatory factor for the alternative pathway, in the AD brain [84,85]. Alternative complement activation has also been reported in other familial dementias with pathologies similar to AD [67]. Therefore, while activation of the classical pathway is an absolute requirement for an optimal animal model of AD-related complement activation, an increase in the alternative pathway is also desirable.
Complement activation in animal models of AD: present knowledge
The examination of complement activation in experimental models of AD has been limited to mice and rats. The extent of complement activation and its relationship to the development of AD-type neuropathology have generally not been determined in these studies.
APP/sCrry mouse
Increased complement activation was induced by overproduction of transforming growth factor beta1 (TGF-β1) in transgenic mice expressing mutations in the human amyloid precursor protein (hAPP) gene. The APP mutations expressed in these mice have been associated with early-onset, familial AD [86]. The TGF-β1 overproduction resulted in a 50% reduction in Aβ accumulation in the hippocampus and cerebral cortex [87]. Because the production of soluble Aβ was unchanged, these results suggested that reduction in Aβ may have been due to its increased clearance by microglia. A subsequent study by the same investigators [88] found that the mRNA level of C3 in the cerebral cortex was 5-fold higher in APP/TGF-β1 mice than in APP mice at 2 months of age (prior to deposition of Aβ) and 2-fold higher at 12–15 months, when senile plaques are present. Thus, in this model, increased CNS synthesis of C3 precedes senile plaque formation. Because C3b, an activation protein produced by cleavage of C3, functions as an opsonin [89], the increased C3 levels together with the reduced Aβ deposition in the APP/TGF-β1 mice suggested a neuroprotective role for complement in this model. To investigate this possibility, the APP mice were crossed with mice expressing soluble complement receptor-related protein y (sCrry), a rodent-specific inhibitor of early complement activation [90]. APP/sCrry mice had a 2- to 3- fold increase in Aβ deposition in the neocortex and hippocampus at 10–12 months of age, together with a 50% loss of pyramidal neurons in hippocampal region CA3. The authors concluded that complement activation may protect against Aβ-induced toxicity, and may reduce the accumulation or promote the clearance of amyloid and degenerating neurons [88]. Neuroprotective functions (protection against excitotoxicity) have been demonstrated in vitro for C3a [52], and the increased neuronal loss in the APP/sCrry mouse may be due to decreased production of C3a as well as the opsonin, C3b. However, whether inhibition of complement activation in the AD brain would similarly result in increased neuropathology is unclear, because complement activation in AD is likely to be more extensive than in the APP mouse. Although no peer-reviewed articles have appeared in which the extent of complement activation in the APP mouse has been examined, two abstracts have dealt with this issue. Yu et al. [91] reported C3, C5, and C6 immunoreactivity to thioflavin-S-reactive plaques, whereas McGeer et al. [92] found only weak complement staining of plaques and slight upregulation of complement proteins. Significantly, neither study reported detection of the MAC. At least two factors, in addition to the lack of NFTs, mitigate against complement activation in the APP mouse being equivalent to that in AD: (a) the mouse complement system is functionally deficient, as mouse C4 lacks C5 convertase activity [93] and many mouse strains have low complement levels relative to other mammals [94], and (b) mouse C1q binds less efficiently to human Aβ than does human C1q, resulting in less activation of mouse complement than of human complement in the presence of human Aβ [95].
PS/APP mouse
In addition to APP, mutations in the gene encoding for presenilin-1 (PS-1) have also been associated with familial AD [96]. The PS/APP mouse carries both of these transgenes and has been extensively used as a model for studying processes relating to the formation of SPs. Aβ deposition occurs more rapidly in these mice than in the single transgenic APP mouse [97]. In neither model does NFT formation occur. Aβ deposition in PS/APP mice is initially detected at 3 months of age, and increases with age; total Aβ burden peaks at one year of age, although the percentage of Aβ that is fibrillar (thioflavin-S reactive) increases up to 2 years of age. Matsuoka et al. [98] described the CNS inflammatory response to Aβ in these animals. Activated astrocytes and microglia increased in parallel with total Aβ and were closely associated with both diffuse and fibrillar plaques. C1q immunoreactivity was detected at both 7 and 12 months of age, co-localizing with activated microglia and fibrillar Aβ. These findings were similar to those in the AD brain in that complement activation was associated with SP formation. The extent of complement activation was not addressed in this study.
APP (Tg2576)/C1q-deficient mouse
Fonseca et al. [99] investigated the role of C1q in AD by crossing Tg2576 (APP) mice [100] and APP/PS1 mice with C1q knockout mice [101]. C1q immunoreactivity was associated with plaque formation in the APP Tg2576 animals, as previously reported by Matsuoka et al. [98]. In both the Tg2576/C1q- and APP/PS1/C1q- animals, lack of C1q did not alter either plaque density or the time course of plaque deposition. Neuronal cell numbers (NeuN+ cells), assessed only in the Tg2576 (APP) mouse, were not changed by the absence of C1q; however, immunoreactivity to MAP-2 (a marker for neuronal dendrites and cell bodies) and synaptophysin (a marker for presynaptic terminals) in the hippocampus (region CA3) was increased 2-fold in the APP/C1q- animals, compared with APP mice. Microglial and astrocytic activation was significantly reduced in the APP/C1q- animals. These results were interpreted to suggest that in these animal models of AD, (1) early complement activation (as indicated by C1q deposition) in response to fibrillar Aβ deposition might be responsible for the chemotactic attraction of activated glial cells, and (2) the activated microglia, while unable to clear fibrillar Aβ, may have contributed to the loss of neuronal integrity indicated by reduced MAP-2 and synaptophysin staining in the APP mice. By recruiting activated microglia, complement activation could potentially contribute to neuronal injury even if full activation (MAC formation) does not occur.
Postischemic hyperthermic rat model
Coimbra and colleagues [102] described progressive neuronal loss in the hippocampus and cerebral cortex in rats subjected to common carotid artery occlusion to produce transient forebrain ischemia, as an animal model for stroke. The post-surgical hyperthermia which occurs spontaneously in these animals was suggested to promote the infiltration of microglia, whose secretory products increased the subsequent neuronal loss. A later study by the same group [103] found that subjecting the rats to post-surgical hyperthermia (38.5 – 40°C) increased microglial and astrocytic infiltration and accompanying neuronal loss, and resulted in the formation of AD-type pathology. Aβ-reactive diffuse plaques were detected in the cerebral cortex at 2 months post-surgery, with more compact plaques in the hippocampus and cortex by 6 months. Increased ubiquitin and phosphorylated tau immunoreactivity was observed at both time points, together with staining for C5b-9 in the somatosensory cortex. The MAC immunoreactivity co-localized with acid fuchsin staining, a marker for neuronal death [104]. Other complement proteins were not evaluated in these studies. This is apparently the only animal model of AD in which full complement activation has been reported. It is noteworthy that while both SPs and neurofibrillary pathology were present in these animals, the MAC apparently did not co-localize with these structures, unlike in AD.
Acute lesioning
Alterations in native complement mRNA and protein levels have been evaluated in the rat hippocampus following experimental induction of acute neuronal injury. These surgical and pharmacological procedures result in neuronal loss in the entorhinal cortex, and deafferentation of hippocampal neurons, similar to that which occurs in AD [105]. Selective damage to the rat hippocampus has been induced by surgical transection of the perforant pathway, which runs between the entorhinal cortex and the molecular layer of the dentate gyrus [106,107], systemic administration of the excitotoxin kainic acid [108,109], or injection of the neurotoxin colchicine into the dorsal hippocampus [109]. Surgical transection of the perforant pathway increased C1qB mRNA in the entorhinal cortex and hippocampus [106] and C9 immunoreactivity in the hippocampus [107]. Injection of kainic acid similarly increased C1qB and C4 mRNA expression and C1q immunoreactivity in the hippocampus [108,109]. Colchicine infusion into the dorsal hippocampus, which selectively damages granule cells of the dentate gyrus, produced elevated mRNA expression of hippocampal C1qB and C4 [109]. Though the acute neuronal damage in these studies differs from the chronic, progressive neurodegenerative process that occurs in AD, these results demonstrated that the neuronal response to injury includes upregulation of native complement protein synthesis. The significance of this upregulation, i.e. whether it promotes neuroprotection or neurotoxicity, was not addressed.
Infusion of Aβ and C1q into rats
Frautschy et al. [56] examined the effects of infusion of human C1q and oral administration of rosmarinic acid on glial cell proliferation (microgliosis and astrocytosis), plaque load, and memory (Morris water maze) in Aβ-infused rats. Rosmarinic acid inhibits both the classical and the alternative complement cascades, by covalent binding to newly formed C3b [110]; it also possesses anti-inflammatory [111,112], anti-oxidative [113], and anti-amyloidogenic properties [114]. Gliosis was greater with C1q and Aβ infusion than with Aβ alone. Plaque density was decreased by C1q infusion (note: this result differs from the in vitro study of Webster et al. [57], in which C1q was found to inhibit microglial phagocytosis of Aβ, and also from the recent study of Fonseca et al. [99] in which C1q deficiency had no effect on plaque density in APP mice), but, curiously, performance in the water maze worsened. Treatment with rosmarinic acid had the opposite effect; though plaque load increased, memory was improved. These findings were interpreted as suggesting that C1q and/or complement activation may, by promoting microglial activation, worsen memory independent of the clearance of Aβ.
Additional animal models for studying AD-related complement activation
TAPP and 3xTg-AD mice
Mutations in the gene encoding for human tau protein have been linked to the development of frontotemporal dementia with parkinsonism [115]. By combining this mutation with the human APP and PS1 mutations associated with familial AD, animal models of AD have been produced in which NFTs as well as SPs are formed. Lewis et al. [116] crossed human APPswe mice (Tg2576) with mice expressing the transgene for a human tau mutation (JNPL3 mice) to generate a double mutant tau/APP mouse (the "TAPP mouse"). These mice develop SPs similar to APP mice (high numbers of plaques are present in older [8.5–15 months of age] mice, in the olfactory cortex, cingulate gyrus, amygdala, entorhinal cortex, and hippocampus), and older TAPP mice have NFTs, in association with increased astrocyte proliferation, in limbic areas. The plaques contain both Aβ40 and Aβ42. Oddo et al. [117] injected the human transgenes for APP and mutated tau into embryos of PS1 "knock-in" mice, generating the "3xTg-AD" mouse which develops both SPs and NFTs in an age-related, region-specific manner. Aβ deposition in these animals precedes NFT formation, with extracellular Aβ (primarily Aβ42) detected in the frontal cortex by 6 months of age, and in other cortical regions and hippocampus by 12 months. Many of the extracellular Aβ deposits are thioflavin-S-positive and are associated with reactive astrocytes. Phosphorylated tau initially appears in the hippocampus and subsequently in cortical regions; it is detected within neurons by 12–15 months and within dystrophic neurites at 18 months. Though Aβ immunoreactivity precedes that of tau, these proteins co-localize to the same neurons. The presence of NFTs as well as SPs suggests that the 3xTg-AD and TAPP models may be more relevant than APP or APP/PS-1 mice for studying the significance of complement activation in the development of AD-type pathology. Potential drawbacks for using these models for complement-related studies include, as discussed earlier, functional deficiencies in activation of mouse complement [93], decreased complement levels in common laboratory mouse strains [94], and the decreased efficiency of binding of mouse C1q by the human Aβ within the SPs in these animals [95]. It is not known whether a similar decrease in the efficiency of activation of mouse complement occurs when mouse C1q binds to human, rather than murine, tau protein.
AD11 (anti-NGF) mouse
Ruberti et al. [118] developed a mouse transgenic model, the AD11 mouse, in which neutralizing antibody to nerve growth factor (NGF) is secreted by neurons and glial cells. NGF exerts trophic effects on basal forebrain cholinergic neurons and is widely distributed in these neurons [119]; the local secretion of anti-NGF antibody in these mice results in marked loss of basal forebrain cholinergic neurons. Aβ-containing plaques, tau hyperphosphorylation, and NFTs are present at 15–18 months of age. CNS production of anti-NGF antibody increases with age in these animals, therefore pathology develops only in adult mice. Extracellular deposition of APP is widespread in the brain, including the cortex and hippocampus. Phosphorylated tau immunoreactivity is present in neurons and glia in the cortex and hippocampus, and intracellular NFTs, extracellular neurofibrillary deposits, neuropil threads, and dystrophic neurites are observed in the cortex. Behavioral abnormalities, including impaired object recognition and spatial learning, are associated with this neuropathology [120]. The Aβ-containing plaques in the AD11 mouse are of murine, rather than human, origin, allowing the problem of the poor efficiency of activation of mouse complement by human Aβ [95] to be overcome. However, it is unclear whether plaques in these animals contain Aβ in the β-pleated sheet conformation, which is thought to be the most effective conformation for activating complement [71]. The distribution of SPs and NFTs in this model is less similar to AD than for 3xTg-AD and TAPP mice, because in addition to the cortex and hippocampus, large numbers of APP-reactive structures are present in the neostriatum (where, in AD, plaques are primarily diffuse [121]), and in other areas of the brain. Despite these concerns, the AD11 mouse is attractive as a potential model for studying the significance of AD-related complement activation.
Chlamydia pneumoniae-infected mouse
C. pneumoniae is an intracellular, gram-negative or gram-variable bacterium long identified as a respiratory pathogen. It has more recently been demonstrated to be a causative agent in reactive arthritis [122] and to be associated with autoimmune disorders including multiple sclerosis [123] and atherosclerosis [124]. Some laboratories have also reported an association of this agent with AD [125-127], although this has not been confirmed by others [128-131]. A recent study by Little et al. [132] examined the hypothesis that experimental C. pneumoniae infection in BALB/c mice could produce AD-like pathology. Intranasal inoculation with C. pneumoniae resulted in deposition of Aβ1–42 in the hippocampus, amygdala, entorhinal cortex, perirhinal cortex, and thalamus by 3 months post-inoculation. The majority of these Aβ deposits appeared similar to diffuse plaques, though a small number of them were thioflavin-S-reactive. NFTs were not detected. The authors suggested that soluble factors such as lipopolysaccharides, which are present in the cell wall of all Chlamydiae [133], may have been responsible for the altered amyloid processing which resulted in Aβ deposition. Because the Aβ within the SPs in these animals is of endogenous origin, and because other chlamydial species have been shown to activate complement [134,135], the C. pneumoniae-infected mouse may offer a novel infectious model for studying the relationship of complement activation to the development of Aβ-containing plaques.
Aged dogs
Old dogs, in particular the beagle, have been extensively investigated as a model for CNS Aβ deposition and associated age-related cognitive dysfunction. Aβ deposits are detectable in the brains of most older dogs [136]. The regional distribution of Aβ in the dog brain resembles that in humans, found initially in the prefrontal cortex, subsequently in entorhinal and parietal cortices, and lastly in occipital cortex [137]. Aβ42 is the predominant type of Aβ deposited in plaques [138]. Canine plaques are nonfibrillar and do not contain neuritic elements; thus, they resemble diffuse Aβ deposits in the human brain, but not the mature plaques predominating in AD. The neuropathological findings in old dogs also differ from AD in that activated glial cells are rarely associated with Aβ deposits, and NFTs are not detected [136,139]. Age-related cognitive impairment, termed "canine cognitive dysfunction syndrome," occurs in some older dogs and correlates with Aβ deposition in the hippocampus and frontal cortex [140,141]. The endogenous nature of the deposited Aβ in old dog brain, and similarities between canine and human Aβ in their patterns of regional deposition, suggest that this model may be useful for studying the relationship between complement activation and plaque formation.
Non-human primates
Age-related formation of SPs has been reported in a variety of non-human primates including the cynomolgus monkey [142], rhesus monkey [143], chimpanzee [144], and marmoset [145]. Aβ within these plaques is predominantly Aβ40 [146]. NFTs apparently do not form in the brains of most aged primates, with a few exceptions. The brain of the aged baboon contains phosphorylated tau protein [147,148], and an age-related accumulation of tau also occurs in the neocortex of the mouse lemur [149-151]. In this latter species, Aβ deposition occurs in the cerebral cortex and amygdala but is not age-dependent [151]. The mouse lemur appears to be the most promising primate species to date for studying the significance of AD-related complement activation because of the presence of NFTs as well as plaques.
Other animal species
Scattered reports of AD-type pathology in other species have also appeared. Adding trace amounts of copper to the water supply of cholesterol-fed rabbits results in Aβ deposition within SP-like structures in the hippocampus and temporal cortex, with associated learning deficits [152]. The neuropathology in the aged cat is similar to that in the old dog in that Aβ is deposited only as diffuse, Aβ42-containing plaques, and NFTs are not detected [138]. A report of AD-type pathology in an aged wolverine [153] described neuritic as well as diffuse plaques in the cortex and hippocampus, and intracellular NFTs containing phosphorylated tau protein in cortical and hippocampal neurons. Finally, the aged polar bear brain also contains both diffuse plaques and NFTs [154]. While the neuropathological findings in the aged wolverine and polar bear resemble AD more closely than in most species examined to date, their inaccessibility to laboratory researchers limits the usefulness of these species for studies of AD-related complement activation.
Conclusions
1. Complement activation has been extensively studied in the AD brain. There is convincing evidence for activation of both the classical and alternative pathways, resulting in full activation as indicated by the presence of the MAC. Both aggregated Aβ (in SPs) and phosphorylated tau (in NFTs) are likely to be responsible for this activation.
2. Because complement activation generates both both neuroprotective and neurotoxic effects, the significance of increased complement activation in the development and progression of AD is unclear.
3. An optimal animal model for studying the significance of complement activation in the development of AD-type pathology would have complete activation of this process, with co-localization of complement activation proteins with SPs and with NFTs (if present). Other desirable features include early complement activation prior to the development of extensive neuropathology, increased CNS production of native complement proteins, and both classical and alternative pathway activation.
4. Surprisingly little is known about the extent of complement activation in animal models of AD. The postischemic hyperthermic rat [103] is the only animal model of AD in which full complement activation has been reported. The few studies with APP-transgenic mice have yielded conflicting results, with one investigation suggesting a neuroprotective role for complement activation [88], while another found that early complement activation (as indicated by C1q deposition) was associated with a loss of neuronal integrity [99]. Transgenic mouse models may be problematic for studies of AD-related complement activation because of inherent deficiencies in mouse complement activation and inefficient activation of mouse complement by the human Aβ present in the SPs in these animals. Other animal models in which SPs (and NFTs, if present) are of endogenous, rather than human, origin offer alternatives to transgenic mice for studying this issue.
5. The extent of complement activation and its association with neuropathology must be determined in animal models of AD to clarify the relevance of these models for investigating the significance of complement activation in the development of AD-type pathology.
Abbreviations used
Aβ, amyloid beta; AD, Alzheimer's disease; APP, amyloid precursor protein; CNS, central nervous system; MAC, membrane attack complex; mRNA, messenger ribonucleic acid; NFTs, neurofibrillary tangles; NGF, nerve growth factor; PS-1, presenilin-1; sCrry, soluble complement receptor-related protein y; SPs, senile plaque; TGF-β1, transforming growth factor beta1.
Competing interests
The author declares that he has no competing interests.
Acknowledgements
Thanks are expressed to Elizabeth Head, Ph.D, Dianne Camp, Ph.D., Stephanie Conant, Ph.D., and Peter LeWitt, M.D., for reviewing the manuscript. This work was supported by a donation from Mrs. Martha Loeffler in memory of Erwin S. Loeffler, Ph.D., and Harold J. Loeffler, Ph.D.
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| 15479474 | PMC529311 | CC BY | 2021-01-04 16:38:19 | no | J Neuroinflammation. 2004 Oct 12; 1:18 | utf-8 | J Neuroinflammation | 2,004 | 10.1186/1742-2094-1-18 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1555098310.1371/journal.pbio.0020399Research ArticleCell BiologyEvolutionGenetics/Genomics/Gene TherapyMicrobiologyMolecular Biology/Structural BiologyEubacteriaAdaptive Amplification and Point Mutation Are Independent Mechanisms: Evidence for Various Stress-Inducible Mutation Mechanisms Adaptive Mutation and AmplificationHastings P. J [email protected]
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Slack Andrew
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Petrosino Joseph F
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Rosenberg Susan M
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1Department of Molecular and Human Genetics, Baylor College of MedicineHouston, TexasUnited States of America2Department of Biochemistry and Molecular Biology and Department of Molecular Virology and Microbiology, Baylor College of MedicineHouston, TexasUnited States of America12 2004 23 11 2004 23 11 2004 2 12 e3998 9 2003 20 9 2004 Copyright: © 2004 Hastings et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
In Times of Stress, Mutate Early and Often
“Adaptive mutation” denotes a collection of processes in which cells respond to growth-limiting environments by producing compensatory mutants that grow well, apparently violating fundamental principles of evolution. In a well-studied model, starvation of stationary-phase lac− Escherichia coli cells on lactose medium induces Lac+ revertants at higher frequencies than predicted by usual mutation models. These revertants carry either a compensatory frameshift mutation or a greater than 20-fold amplification of the leaky lac allele. A crucial distinction between alternative hypotheses for the mechanisms of adaptive mutation hinges on whether these amplification and frameshift mutation events are distinct, or whether amplification is a molecular intermediate, producing an intermediate cell type, in colonies on a pathway to frameshift mutation. The latter model allows the evolutionarily conservative idea of increased mutations (per cell) without increased mutation rate (by virtue of extra gene copies per cell), whereas the former requires an increase in mutation rate, potentially accelerating evolution. To resolve these models, we probed early events leading to rare adaptive mutations and report several results that show that amplification is not the precursor to frameshift mutation but rather is an independent adaptive outcome. (i) Using new high-resolution selection methods and stringent analysis of all cells in very young (micro)colonies (500–10,000 cells), we find that most mutant colonies contain no detectable lac-amplified cells, in contrast with previous reports. (ii) Analysis of nascent colonies, as young as the two-cell stage, revealed mutant Lac+ cells with no lac-amplified cells present. (iii) Stringent colony-fate experiments show that microcolonies of lac-amplified cells grow to form visible colonies of lac-amplified, not mutant, cells. (iv) Mutant cells do not overgrow lac-amplified cells in microcolonies fast enough to mask the lac-amplified cells. (v) lac-amplified cells are not SOS-induced, as was proposed to explain elevated mutation in a sequential model. (vi) Amplification, and not frameshift mutation, requires DNA polymerase I, demonstrating that mutation is separable from amplification, and also illuminating the amplification mechanism. We conclude that amplification and mutation are independent outcomes of adaptive genetic change. We suggest that the availability of alternative pathways for genetic/evolutionary adaptation and clonal expansion under stress may be exploited during processes ranging from the evolution of drug resistance to cancer progression.
Cells can respond to stress by apparently increasing their mutation rate. This study provides evidence that there is more than one pathway by which cells achieve such a response
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Introduction
Adaptive mutation was first brought to wide attention by Cairns et al. (1988), and encompasses a collection of processes whereby cells adapt genetically in response to growth-limiting environments. The phenomenon is reported in several different microbial systems, and appears to occur via different molecular mechanisms (reviewed by Rosenberg 2001; Hersh et al. 2004). The Lac frameshift assay system of Escherichia coli (Cairns and Foster 1991) is the best understood in terms of mutation mechanism (reviewed by Foster 1999; Rosenberg 2001; Hersh et al. 2004). In this system, cells carrying a +1 frameshift mutation in lac genes on an F′ conjugative plasmid are starved on solid medium with lactose as the sole carbon source, selecting Lac+ revertants. Lac+ clones appear over time during selection in a population of cells that shows no net growth (i.e., is in stationary phase). In most clones, Lac+ is conferred by a compensatory −1 frameshift mutation in the lac gene (Lac+ point mutants). Lac+ point mutation during stationary phase differs from mutation during rapid growth in that it requires the recombination proteins RecA, RecBC, RuvA, RuvB, and RuvC (Harris et al. 1994, 1996; Foster et al. 1996), and induction of the SOS DNA damage response regulon (Cairns and Foster 1991; McKenzie et al. 2000), particularly error-prone DNA polymerase (Pol) IV (DinB) (McKenzie et al. 2001, 2003; Wolff et al. 2004). The lac frameshift allele is leaky, conferring 1%–2% of the wild-type level of β-galactosidase (Foster 1994; Andersson et al. 1998), and Lac+ colonies can also occur by amplification of the lac locus into a tandem array of 20–100 copies (Foster 1994; Andersson et al. 1998; Hastings et al. 2000). Adaptive amplification requires RpoS, the transcriptional activator of approximately 50 stationary-phase/starvation- and general-stress-response-specific genes, which is also required for point mutation (Lombardo et al. 2004). Amplification does not require DinB or induction of other SOS proteins (McKenzie et al. 2001), and whether or not it requires recombination proteins has not been determined unambiguously (Tlsty et al. 1984; Hastings and Rosenberg 2002).
Consistent with Darwinian principles of evolution by selection of undirected and random genetic changes, adaptive point mutation in the Lac system is accompanied by a high level of mutation in genes unrelated to lactose catabolism (secondary mutation). Lac+ point mutants show about 50-fold more secondary mutations than do cells starved on the same plate that did not become Lac+ (Torkelson et al. 1997; Rosche and Foster 1999; Godoy et al. 2000). This indicates that a subpopulation of the starved cells experiences a transient episode of hypermutation (adaptive point mutants are not mutator mutants; Torkelson et al. 1997; Rosenberg et al. 1998; Rosche and Foster 1999). In keeping with their independence of DinB and SOS, the amplified clones do not show hypermutation of unrelated genes (Hastings et al. 2000). These differences, together with the finding that cells carrying lac amplification (“lac-amplified cells”) do not readily yield Lac+ point mutants when resubjected to selection on lactose medium, led us to propose that amplification reflects a pathway of genetic change wholly or partly separate from point mutation (we suggested an alternative outcome of a branched pathway; Hastings et al. 2000; Rosenberg 2001; Hastings and Rosenberg 2002).
Two general models for the adaptive point mutation mechanism in this system are currently at odds, each focused on the role of DNA amplification, and each representing a different view of evolution (e.g., Rosenberg and Hastings 2004a, 2004b; Roth and Andersson 2004a, 2004b). In “hypermutation models,” the observed high frequency of Lac+ point mutants is proposed to result from transient hypermutation as part of a stress response, one consequence of which is acceleration of genetic change (and so potentially of evolution) (e.g., see Rosenberg 2001, and below, where specific molecular mechanisms are suggested). In this view, amplification is an adaptive pathway alternative to point mutation that allows growth of cells, relieving the stress of starvation, and so deflects them from the point mutation route. This view is compatible with Darwinism, which allows for changing rates of generation of heritable variations (Darwin 1859).
In an alternative model, called “amplification–mutagenesis,” amplification is an intermediate in the formation of Lac+ point mutants, and its most important role is to provide more lac copies per cell so that point mutation can occur without increase in mutation rate per copy of lac (Andersson et al. 1998; Hendrickson et al. 2002). This adheres to conservative neo-Darwinist ideas of constant and gradual evolutionary change (e.g., Mayr 1982).
Here, we report several experiments that demonstrate that amplification and point mutation are separate adaptive outcomes, compatible with hypermutation models, and not part of a sequential pathway leading to mutation (as in the amplification–mutagenesis model). We also report the first genetic requirement specific to the adaptive amplification pathway, Pol I.
Results/Discussion
Colony Phenotypes
Experiments described below make use of the different colony color phenotypes of point-mutant and lac-amplified clones grown on rich medium with the dye 5-bromo-4-chloro-3-indoyl β-D-galactoside (X-gal) (e.g., Tlsty et al. 1984; Andersson et al. 1998; Hastings et al. 2000) (Figure 1). We use the word “sectored” to describe the appearance of a colony of cells on X-gal medium (e.g., Figure 1B and 1C). The word “unstable” is used to denote continued sectoring when cells from sectored colonies are replated. This instability has been shown in many examples to reflect tandem amplification of the lac region (Tlsty et al. 1984; Foster 1994; Andersson et al. 1998; Hastings et al. 2000). Sectoring that is not unstable (Figure 1C) may reflect incidental juxtaposition of blue and white colonies on a plate (see below). “Stable” describes the absence of visible sectoring that indicates Lac+ point mutation (Tlsty et al. 1984; Hastings et al. 2000; see Figure 1A).
Figure 1 Colony Morphologies
(A) Point-mutant Lac+ colony showing solid blue color (the pale colonies are derived from Lac− cells).
(B) lac-amplified colonies showing sectoring caused by the instability of the amplified array. Cells from these colonies grow either into sectored blue colonies or, if they have lost the amplification, into white colonies, a phenotype that we call “unstable.”
(C) A sectored colony that is not unstable in that it was found to contain only stable blue and stable white cfu upon retesting.
(D) An example of a microcolony of the sort used in this work. The visible colony on the lower edge of the field has a diameter of 1.4 mm (>108 cells).
(E and F) Phase contrast (E) and green fluorescence (F) of the same field, showing two of 30 SMR6039 cells fluorescing.
Most Microcolonies Are Pure, Not Mixed
If most adaptive point mutants arise by mutation in young colonies carrying lac amplification (Andersson et al. 1998; Hendrickson et al. 2002), then young colonies should contain both the lac-amplified progenitor cells and their point-mutant descendants, as reported by Hendrickson et al. (2002) and reexamined here. To determine whether a microcolony carries point-mutant or lac-amplified cells or both, in principle, one picks the microcolony from the lactose-minimal selection medium and replates its cells onto rich X-gal medium to observe whether the resulting colonies are pure blue (point mutant), unstable sectoring (lac-amplified), or both. A significant problem in interpreting data from such analyses is that the picked microcolony carries with it many Lac− background cells from the selection plate (Figure 2C) and may contain cells from unrelated microcolonies, because, of necessity, these microcolonies cannot be streaked to purify them before analysis.
Figure 2 Stringent Analysis of Whole Microcolonies: Analyzing All Cells in a Microcolony and Reducing Contamination by Unrelated Neighboring Bacteria
To analyze all cells in a microcolony, very young microcolonies were harvested (103–104 cfu/microcolony; see Figure 1D; Table 1). To reduce contamination with neighboring Lac− bacteria and unrelated Lac+ microcolonies from the minimal-lactose selection plate, first, a minority of the Lac− cells plated carried an antibiotic-resistance marker, and only resistant microcolonies were analyzed, and, second, sectored colonies observed were retested for instability (to eliminate sectored colonies that are not unstable, shown in Figure 1C, which may result from accidental overlap of blue and white cfu). Procedures described in Materials and Methods, “whole microcolony analysis.” CamR, chloramphenicol resistant; KanR, kanamycin resistant; TetR, tetracycline resistant. Streptomycin resistance (not shown) was also used.
Table 1 Composition of Whole Microcolonies
Microcolonies were harvested from five adaptive mutation experiments and analyzed as described in Figure 2 and Materials and Methods
a Cells derived from these two cfu were Lac− (see text)
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We have improved the sensitivity of microcolony analysis substantially, first, by analyzing all cells in a microcolony (rather than a sample) and, second, by imposing stringent methods to reduce contamination of microcolonies with neighboring cells and microcolonies from the selection plate (Materials and Methods; Figure 2). First, frameshift-bearing (tester) cells were mixed with 3% each of three or four different derivative testers that carry antibiotic resistance markers (Figure 2A), and only microcolonies that carried one of these minority antibiotic resistance markers were analyzed to search for sectored colonies on medium carrying the antibiotic. This reduces, but does not eliminate, accidental overlap with Lac− background cells and with unrelated Lac+ microcolonies accidentally harvested in the same sample (shown below). Second, because some sectored colonies are not unstable (e.g., mixtures of stable, white Lac− background cells, and blue colony formers that overlapped), all sectored colonies found were retested by plating a sample on the same X-gal antibiotic medium to determine whether there was continued instability indicative of amplification (Figure 2E). Third, because the sequential model predicts that the younger the microcolony is, the greater the proportion of lac-amplified cells (colony-forming units [cfu]) it should contain (Hendrickson et al. 2002), and because one might miss the putative lac-amplified cfu by not examining enough cfu per microcolony, we analyzed very young microcolonies of 102–104 cfu/microcolony (as compared with approximately 105 cfu/microcolony used by Hendrickson et al. [2002]) and analyzed all cfu in each of them (rather than a sample) (Figure 2
Table 1).
The results of analyzing every cell in 18 whole microcolonies of stable cells, isolated on days 3 through 6 of adaptive mutation experiments, are shown in Table 1. Although we found sectored colonies at frequencies of 10−2–10−3 in almost all microcolonies (as reported by Hendrickson et al. 2002), these sectored colonies, when replated, were almost all found to consist of cells giving rise to stable white and stable blue colonies only, indicating that these were not from lac-amplified clones (also demonstrated below). In general, we looked at 50 to 100 cells from these replated sectored colonies, but, for three of them, we screened 3,000–4,000 cells with the same result: we could find no unstable cfu within them. In contrast, cells from lac-amplified colonies, upon replating, again give rise to sectored colonies. Therefore, most of the sectored colonies that we found give no evidence of carrying amplification of the lac locus. Sectoring in these colonies may occur by chance juxtaposition of Lac+ point-mutant cells with non-mutated background cells (which are numerous) during the plating (Figure 2). Other explanations might also be possible; however, these colonies are not lac-amplification bearers by this test and also as demonstrated in the following section. An example of such a colony is shown in Figure 1C. Such colonies may account for the report of Hendrickson et al. (2002) that more than 10−3 of cells in every Lac+ microcolony are sectored colony formers. These sectored, non-lac-amplified colonies have also been reported by another laboratory (Poteete et al. 2002).
Of the 18 whole microcolonies examined (Table 1), 16 were pure stable. One day-6 microcolony was found to contain seven unstable cfu. (Another, found on day 5, contained two cfu showing sectors of an extremely pale blue color along with sectors of the same color as the Lac− parental cells.) Those unstable cfu might be clonally related to the rest of the microcolony, or might result from chance juxtaposition of two different clones carrying the same antibiotic resistance marker. To determine the frequency at which such chance juxtapositions are expected, we analyzed the cells carried on the agar plugs on which microcolonies were harvested. We did this by plating the microcolony suspensions on medium containing an antibiotic to which cells of the microcolony were sensitive, to ascertain the frequency of marked Lac+ cells that are unrelated to a microcolony but get harvested with it. The microcolony plugs contained from 900 to 7,000 antibiotic-resistant lac
− background cells each. Of eleven such plugs analyzed, one contained three unrelated (different antibiotic resistance) point-mutant Lac+ cfu, one contained one unrelated unstable cfu, and three contained unrelated cfu of the very pale sectored phenotype described above. The presence on agar plugs of cells giving rise to sectored colonies that are unrelated to the microcolony, but occur at a frequency comparable to the frequency of unstables in a microcolony of the same antibiotic-resistance genotype (Table 1, last line), implies that the presence of unstable cfu was independent of the microcolony, and thus not relevant to the origin of the microcolony.
The very pale sectored colonies that we found were examined further and were shown to be unstable upon retesting but contained no cells capable of forming colonies on lactose medium under the conditions of an adaptive mutation experiment. Presumably they contain too few copies of the lac region to allow growth on lactose medium. They might carry lac gene duplication, or tandem amplification at a very low level. In support of this possibility, we note that their pale appearance on X-gal medium is similar to known lac gene duplications of this frameshift allele that we have constructed (A. Slack and P. J. Hastings, unpublished data). Another possibility is that they contain multiple F′ plasmids (Foster and Rosche 1999). Their failure to form adaptive point-mutant or lac-amplified colonies shows that they do not advance to a Lac+ phenotype efficiently (so are not obvious precursors to point mutation).
Thus, colonies composed of a mixture of lac-amplified and point-mutant cells, as reported by Andersson et al. (1998) and Hendrickson et al. (2002), are not common when stringent methods of scoring are imposed, and such mixed colonies are certainly not every microcolony in which a mutation has occurred. Those apparent mixed colonies that do occur are likely to be unrelated mixtures and not relevant to the origin of the Lac+ point mutants in the colony.
Selection for lac-Amplified Cells in Point-Mutant Microcolonies
As an independent test for the presence of rare lac-amplified cells in point-mutant microcolonies, we devised a genetic selection capable of revealing single lac-amplified cells among numerous non-amplified cells. The selection depends on the quantitative relationship between the number of copies of the chloramphenicol acetyl transferase gene (cat) and the concentration of the antibiotic chloramphenicol that cells resist (Petit et al. 1992). We inserted the cat gene into codA, about 5 kb from lac. Control experiments established that strains with lac and cat amplification (as shown by unstable phenotypes and Southern blot analyses) could grow on rich Luria-Bertani-Herskowitz medium (LBH) containing 100 μg/ml chloramphenicol, whereas Lac+ point-mutant cells, and those amplified at lac but not at cat, did not form colonies at this concentration. We determined that 61% or more of all lac-amplified cells are detected by this selection (Materials and Methods).
The data are shown in Table 2. Among 75 Lac+ point-mutant microcolonies isolated from days 4, 5, and 6 of an adaptive mutation experiment, three (4%) were found to contain a few chloramphenicol-resistant (CamR) cfu (Table 2). All of these showed the lac-amplified colony phenotype. Correction for those that would not be detected by this selection (above; Materials and Methods) shows that, at most, 6.6% of point-mutant microcolonies contain any lac-amplified cells (and probably fewer; see below). As discussed above, this contrasts with the report of Hendrickson et al. (2002) that all Lac+ microcolonies contain lac-amplified cells. Also, these data verify independently that the high levels of sectored, but not unstable, cfu found among microcolonies (see Table 1) were not lac-amplification bearers, because most lac-amplified cfu would have scored positively in this selection.
Table 2 Selection for Rare Amplified Cells in Colonies of Point-Mutant Cells
Results of selection for CamR cells in 152 microcolonies (103 to 2 × 104 cells each) and 70 control samples with no visible microcolony. The data show that amplified cells are no more common in microcolonies of point-mutant cells than they are in the negative control carrying only background cells (no microcolony). Numbers in parentheses are the numbers of CamR cfu found in each case. For unstable microcolonies, all cfu in any microcolony were either CamR or chloramphenocol sensitive
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A negative control was performed by plating samples of cells from agar plugs that did not carry a microcolony visible on a dissecting microscope. Five of 70 such samples yielded one or a few cells that were lac-amplified and showed CamR. The similarity of this proportion to that for lac-amplified cells in point-mutant microcolonies suggests that none of the mixtures detected represents an occurrence of lac-amplified cells that arose in the same cell clone as the point mutation. Thus, we conclude that few, if any, point-mutant microcolonies include lac-amplified cells.
Nascent Lac+ Point-Mutant Colonies Do Not Contain lac-Amplified Cells
The selection method described above allowed us to detect amplification in samples of stressed cells that did not include a visible microcolony. This suggested the possibility of analyzing point mutation and amplification in nascent Lac+ colonies as young as the two-cell stage and up to about 40 cells. The sequential amplification–mutagenesis model demands that all colonies as young as 2–40 cells must consist only of lac-amplified cells, because these are proposed not to generate Lac+ point mutations until the number of lac copies reaches 107 or more (e.g., a colony of 105 cells with 100 lac copies per cell caused by amplification; Hendrickson et al. 2002).
Cells from 600 sample plugs, harvested on days 3 to 5 in two experiments, that carried no microcolony visible at 20-fold magnification were analyzed by selecting for CamR in half the sample, and Lac+ phenotype in the other half. The samples were found to contain 1 × 104 to 5 × 104 total cells. We detected 22 events by either Lac+ or CamR selection (3.7% of the samples). This frequency of events is compatible with the observation of 30–60 Lac+ colonies per 108 cells per day, which we see routinely in adaptive mutation experiments (e.g., Hastings et al. 2000). These events are listed in Table 3.
Table 3 Nascent Lac+ Point-Mutant Colonies at the Few-Cell Stage Do Not Include Amplified Cells
Number of cfu per sample appearing in two selections, half of each sample applied to each selection. Data for 600 samples of stressed cells that did not include a microcolony
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Twelve samples gave lac-amplified cells (cfu) detected as CamR. The cell numbers ranged from one to 16 cells (from half the sample). Two amplification events gave Lac+ cells that were chloramphenicol sensitive, detected on lactose-minimal medium plates. Many more amplification events were detected by CamR selection than by growth on lactose medium (even though all were lac-amplified because of their being selected on lactose medium), implying that the CamR test is more sensitive, and capable of detecting levels of amplification that are too low to allow colony formation on lactose medium.
Eight samples gave stable Lac+ cells (cfu), ranging in number from one to 20 cells from half the sample (implying colonies ranging in size from 2–40 cells). None of these eight contained amplified cells, as determined by CamR, or by rescoring the lactose plates after 7 d, by which time any pre-existing lac-amplified cells would have formed colonies (Hastings et al. 2000). Thus, we see that, even at the 2- to 40-cell stage of colony growth, we are unable to detect a correlation between mutation and amplification events, and we conclude that these are independent processes. These results are incompatible with the idea that Lac+ adaptive point mutants result from point mutation events in young colonies of lac-amplified cells (Andersson et al. 1998; Hendrickson et al. 2002).
lac-Amplified Microcolonies Arise Later than Point-Mutant Microcolonies
If point-mutant colonies arose from lac-amplified microcolonies, the majority of microcolonies of sufficiently small size would be expected to consist of lac-amplified cells. For example, in the sequential model of Hendrickson et al. (2002), Lac+ point mutation is expected to occur when a colony of lac-amplified cells grows to 105 cells (with a mutation rate of 10−8 mutations per cell per generation that is enhanced 35-fold by SOS induction and acts on an additional 30 copies of lac; 35 × 30 × 105 × 10−8 = 1 mutation per microcolony of 105 cells). This model predicts that lac-amplified microcolonies of approximately 105 cells should precede point-mutant colonies. As shown in Figure 3, the microcolonies harvested in the course of these experiments, all much smaller than 105 cells, show a very distinct pattern that conflicts with this expectation. Only 5% of microcolonies isolated on day 3 consisted of lac-amplified cells (the rest were stable point mutant). The proportion of unstable microcolonies increases on later days. This is the pattern of arising of lac-amplified visible colonies, which are rare in the first few days of an experiment and increase in frequency later (Hastings et al. 2000; Figure 3). The proportion of unstable microcolonies appears to reflect the proportion of visible colonies that would consist of lac-amplified cells 1 to 4 d later (Figure 3), as expected if lac-amplified microcolonies produce lac-amplified (not point-mutant) visible colonies.
Figure 3 Temporal Distribution of lac-Amplified Microcolonies
Data from six adaptive mutation microcolony experiments were pooled to give the distribution of point-mutant and lac-amplified phenotypes. Squares, the mean percentage of Lac+ microcolonies with unstable phenotype (± SEM; n = 3 experiments on day 3 and n = 4 on days 4, 5, and 6); diamonds, the percentage of lac-amplified visible colonies on the same days included for comparison (data from Hastings et al. 2000). Note that the observed proportion of unstable microcolonies is higher than the actual proportion because lac-amplified clones are slower growing than point mutants (Hastings et al. 2000), and so spend more time as microcolonies before becoming visible colonies. Thus, the lack of unstable microcolonies on early days is even more severe than the data show.
lac-Amplified Microcolonies Produce lac-Amplified, Not Point-Mutant, Visible Colonies
The sequential amplification–mutagenesis model states that mutation occurs in a cell in a microcolony of lac-amplified cells (Hendrickson et al. 2002). Growth of the point-mutant cell clone is proposed to overtake the lac-amplified cells to give a visible colony consisting mainly of point-mutant cells. We looked for this change from lac-amplified to point-mutant phenotype by removing a few cells from microcolonies to determine which microcolonies consisted of lac-amplified cells, and sampling them again after they had grown to form visible colonies (Materials and Methods). The data in Figure 4 show that we did not see this change occur. lac-amplified microcolonies grew into lac-amplified visible colonies, and point-mutant microcolonies remained point mutant. We conclude that microcolonies of lac-amplified cells do not become visible colonies of point-mutant cells.
Figure 4 Fate of Microcolonies Developing In Situ
Microcolonies on days 4 and 5 of separate adaptive mutation experiments were sampled by touching with a needle, then the sample was spread on rich X-gal medium and scored for point-mutant or sectored Lac+ phenotype (see Materials and Methods). The microcolonies were then left to grow into visible colonies (∼107 cells), and the colonies sampled again and samples plated to score for sectoring (hatched bars, day 4 microcolony samples; black bars, day 5 microcolony samples). The numbers of each type of colony observed are shown next to the bars. One single stable microcolony produced a mixed visible colony, which may have been caused by overlap with another colony or microcolony.
Hendrickson et al. (2002) reported a seemingly similar experiment that gave the opposite result. They scored small (presumed young) and large (old) colonies and found many more unstable cfu in small colonies than in larger colonies. We note that they did not follow the same small colonies to score later, when those small colonies had grown larger, as we have done. Their result is probably caused by the slow growth rate of lac-amplified cells, which causes microcolonies of lac-amplified cells to remain at the microcolony size for longer than do microcolonies of point-mutant cells, such that many more small colonies are lac-amplified. We have found that point-mutant microcolonies stay small for a much briefer period of time than do lac-amplified ones (data not shown). We overcame this problem by scoring the same colonies as microcolonies and found that these small lac-amplified colonies do not grow to become point mutant (Figure 4). An additional explanation for why the small colonies of Hendrickson et al. (2002) contained more lac-amplified cfu is that, in most of their experiments, these authors did not employ the stringent methods that we devised to avoid scoring unrelated mixtures of cells in microcolony samples. These include the antibiotic-resistant-minority-scoring method outlined in Figure 2. Thus, when they picked small colonies in a fixed volume of agar, a greater fraction of the cells in their sample would be unrelated background cells than when they picked larger colonies (same number of background cells each time, but fewer or more cells from the colony they intended to sample). This, too, should result in the appearance of more mixtures of lac-amplified and point-mutant cfu in the small colony samples and more pure point mutant in the large colony samples; but the mixtures in the small colony samples would be unrelated. We conclude that when stringent colony-fate experiments are performed, lac-amplified microcolonies are seen to produce lac-amplified and not point-mutant visible colonies (Figure 4).
Point Mutants Do Not Overgrow lac-Amplified Cells in Competition Experiments
We tested experimentally whether point-mutant cells occurring in a lac-amplified microcolony would be able to overgrow and mask the lac-amplified cells on the timescale of adaptive mutation experiments, as is postulated in sequential models (Hendrickson et al. 2002). Cultures of lac-amplified cells were seeded with Lac+ point-mutant cells in various proportions and grown for a known number of cell generations, as spots on lactose plates, to form pseudocolonies. The proportion of lac-amplified and point-mutant cells was determined after 3 d, by which time the pseudocolony had reached 8 × 107 to 1.6 × 108 cells (Table 4). This is three to four more generations than are needed to form a visible colony (approximately 0.5–1 × 107 cells; Hastings et al. 2000). These experiments were performed with two different lac-amplified isolates that have 2.0 and 4.2 times the generation time on lactose medium of the point-mutant Lac+ strain used. That is, these strains can produce 2 and 4.2 times fewer generations than point mutants, and so might, in principle, be overtaken by point mutants during colony growth. However, this was not observed under the real-life conditions of growth in colonies under selective conditions. Only when point-mutant cells were introduced at a ratio of one to ten was the proportion of lac-amplified cells reduced to below 1% (the level reported in mixed colonies by Hendrickson et al. [2002]), and then only with the slower growing lac-amplified isolate (Table 4). We did not see the point-mutant cells take over when the point mutant started at 10−5 of the cells, the predicted time of occurrence of a mutation (Hendrickson et al. 2002). Similar results were obtained when the experiment was performed in liquid culture (data not shown).
Table 4 Competition between Two lac-Amplified Isolates and a Point-Mutant Lac+ Strain
Lac+ point-mutant cells were seeded into suspensions of lac-amplified cells of two different isolates with different growth rates, at dilutions from 10−5 to 10−1. Cell numbers ranging from 102 to 105 cells were spotted as 0.5-μl spots on M9 lactose plates to form pseudocolonies. After 3 d of growth, the pseudocolonies were suspended, and dilutions of cells plated to determine cell number and the proportion of Lac+ point-mutant and lac-amplified cells. Each number is the mean ± SEM of three parallel cultures with two pseudocolonies per culture. This experiment was performed three times on plates (as above) and twice in liquid culture, giving comparable results each time
10.1371/journal.pbio.0020399.t004
We conclude that overgrowth of colonies of lac-amplified cells by point-mutant cells does not occur as postulated in the sequential, amplification–mutagenesis model (in which one point-mutant cell is proposed to overgrow 105
lac-amplified cells; Hendrickson et al. 2002). The only circumstance under which overgrowth might be seen would be if the mutation occurred in the first ten cells of a microcolony. With only approximately 300 lac copies present at the ten-cell stage, this mutation frequency, approximately 3 × 10−3 mutations per lac copy, would be untenably high in any current mutation model.
Amplification and Not Point Mutation Requires DNA Pol I
If adaptive amplification were a prerequisite for point mutation, then all genetic requirements for amplification should also be required for point mutation. We report the first genetic requirement found for amplification that is not also required for point mutation: Pol I. Pol I, encoded by polA, is required for most amplification (Figure 5A), but adaptive mutation is increased in a polA mutant (Figure 5C). We used the temperature-sensitive polA12 (polA[Ts]) allele incubated at semi-permissive temperature, because polA null mutation causes inviability in this strain background (Harris 1997). In repeat experiments, we saw a 2- to 6-fold reduction in amplification rate. In the same experiments, adaptive mutation was unchanged or increased by up to 8-fold.
Figure 5 Pol I Is Required for Adaptive Amplification and Not Point Mutation
Strains were plated on lactose-minimal medium and Lac+ colonies counted daily (see Materials and Methods). The plots are cumulative, showing the mean of 3–4 cultures with one SEM. Strains used: FC40 dinB+ polA+
fadAB::Tn10Kan, SMR3490 (squares); FC40 dinB+ polA12(Ts)fadAB::Tn10Kan, SMR3491 (diamonds); FC40 dinB10 polA12(Ts)fadAB::Tn10Kan, PJH308 and PJH309 (triangles, inverted triangles); and FC40 dinB10 pol+ fadAB::Tn10Kan, PJH310 (circles). All cultures were grown at 30 °C and the experiments conducted at 37 °C.
(A) An example of the effect of polA(Ts) on the yield of lac-amplified colonies, showing a partial requirement for polA at a semi-permissive temperature. (B) and (C) show the effect of the dinB10 mutation on adaptive lac-amplification and point mutation, respectively. The dinB polA(Ts) cells display the decreased lac amplification of the polA mutant (B), and the decreased point mutation characteristic of the dinB mutant (C), demonstrating that the decrease in lac amplification rate does not result from channeling of lac-amplified cells into a point mutation pathway. These data (C) also show that the absence of Pol I increases point mutation (reported previously, Harris 1997) in a completely DinB-dependent manner. This could occur via the absence of Pol I leading to SOS induction (Bates et al. 1989) and more DinB/Pol IV, or via relief of a competition between high-fidelity Pol I and error-prone Pol IV at the replisome. Neither of these ways should affectlac amplification, which is Pol IV–independent, as observed (B).
The possibility that amplification occurs in the Pol I–deficient strain but that the Lac+ colonies are unable to grow to visibility in the time-span of an adaptive mutation experiment is ruled out by reconstruction experiments, in which three independent lac-amplified isolates carrying polA(Ts) or polA+ formed visible colonies in 5.25 ± 0.17 d and 5.39 ± 0.24 d, respectively (mean of three strains ± standard error of the mean [SEM]). Another possibility that we considered is that the polA mutation, which causes an increase in SOS induction (Bates et al. 1989), might channel amplified DNA into the point mutation pathway by increasing SOS-induced mutagenesis, so that there is an increase in mutation concomitant with a decrease in amplification. We were unable to test this directly by preventing SOS induction, because the non-inducible allele of lexA confers low viability in combination with polA(Ts) (data not shown). However we tested this possibility using dinB-defective cells. DinB/Pol IV accounts for most, if not all, of the requirement of adaptive mutation for SOS induction (McKenzie et al. 2001). The results are shown in Figure 5 Two independent isolates of the double mutant dinB polA(Ts) show the reduction in point mutation (Figure 5C) expected of dinB (McKenzie et al. 2001) and the reduction in amplification (Figure 5B) reported above for polA(Ts). This demonstrates that the decrease in lac-amplified colonies in polA(Ts) strains is not due to conversion/channeling of lac-amplified cells into point mutants, but rather is caused by reduction of the number of lac-amplified cells formed. We conclude that Pol I is required specifically for adaptive amplification and not point mutation. These data are incompatible with models in which amplification is a prerequisite for point mutation, and support models in which point mutation is an independent mechanism.
Amplification Does Not Induce SOS
A final aspect of the sequential model of Hendrickson et al. (2002) was tested. To explain the high frequency of unselected secondary mutations observed in Lac+ adaptive point mutants (Torkelson et al. 1997; Rosche and Foster 1999; Godoy et al. 2000), Hendrickson et al. proposed that amplified DNA itself is sufficient to induce a chronic SOS response leading to increased mutation, and that, therefore, the SOS regulon should be induced in all or most cells carrying amplification. By contrast, hypermutation models (e.g., Rosenberg 2001; Hersh et al. 2004; Rosenberg and Hastings 2004a) suggest that a hypermutable state, possibly an SOS response, is induced only transiently in a subpopulation of starved cells, and that this condition will not persist for long in cells that are able to utilize lactose because they no longer experience the stress of starvation. To determine whether the involvement of SOS in adaptive point mutation (Cairns and Foster 1991; McKenzie et al. 2000) is a consequence of amplification, we constructed a strain in which the green fluorescent protein (GFP) gene is expressed under the control of the SOS-regulated sulA promoter (see Figure 1E and 1F). sulA is one of about 40 SOS (DNA damage inducible) genes in E. coli (Courcelle et al. 2001). Figure 6A shows that about 2% of cells in the growing cultures showed spontaneous SOS induction, as revealed by GFP fluorescence. There are no detectable differences in this proportion between Lac+ point-mutant and lac-amplified cultures, or between cultures grown with (lactose medium) or without (glycerol medium) selection for lactose utilization. Control experiments showed that 3 h after exposure to 20 J/m2 of ultraviolet light, all cells were induced for SOS (data not shown). Thus, amplification per se does not induce an SOS response.
Figure 6 DNA Amplification Does Not Induce the SOS Response
(A) Known lac-amplified and point-mutant (control) derivatives of SMR6039 were grown in liquid M9 medium containing either lactose or glycerol. Mid-logarithmic phase cells were harvested and scored microscopically for GFP fluorescence, using an Olympus BL51 microscope mounted with a mercury lamp UV source and a High Q Endow GFP emission fluorescence filter cube. Some 1,000–2,000 cells from each of 4–10 fields were scored per determination. Error bars indicate one SEM for 8–13 cultures as indicated.
(B) Microcolonies were harvested and suspended in 500 μl of buffer, 50 μl of which was spread on LBH X-gal rif solid medium to determine sectoring in resulting colonies. The remainders were concentrated and examined microscopically for GFP fluorescence, counting 60–400 cells per microcolony. These experiments were plated at very low cell density to avoid significant numbers of background Lac− cells being harvested with the microcolonies. Open bars, fraction of stable Lac+ isolates (32 microcolonies); black bars, fraction of sectored isolates (11 microcolonies). The two distributions do not differ (p = 0.8 by Student's t-test). These experiments were repeated, giving similar results.
We also studied the persistence of SOS induction in microcolonies during an adaptive mutation experiment. We isolated microcolonies from lactose plates, spread a sample on LBH (rich) X-gal rifampicin (rif) plates to determine which colonies were point mutant and which lac-amplified, and screened other cells from the microcolonies for GFP production. The data presented in Figure 6B show that there is no difference between point-mutant and lac-amplified microcolonies in the distribution of cells producing GFP (p = 0.8 by Student's t-test) and that neither shows persistent SOS induction. Thus, even under adaptive mutation experimental conditions, amplification is not sufficient to induce the SOS regulon. The results also show that SOS induction does not persist for many generations after stress has been released. The data are compatible with hypermutation models for adaptive mutation (Rosenberg 2001; Figure 7), wherein the SOS response is induced transiently during starvation, and are incompatible with the postulate that chronic SOS induction is caused by amplified DNA.
Figure 7 Stress-Response Models for Adaptive Amplification and Point Mutation
Modified from Lombardo et al. (2004) and Rosenberg and Hastings (2004a). DSBR, DSB repair; hypermutation, increased mutation at lac and elsewhere. The origins of DSEs during starvation on lactose medium in this assay system are unknown, and possibilities are reviewed elsewhere (Rosenberg 2001). On the F′ plasmid carrying the lac gene, DSEs may be frequent and may be derived from chronic single-strand nicks at the origin of transfer. However chromosomal mutation during starvation on lactose medium in these cells also requires DSB repair proteins (Bull et al. 2001), and so probably results from (lower levels of) DSEs generated in the chromosome, independently of the transfer origin, or perhaps by transient integration of the F′ into the chromosome (Hfr formation). Both adaptive point mutation and amplification are proposed to be outcomes of RpoS-dependent stress response, which both mechanisms require (Lombardo et al. 2004), and to result from alternative error-prone ways of repairing DSBs.
Further Discussion
Amplification is a separate genetic endpoint that allows growth under selection
The experiments reported here were designed to determine whether DNA amplification is an intermediate in the formation of adaptive point mutations in the Lac system, or whether the two are independent end points of genetic change under stress, each of which adapts cells to their environment. All of the data presented support the idea that lac amplification and point mutation are alternative end points, either of which allows growth on lactose. These data are reviewed conveniently by contrast with the sequential, amplification–mutagenesis model, as follows.
Significance and predictions of amplification–mutagenesis
The experiments reported test the amplification–mutagenesis model for the mechanism of adaptive mutation in the E. coli Lac system (Andersson et al. 1998; Hendrickson et al. 2002). The model's significance lies in its apparent explanation of adaptive mutation phenomenology without invoking an environmentally induced increase in mutation rate, in adherence to conservative neo-Darwinist ideas about constant, gradual evolutionary change (e.g., Mayr 1982). In the model, spontaneous amplification of lac allows growth of cells into microcolonies on lactose medium; replication of the multiple lac copies generates a point mutant at about the 105-cell stage; and the point-mutant cell overgrows the lac-amplified cells, leading to colonies consisting mostly of point-mutant cells with a few (10−2–10−3) lac-amplified cells. This model derives most of its support from the observation of sectored-colony-forming cells in colonies of point-mutant cells (Andersson et al. 1998; Hendrickson et al. 2002).
Independent point mutation and amplification mechanisms supported
By contrast, we found, first, that when stringent scoring methods are applied, and all cells in very young microcolonies analyzed, most point-mutant Lac+ colonies are not mixtures of stable point-mutant cells with 10−2–10−3 unstable (amplification bearing) cells, but rather are pure (see Table 1). This was verified with sensitive genetic selection methods that can detect single lac-amplified cells among microcolonies (see Table 2), and was found to be true even at the two- to 40-cell stage of colony growth (see Table 3). We conclude that adaptive point mutants arise directly from lac− single cells without an intermediate stage involving colonies of lac-amplified cells, and that the lac-amplified cells are a separate adaptive outcome.
Previous observations of mixed colonies (Andersson et al. 1998; Hendrickson et al. 2002) seem likely to have arisen from accidental overlap of point-mutant and Lac− colonies that were not related, which would cause sectored, but not unstable colonies. We observed these, recapitulating the previous results (see Table 1; Figure 1C; Poteete et al. 2002), only when we failed to apply stringent criteria for identification of lac-amplified cells. Such colonies might also have arisen from overlap of stable with unrelated unstable microcolonies, which were not screened out stringently (Hendrickson et al. 2002), as done here (see Figure 2; Table 1).
Second, we found that microcolonies smaller than 105 cells are not mostly lac-amplified, as the amplification–mutagenesis model demands, but rather that microcolonies carrying amplification are rare initially and frequent later, with kinetics that anticipate the arising of lac-amplified visible colonies (See Figure 3). Third, when sampled early and late, young microcolonies with lac amplification give rise to lac-amplified, not point-mutant, visible colonies (see Figure 4). Fourth, artificial mixtures of point-mutant with lac-amplified cells do not show the predicted takeover of the amplification carriers by the point mutants (one point mutant overtaking 105
lac-amplified, as predicted Hendrickson et al. [2002] and Roth et al. [2003]) unless the point mutants comprise a very large fraction (10%) of the total (see Table 4). Fifth, we show that DNA Pol I is required for lac amplification and not point mutation (see Figure 5), demonstrating that lac amplification is not a prerequisite for point mutation. We conclude that amplification is an independent outcome that adapts cells, not a transient intermediate en route to point mutation.
Amplification does not induce SOS
To explain the genome-wide hypermutation observed in Lac+ point mutants (Torkelson et al. 1997; Rosche and Foster 1999; Godoy et al. 2000) with a sequential model, Hendrickson et al. (2002) suggested that amplified DNA per se induces an SOS response and increased mutability. Using an SOS-inducible gfp reporter gene, we find that amplified DNA does not induce SOS (see Figure 6). This conclusion was also supported previously by the observation that cells carrying amplification do not display general hypermutation (Hastings et al. 2000).
Apparent support for sequential model compatible with independent pathways
An additional result previously interpreted as support for sequential models was that when a gene counter-selectable at more than one copy was placed next to lac, point mutation was decreased (Hendrickson et al. 2002). However, we note that this is predicted by our non-sequential hypermutation model for point mutation (below) as well, in which point mutation is proposed to result from error-prone DNA double-strand-break (DSB) repair, because more than one copy of lac must be present to allow repair by homologous recombination. This copy could be on another DNA molecule (a sister chromosome) or the same one (a duplication), but either way it is required for error-prone repair leading to mutation, and its counter-selection would decrease point mutation, even though amplification is not an intermediate in the process.
Branched- or independent-pathway, stress-response models for adaptive amplification and point mutation
In Figure 7, we outline a model supported by previous and current data. In this model, amplification and point mutation result from alternative modes of error-prone DNA DSB repair caused as stress responses to starvation. Adaptive point mutation requires homologous recombination proteins, including those specific to DSB repair, implicating DSBs or double-strand ends (DSEs) as a molecular intermediate (Harris et al. 1994). We suggest that adaptive point mutations form by homologous recombinational DSB/DBE repair that is error prone due to use of the SOS-inducible (Kim et al. 1997) and starvation-inducible (Layton and Foster 2003) error-prone DNA polymerase, Pol IV/DinB. The SOS response (Cairns and Foster 1991; McKenzie et al. 2000) and DinB are required for most (85%) adaptive point mutation and not for lac amplification in E. coli (McKenzie et al. 2001). Point mutants, but not lac-amplified cells (Hastings et al. 2000), carry high levels of mutation of genes throughout their genome (Torkelson et al. 1997; Rosche and Foster 1999; Godoy et al. 2000). Because these levels of mutation are not found in most Lac− cells starved on the same plates, we infer that only a subpopulation of the starving cells experiences this transient hypermutation (Torkelson et al. 1997), perhaps those that are induced for the SOS response (McKenzie et al. 2000) or the SOS and RpoS stress response (which is required, discussed below) (but see Foster 2004; Rosenberg and Hastings 2004a). Mismatch repair, which corrects replication errors, also becomes limiting specifically during adaptive mutation, leading to increased mutagenesis (Harris et al. 1997); this might occur by titration of mismatch repair proteins by excess DNA polymerase errors (Harris et al. 1997) made by Pol IV (McKenzie et al. 2001). This model, which includes elevated rates of general (not lac-specific) genetic change in response to stress, is compatible with Darwinism, which also encompasses changing rates of heritable variations (Darwin 1859), but not with the conservative neo-Darwinist constraint of gradual evolutionary change (e.g., Mayr 1982), a major impetus for the sequential, amplification–mutagenesis model (Andersson et al. 1998; Hendrickson et al. 2002).
Amplification mechanism and the role of Pol I
Amplification, on the other hand, could be formed by non-homologous repair of the DSEs (Figure 7), leading to a gene duplication or rolling-circle replication, either of which might amplify rapidly to produce the 20–100 copies of lac that allow growth without a point mutation (Hastings and Rosenberg 2002). Alternatively, homologous DSE repair might produce replication forks that lack some of the control of origin-initiated replication and stall in the nucleotide-depleted milieu of starvation. We suggest that template switching can occur at a stalled fork and produce a duplicated-DNA segment or a rolling-circle intermediate from which amplification results. This is similar to template-switching recombination models (Bzymek and Lovett 2001). This template-switching event might occur preferentially with Pol I, which is capable of template switching in vitro (Kornberg and Baker 1992). The partial requirement for Pol I might result from the use of semi-permissive conditions for a conditional mutant. Acquisition oflac amplification, and thus the ability to grow on lactose, alleviates the stress and deflects lac-amplified cells from the point mutation pathway, making it a (more or less) stable, alternative outcome (Figure 7).
There is no appearance of directed mutation in the lac system
Unlike the hypermutable state model of Hall (1990), our model (Figure 7) does not specify that cells that do not acquire an adaptive point mutation or amplification die. That feature would be necessary for explaining a lack of mutations in genes other than lac (“directed” mutation; Cairns et al. 1988). However, in the E. coli Lac assay, exposure to selection conditions generates (non-adaptive) mutations in other genes efficiently—and by the same recombination-protein- and DinB-dependent mechanism as Lac+ adaptive point mutation—both in the F′ that carries lac (Foster 1997) and the bacterial chromosome (Bull et al. 2001). A report to the contrary used a different bacterium (Salmonella) with many different genetic features that might not parallel the E. coli Lac assay (Slechta et al. 2002).
Adaptive point mutation and amplification in the lac system are stress responses
That both adaptive point mutation and amplification are outcomes of a stress response is supported by their requirements for the general-stress-response and stationary-phase transcription factor RpoS (σS) (Lombardo et al. 2004). RpoS upregulates approximately 50 genes in response to starvation, oxidation, pH, heat, and osmotic stresses. RpoS upregulates error-prone Pol IV (Layton and Foster 2003), which might explain its role in the Pol IV–dependent process of adaptive point mutation (McKenzie et al. 2001). However RpoS must play some other role in adaptive amplification, which is Pol IV–independent (McKenzie et al. 2001). RpoS is also required for a Pol IV–independent, starvation-induced hypermutation mechanism in a natural isolate of E. coli (Bjedov et al. 2003) and in other adaptive mutation pathways (Gomez-Gomez et al. 1997; Saumaa et al. 2002), implying that this is a general feature of starvation-induced mutability. Thus, several mechanisms of adaptive genetic change appear to require a differentiated, stress-response condition of the cells, most probably stationary-phase cell physiology. This further supports the idea of stress-induced increases in mutation rate generally.
Mutation rates are increased in starving/stressed cells generally
Although it has been argued that increasing mutation generally in response to stress is implausible (Roth et al. 2003), a recent study found that starvation-induced general mutability is the norm in natural isolates of E. coli (Bjedov et al. 2003). The authors found that the vast majority of 787 natural isolates from diverse habitats worldwide produced random, unselected mutations in response to starvation. Multiple mechanisms of mutation in the natural isolates are implied, but at least one of them has similarities to that of the Lac system, and to a different stationary-phase-mutation model system (reviewed by Rosenberg and Hastings 2003), in that these mutation responses require RecA, an SOS-regulated DNA polymerase, and RpoS (Bjedov et al. 2003). Moreover, in the eukaryote Caenorhabditis elegans, a new study of mutation (Denver et al. 2004) suggests that cellular stress responses might provoke hypermutation generally, and also lead to a mismatch-repair-compromised transient state (Rosenberg and Hastings 2004c) similar to that suggested here. These systems support the idea that evolution might be hastened during stress. They promise to reveal mutation mechanisms that are likely to pertain to cancer formation and progression, acquisition of drug resistance in pathogens and tumors, and many processes in which clonal expansion under stress or growth limitation follows from an adaptive genetic change.
Materials and Methods
Strains
E. coli strains used are isogenic with FC40 (Cairns and Foster 1991) (also SMR4562, an independent construction of FC40; McKenzie et al. 2000), the lac-frameshift-bearing strain in which adaptive mutation and amplification are assayed, and FC29, a non-revertible lac deletion strain used to scavenge other carbon sources from the lactose plates (Cairns and Foster 1991). Antibiotic-resistant derivatives SMR4481 (FC40 malB::Tn10dTet), SMR3598 (FC40 zff3139::Tn10Kan), SMR533 (FC40 malB::Tn9), and PJH223, a spontaneous streptomycin-resistant mutant of SMR4562, are resistant to 10 μg/ml tetracycline, 30 μg/ml kanamycin, 25 μg/ml chloramphenicol, and 100 μg/ml streptomycin, respectively. SMR4481 was made by transposition of Tn10dTet from lambda NK1323 (Kleckner et al. 1991). SMR533 and SMR3598 were constructed by transduction of FC40 with phage P1 grown on FS2055 (F.W. Stahl, University of Oregon) carrying malB::Tn9, and STL1605 (S.T. Lovett, Brandeis University) carrying zff3139::Tn10Kan, respectively. SMR4562 codA::cat (PJH220) was constructed by linear replacement into SMR4562[pKD46] (plasmid from Datsenko and Wanner 2000) using the cat gene from pKRP10 (Reece and Phillips 1995). The isogenic strain pair FC40 fadAB3165::Tn10Kan (SMR3490) and FC40 polA12(Ts)fadAB3165::Tn10Kan (SMR3491) were constructed by P1 transduction of FC36 (Cairns and Foster 1991) with a P1 lysate carrying fadAB3165::Tn10Kan from CAG18495 (Singer et al. 1989), or with P1 grown on a strain with fadAB3165::Tn10Kan from CAG18495 linked with polA12(Ts) (Monk and Kinross 1972, via F.W. Stahl, University of Oregon), respectively, followed by transfer of F′128 from FC40 by conjugation. PJH308 and PJH309 were made by P1 transduction of SMR5380 (McKenzie et al. 2001) with lysate from SMR3491 and screened for sensitivity to ultra-violet light (UV) to give two separate UV-sensitive isolates of FC40 polA(Ts)fadAB3165::Tn10KandinB10 and the UV-resistant isogenic control strain PJH310 (FC40 fadAB3165::Tn10KandinB10). polA mutant strains were grown at 30 °C, and experiments were carried out at 37 °C (a semi-permissive temperature). SMR6039 is a derivative of SMR4562 carrying Δattλ::PsulAΩgfp-mut2: the gfp-mut2 reporter gene (Cormack et al. 1996) under the control of the LexA-regulated sulA promoter replacing the phage attλ site in the E. coli chromosome (as per Gumbiner-Russo et al. 2001; see below).
Construction of an SOS-GFP reporter
The gfp-mut2 gene (Cormack et al. 1996) was inserted into plasmid pACYC184 (New England Biolabs, Beverly, Massachusetts, United States), at the BamHI and HindIII restriction sites, to create pJP21. To fuse the sulA promoter to the gfp gene, two 5′ phosphorylated oligonucleotides corresponding to the top and bottom DNA fragments of the sulA −1 to −67 region were synthesized: BspHI-pSulA-BamHI-top, 5′-P-CATGAGGGTTGATCTTTGTTGTCACTGGATGTACTGTACATCCATACAGTAACTCACAGGGGCTGGATTGATTG-3′ and BspHI-pSulA-BamHI-bot, 5′-P-GATCCAATCAATCCAGCCCCTGTGAGTTACTGTATGGATGTACAGTACATCCAGTGACAACAAAGATCAACCCCT-3′. When these are annealed, a BspHI-complementary end is reconstituted at one end and a BamHI-complementary end at the other. This fragment was ligated into BspHI /BamHI-digested pJP21 to create pJP23. A SphI fragment from pJP23, containing the PsulAΩgfp-mut2 fusion, was isolated and ligated into SphI-digested pTGV-Light (Gumbiner-Russo et al. 2001) to create pJP30. pJP30 was digested with AscI to generate a linear fragment for chromosomal integration replacing the attλ site (Gumbiner-Russo et al. 2001) with the PsulAΩgfp-mut2 fusion, Δattλ::PsulAΩgfp-mut2. Phage P1 grown on this strain was used to construct SMR6039, an SMR4562 derivative carrying Δattλ::PsulAΩgfp-mut2, by transduction into SMR5084, genotype SMR4562(λxis1 cIts857Ind), as per (Gumbiner-Russo et al. 2001).
Adaptive mutation experiments
Adaptive mutation experiments were performed as per Harris et al. (1996), with the following variations for microcolony experiments: lactose plates were prescavenged by plating 5 × 109 FC29 “scavenger” cells on them and incubating at 37 °C for 24 h, then washing the cells off with M9 buffer; plating of frameshift-bearing cells was not in top agar, but on the plate's surface; growth of Lac− cells throughout the experiments was monitored either by sampling the lawn as described (Cairns and Foster 1991; Harris et al. 1994, 1996) or by microscopic examination. Microscopic examination used 50-fold magnification on a dissecting microscope. The lawn was seen to be smooth at this magnification when less than about 2-fold growth had occurred (number of viable cells relative to starting viable cell measurement). Plates on which growth occurred acquired a punctate appearance. This method was validated by using the two methods in parallel in two experiments, the largest of which involved 24 plates from two cultures. The correlation between growth detected as a doubling in cell number obtained from an agar plug, and the appearance of puncti was complete. No growth (≤ 2-fold) was detected during the relevant times in the experiments reported.
For quantitative measure of mutation and amplification, three or four parallel cultures of each genotype were scored for Lac+ colonies each day. Cells from 20 colonies from each culture, or 40 colonies in the case of SMR3491, were plated on LBH rif X-gal to determine the proportion amplified.
Whole microcolony analysis
Frameshift-bearing (tester) cells were mixed with 3% each of three or four different derivative testers that carried antibiotic-resistance markers (see Figure 2). Microcolonies were found by use of a dissecting microscope, and harvested on a plug of agar with a capillary tube (see Figure 2). Working at 25× magnification on a dissecting microscope, we were able to find and harvest very young colonies (see Figure 1D), down to a size of 102 cells. The cells were suspended in 500 μl M9 buffer. One percent of the suspension was plated on LBH plates (Torkelson et al. 1997) containing 40 μg/ml X-gal and 100 μg/ml rif (to prevent growth of scavenger cells). The remainder of the suspension was diluted with glycerol (to 12.5%) and stored at −80 °C. Many of the cells in any plating of a harvested microcolony were background (white colony forming) Lac− tester cells that were on the agar plug (see Figure 2). The cells from the microcolony either formed solid blue colonies on LBH X-gal rif plates (see Figure 1A), indicating a point-mutant colony, or sectored blue colonies (see Figure 1B), indicating an unstable lac-amplified colony (Hastings et al. 2000). The colonies were scored for sectoring, counted, and patched from the LBH X-gal rif plates onto plates containing antibiotics. Those microcolonies that showed a stable Lac+ point-mutant phenotype, had a suitable cell number, and carried one of the minority antibiotic-resistance markers were then plated in toto from the frozen suspension onto LBH X-gal rif medium with the appropriate antibiotic, to search for sectored colonies. Because some sectored colonies are not unstable (e.g., mixtures of stable white and blue colonies that overlap; see Results/Discussion and Table 1), all sectored colonies found were retested by plating a sample on the same LBH X-gal antibiotic medium to determine whether there was continued instability indicative of amplification.
Harvesting the microcolony on a plug of agar with a 25-μl capillary pipette also picks up many Lac− tester cells. The number of these that form colonies during the analysis is reduced by use of antibiotics (as described above) because most cells are not resistant, but the background of tester cells may still be equal in number to those in the microcolony (see Figure 2C). Use of a smaller caliber tube was found to risk losing microcolony cells on the wall of the tube.
Selection for lac-amplified cells
Microcolonies of PJH220 were harvested as above and suspended in 200 μl of buffer. One microliter (0.5%) of the suspension was spread on LBH X-gal rif medium to determine cell number and which microcolonies were point mutant. Point-mutant microcolonies were then plated to select CamR on LBH plates containing X-gal, rif, and 100 μg/ml chloramphenicol. Plates were scored after 15 to 20 h at 37 °C.
To analyze background cells without microcolonies, plugs of agar were taken from areas of the plates where no microcolony was visible at 20× magnification and the cells suspended in 200 μl of buffer. Half of each suspension was plated on chloramphenicol-containing medium as above, and the other half was plated with scavenger cells onto lactose-minimal medium. Chloramphenicol plates were scored between 15 and 18 h, and lactose plates were scored at 2 d, and again at 7 d. Colonies appearing on lactose medium were scored for amplification by plating a sample of the cells on LBH X-gal rif medium. Colonies appearing on chloramphenicol-containing medium were scored for lac amplification (blue and white sectoring) directly on that plate. All CamR colonies were seen to be lac-amplified.
The fraction of lac-amplified cells detectable by CamR selection was determined as follows. There are two reasons why lac amplification might fail to be detected with the CamR selection: first, because some lac amplifications have not co-amplified the neighboring codA::cat gene, and, second, because some cat-amplified microcolonies obtained from the lactose plates (grown selectively for Lac+ and not for CamR) might have too few cat copies for growth on 100 μg/ml chloramphenicol. We determined each of these separately. We found that 59 of 77 lac-amplified microcolonies were CamR (at least 77% had co-amplified cat), and that 84% of cells from CamR (cat-amplified) isolates grown on lactose without chloramphenicol were then able to form colonies on chloramphenicol medium. The cells that did not form colonies on chloramphenicol presumably had lost or reduced their amplification of cat and lac. This would indicate that 77% of 84% (65%) of all lac-amplified cells should be detected by the CamR selection. An independent determination of the number of cat-amplified cells that grow on chloramphenicol after growth on lactose was obtained in a reconstruction experiment in which a few CamR cells were plated with a 104-fold excess of Lac+ point-mutant cells, mimicking more closely the conditions of the experiments in which microcolonies are obtained. A total of 79% of the cat-amplified cells formed colonies on chloramphenicol medium, so 77% of 79% (61%) of all lac-amplified cells should be detected. The selection for CamR is actually a more sensitive assay for lac amplification than is selection for Lac+, as shown by the fact that only 60% of cells from the cultures that showed CamR were able to form colonies on lactose medium under the conditions of an adaptive mutation experiment, though all carriedlac amplification (indicating that 40% had too few lac copies for growth on lactose), whereas 79% were detected on chloramphenicol medium.
Colony-fate experiments
Microcolonies were touched with the point of a needle, and cells were transferred to LBH X-gal rif plates, where they were spread by streaking for scoring. Control experiments (not shown) showed that this procedure removed, on average, 5% of the microcolony from the plate. About 50 separate colonies from each microcolony were scored for sectoring. The remainder of the colony was left for 1 to 4 d to form a visible colony in situ. The visible colony was then sampled, plated, and scored for sectoring (see “whole microcolony analysis” above).
Competition experiments
M9 lactose liquid cultures of Lac+ point-mutant and lac-amplified cells were grown (separately) to saturation, then mixed in various proportions, and 0.5 μl of the mixed cell suspensions was put onto the surface of prescavenged lactose plates and grown into visible colonies. These “pseudocolonies” were suspended, and cells were spread on LBH X-gal rif plates at 100 to 200 cells per plate and scored for sectoring blue phenotypes after 2 d growth.
Supporting Information
Accession Numbers
The Swiss-Prot ( http://www.ebi.ac.uk/swissprot/) and Ecogene ( http://bmb.med.miami.edu/EcoGene/EcoWeb/) accession numbers for the genes and gene products discussed in this paper are cat (Swiss-Prot P62577), codA (Swiss-Prot P25524), DNA Pol I (Swiss-Prot P00582), DNA Pol IV/dinB (Swiss-Prot Q47155), fadAB (Swiss-Prot P21177), malB (Swiss-Prot P02943), gfp-mut2 (Swiss-Prot P42212), lac (Swiss-Prot P00722 and P03023), recA (Swiss-Prot P03017), recBC (Swiss-Prot P08394 and EcoGene EG10825), rpoS (Swiss-Prot P13445), ruvA (Ecogene EG10923), ruvB (Ecogene EG10924), ruvC (Ecogene EG10925), and sulA (Swiss-Prot P08846).
We thank H. J. Bull, M.-J. Lombardo, G. J. McKenzie, M. Radman, and S. Rutherford for discussion and valuable comments on the manuscript, and J. M. Pennington for cell sorting. This work was supported by National Institutes of Health grants R01-GM64022 (PJH) and R01-GM53158 (SMR), and postdoctoral fellowships from the Department of Defense Breast Cancer Research Program (JFP) and the Natural Sciences and Engineering Research Council of Canada (AS).
Conflicts of interest. The authors have declared that no conflicts of interest exist.
Author contributions. PH and SMR conceived and designed the experiments. PH, AS, and JFP performed the experiments. PH, AS, and JFP analyzed the data. SMR contributed reagents/materials/analysis tools. PH and SMR wrote the paper.
Academic Editor: Susan Lindquist, Whitehead Institute
Citation: Hastings PJ, Slack A, Petrosino JF, Rosenberg SM (2004) Adaptive amplification and point mutation are independent mechanisms: Evidence for various stress-inducible mutation mechanisms. PLoS Biol 2(12): e399.
Abbreviations
CamRchloramphenicol resistant/resistance
catchloramphenicol acetyl transferase gene
cfucolony-forming units
DSBdouble-strand break
DSEdouble-strand end
GFPgreen fluorescent protein
LBHLuria-Bertani-Herskowitz medium
Polpolymerase
polA(Ts)temperature-sensitive polA12
rifrifampicin
SEMstandard error of the mean
UVultra-violet light
X-gal5-bromo-4-chloro-3-indoyl β-D-galactoside
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| 15550983 | PMC529313 | CC BY | 2021-01-05 08:21:17 | no | PLoS Biol. 2004 Dec 23; 2(12):e399 | utf-8 | PLoS Biol | 2,004 | 10.1371/journal.pbio.0020399 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1555098410.1371/journal.pbio.0020402Research ArticleAnimal BehaviorBirdsState-Dependent Decisions Cause Apparent Violations of Rationality in Animal Choice Economic Rationality in Animal ChoicesSchuck-Paim Cynthia
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¤Pompilio Lorena
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Kacelnik Alex [email protected]
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1Zoology DepartmentUniversity of OxfordUnited Kingdom12 2004 23 11 2004 23 11 2004 2 12 e40223 6 2004 22 9 2004 Copyright: © 2004 Schuck-Paim et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Are Animals As Irrational As Humans?
Normative models of choice in economics and biology usually expect preferences to be consistent across contexts, or “rational” in economic language. Following a large body of literature reporting economically irrational behaviour in humans, breaches of rationality by animals have also been recently described. If proven systematic, these findings would challenge long-standing biological approaches to behavioural theorising, and suggest that cognitive processes similar to those claimed to cause irrationality in humans can also hinder optimality approaches to modelling animal preferences. Critical differences between human and animal experiments have not, however, been sufficiently acknowledged. While humans can be instructed conceptually about the choice problem, animals need to be trained by repeated exposure to all contingencies. This exposure often leads to differences in state between treatments, hence changing choices while preserving rationality. We report experiments with European starlings demonstrating that apparent breaches of rationality can result from state-dependence. We show that adding an inferior alternative to a choice set (a “decoy”) affects choices, an effect previously interpreted as indicating irrationality. However, these effects appear and disappear depending on whether state differences between choice contexts are present or not. These results open the possibility that some expressions of maladaptive behaviour are due to oversights in the migration of ideas between economics and biology, and suggest that key differences between human and nonhuman research must be recognised if ideas are to safely travel between these fields.
Decisions made by European starlings in certain choice contexts are shown to depend on the training regimes rather than being inherently irrational behavior
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Introduction
The study of animal behaviour has often incorporated concepts from economic theory. This was the case, for instance, with the introduction of game theory to the study of animal conflict (Maynard-Smith and Price 1973; Maynard-Smith 1974). Similarly, optimal foraging theory (Charnov 1976; Stephens and Krebs 1986) was based on viewing animals as maximisers, with utility often being replaced by rate of energy gain as a proxy for Darwinian fitness, and natural selection playing the role of the short-sighted architect of the decision mechanisms followed by individuals. The foundation for this migration of ideas between fields is the notion that optimal choice is defined by the value of the consequences of each option, and that this value is jointly determined by the option's properties and the chooser's state. This is clear within models, but presents considerable difficulties for empirical tests, and we address some of these problems in this paper.
One consequence of expecting individuals to behave as if they maximised the expected value of a particular function (say, inclusive fitness) is captured in the economic concept of rationality. Since “rationality” is used with very different meanings in different fields (see Kacelnik [2004] for a discussion of rationality and its meanings), it is important to point out that here we will use the term only in its economic sense. Rationality, in this restricted sense, encapsulates several principles that are necessary conditions for the existence of a scale of value consistent across contexts (Mas-Collel et al. 1995). Transitivity, for instance, is a hallmark of rational choice theories. It states that if “a” is preferred to “b”, and “b” to “c”, then “a” should also be preferred over “c”. If, say, “c” were to be preferred to “a”, it would not be possible to place the three options on an ordinal scale. Another principle included in economic rationality is that of independence from irrelevant alternatives (IIA; Arrow 1951), namely, the expectation that preference between a pair of options should be independent of the presence of inferior alternatives. There are different versions of IIA, depending on how demandingly one defines “preference”. A strong probabilistic version, known as the “constant-ratio rule” (Luce 1959), states that the relative proportion of choices made between two options should be the same (as opposed to merely maintaining the same order), regardless of whether they are on their own (binary choices) or in the presence of a third (less preferred) option (trinary choices). A weaker version, known as “regularity”, states that rationality is violated if the proportion of choices for any preexisting option is increased after the addition of a new alternative to the choice set (Luce and Suppes 1965).
Breaches of rationality are well documented in observational or experimental studies on human choice (Tversky 1969; Huber et al. 1982; Payne et al. 1992; Simonson and Tversky 1992; Tversky and Simonson 1993; Wedell and Pettibone 1996; Gigerenzer et al. 1999), and have forced a reinterpretation of much of the existing data and models. In many studies, these violations are taken to imply context-dependent valuation, namely the notion that the (subjective) value of each option is not determined only by its properties and consequences, but instead is constructed at the moment of choice as a function of the number and nature of other options available—a finding used, for example, in marketing and political campaigning for manipulating consumer preferences through the strategic presentation of products and candidates.
An alternative view (Kacelnik and Krebs 1997; Gigerenzer et al. 1999) is that although these mechanisms can cause costly choices, they (the mechanisms) are evolutionarily and/or ecologically rational, meaning that on average in the environment where they evolved or were individually acquired they generate stochastically optimal outcomes. Whichever the interpretation, however, locally costly deviations from rationality do occur, and can offer significant insights in the development of theoretical models of decision-making.
A number of psychological mechanisms have been proposed to explain the effect of inferior alternatives on choice and other examples of irrationality. According to them, the observed failure to exhibit consistent preferences across contexts would be attributable to the dependence of the information-processing mechanisms used by individuals, or of the heuristics used for making choices, on the nature of the choice problem and available alternatives (Shafir et al. 1989; Wedell 1991; Payne et al. 1992). While normative microeconomic theory is independent of process and focuses on revealed preferences, these developments relate the theory to cognition and give weight to the process by which agents reach decisions (Kahneman and Tversky 2000).
It is worth remembering that consistency of preference is accepted by all parties to be only relevant when constancy in the state of the subjects and in the properties of the options is assumed. A subject that prefers lamb to ice cream before dinner, ice cream to coffee immediately after dinner, and coffee to lamb a few minutes later is not considered to be showing intransitivity or violating any principle of rationality, because she is (trivially) changing state between the choices. Similarly, a subject that takes a mango when presented with a basket of many mangoes and only one apple, but takes an apple when faced with equal numbers of both fruits may not be considered irrational, because the value of an option may change when it is the last one, as there is a reputation cost of being impolite and taking the last available fruit of any kind (Sen 1997). Many human experiments are comparisons between groups of subjects that can be assumed to be in equal states at the time of testing but, as we shall see, this is often not ensured in nonhuman animals.
Violations of rationality by animals have also been reported (Shafir 1994; Hurly and Oseen 1999; Waite 2001a; Bateson 2002; Bateson et al. 2002, 2003; Shafir et al. 2002). If these observations are corroborated and found to be systematic, the predictive power of the normative approach to animal behaviour should be questioned. Additionally, the observation of similarly irrational behaviour by animal and human subjects raises the possibility that the same cognitive mechanisms or processes operate in both cases. In fact, explanations for irrationality based on phenomena such as regret and overconfidence (e.g., Loomes and Sugden 1982), proposed with humans in mind, could be tested by examining whether the same circumstances elicit the expression of the same type of paradoxical behaviour in human and nonhuman subjects. If they do, and the mechanisms seem unlikely to operate in nonhuman agents, one may be advised to seek alternative explanations that work well for all kinds of subjects.
Although these possibilities make the study of rationality valuable, critical procedural differences between the two fields have not been sufficiently acknowledged. One crucial distinction derives from the fact that, while human subjects can be verbally instructed about the properties of the alternatives, animals must be exposed to the contingencies to experience or learn about them. This difference hinders the comparison of the mechanisms underlying human and animal choices, since repeated exposure to different contexts often affects the organism's state, thus removing the justification for expecting transitivity, regularity, or any other principle of consistency. In the case of foraging research, different contexts can alter the subjects' net rate of intake during training, so that at the time of choice the resulting state differs and it may be unjustified to expect consistency of preferences. The fact that optimal decisions should be contingent upon state (Houston and McNamara 1999) has been indeed an essential part of normative modelling in biology. As a consequence, apparent violations of rational principles by animals could also result from straightforward state-dependent optimality, the very framework being questioned. It may be added that, although we focus on changes in energetic state that could unwittingly be caused by training, these are not the only possible state consequences of instruction by exposure to the contingencies during training. A subject may be in a different state if the consumption of food items during training affects its nutritional requirements for the achievement of a balanced diet in future choice opportunities (Simpson and Raubenheimer 1993; Raubenheimer and Simpson 1997), or if changes in the context of choice provide it with different information about its future options (Houston 1997).
Here we further develop the basis upon which to compare economic rationality between humans and nonhumans, and test whether state-dependent decision-making can be responsible for apparent violations of economic rationality in animal choices. To this end, we compare the foraging preferences of European starlings (Sturnus vulgaris) between members of a fixed, focal pair of options across different choice contexts. Our basic paradigm is defined in Figure 1. The members of the focal pair of options differed in that while one of them (focal amount [FA]) offered a higher amount of food, the other (focal delay [FD]) was associated with a better (shorter) delay to food. These two attributes (amount and delay) were counterbalanced between the focal options so as to preserve their ratio (amount/delay), which is known to be (other factors remaining the same) a strong predictor of preference. A third option, or “decoy”, was also available during training and in some of the choice trials. The decoy could be either decoy amount (DA) or decoy delay (DD), depending on treatment (“High Intake” and “Low Intake” respectively, see Figure 1). We refer to the third option as “decoy” because its ratio of amount to delay was lower than in the focal options, and hence it is expected not to be preferred over either (in economic nomenclature, the decoys were “dominated” by the focal options). As postulated by context-dependent (or “comparative”) models of choice (Shafir et al. 1989, 1993; Wedell 1991; Tversky and Simonson 1993), a decoy can potentially affect preferences between a pair of options whenever subjective values are assigned comparatively, namely whenever an option's subjective value depends on the interaction of its properties with those of the remaining alternatives in a set, as well as when the decoy affects a subject's perception of the choice problem. We thus test whether each animal's preference between the focal options changes between two treatments that differed with respect to which of the two decoys was present. To increase comparability with previous research, the parameter values of the decoys were chosen to maximise their putative effect upon preference within the focal pair as postulated by psychological models purported to explain irrational choice (see Materials and Methods for details).
Figure 1 Amount and Delay to Food Corresponding to Each Option
The figure shows the parameters of the experiments using the conventional representation used in foraging theory, with energy gains in the ordinate and time in the abscissa. The origin of coordinates is the point of choice, so that time to the right indicates the delay between choice and reward, while time to the left represents all other times in the cycle, in this case the ITI. The options forming the focal choice pair are shown as white circles while those used as decoys are shown by black circles. FA and FD offer the same short-term rate of food intake (slope of the solid lines) of 0.5 units/s, whereas DA and DD offer the same short-term rate of 0.25 units/s. The slopes of the dashed lines (interrupted for space economy) indicate long-term rate of intake, considering the inter-trial interval of 60 s between consecutive feeding opportunities. “High intake” (horizontally adjacent rectangles) and “low intake” (vertically adjacent rectangles) denote the treatments in which decoy DA and DD (or their simulated energetic consequences), respectively, were present in addition to the focal pair. Since DA offers a higher long-term rate of gain than DD, intake is higher in the treatment where DA is present (“High Intake”). The reverse rationale applies to treatment “Low Intake.”
Figure 1 also shows that, although the two focal options and the two decoys were equated in the ratio of amount to delay, they were not equated in terms of their energetic consequences. When all times in the cycle are included, the order in terms of energetic rate of return (the slope of the broken lines in Figure 1) is FA > DA > FD > DD. This means that differences in energetic state as a consequence of training with either of the two decoys could be a confounding factor in interpreting putative differential effects of the decoys. Specifically, repeated exposure to DA could lead to a higher cumulative intake, hence changing choices due to the expression of state-dependent preferences instead of the use of a comparative cognitive mechanism of choice. Our study is aimed at separating these possibilities.
We tested preference between the focal options under three conditions: (1) treatments differing in energetic states when the decoys are absent; (2) treatments differing in which of the two decoys is present, when the energetic consequences caused by each decoy are not controlled; (3) treatments differing in which decoy is present, when the energetic differences they may cause are abolished by supplementary feeding. Our rationale for this design is that if the effects of the decoys are independent of their energetic consequences (i.e., differences in preference between the treatments are observed in all conditions), then these effects may indeed be evidence for a comparative cognitive mechanism of valuation, possibly caused by the same types of cognitive biases and heuristics reported in the human literature. However, if the effects of the decoys are abolished by controlling for energetic consequences and are generated by imposing state changes in the absence of decoys, it would be more parsimonious to explain the effects as state-dependent decision-making. Our results strongly favoured the latter hypothesis.
Results
Discrimination of Amounts
We started by testing whether the birds could discriminate between the amounts of reward associated with each of the foraging options shown in Figure 1 (there is already strong evidence that they are able to discriminate between the delays used [Brunner et al. 1992]). Figure 2 shows the proportion of choices of each bird to the option offering the largest amount of food. All birds in both groups significantly preferred the option offering the larger amount (binomial tests, p < 0.01 in all cases), confirming that the birds discriminate between these amounts.
Figure 2 Discrimination Test
Proportion of choices (± standard error [s.e.]) made by birds for the option offering the largest amount of food when time parameters were held constant. Choice proportions are significantly different from random for all birds (binomial test, p < 0.01). Birds 1, 2, and 3 (white bars) were presented with choices between one and two units of food, and birds 4, 5, and 6 (black bars) with choices between two and five units of food.
Effects of Intake Rate without Decoys
The proportion of choices made for each focal option in the absence of decoys when energetic state was manipulated experimentally is shown in Figure 3A. Although the purpose of this experiment was to examine how the strength of preference between the focal options was affected by differences in energetic state, it is worth pointing out that there was an overall preference for FA (the option with higher long-term rate of gain) over FD even though the ratio of amount to delay was the same for both options. This preference for FA is not caused by the accumulated energetic consequences of exposure to each option, because the two focal options were experienced in mixed sessions. In Mazur's (1987) “hyperbolic” model, which is widely used in the behavioural analysis literature, the time between feeding events (or inter-trial interval ) is not included, but instead a constant with the value of 1 s is added to the delay in the denominator. The effect of this term is also to make the value of FA higher than that of FD, consistent with the observed trend.
Figure 3 Individual Proportion of Choices for FA Relative to FD in Treatments “High Intake” and “Low Intake”
(A) Effect of intake on choices without decoys. Here, extra food simulates the intake consequences that the two decoys cause when they are present and consumed on 25% of the feeding opportunities (p < 0.01).
(B) Results of an experiment with decoys when energetic consequences of the decoys were allowed to take effect (group NC) (p = 0.06).
(C) Results of an experiment with decoys, similar to (B), in which the energetic consequences of the decoys were abolished (group C).
In (B) and (C), each symbol corresponds to each of the subjects. The dashed lines show the mean values in each of the cases.
The critical observation for the present purposes, however, is that the magnitude of the preference between the focal options differed significantly between intake treatments. Specifically, preference for FA over FD was higher in treatment “Low Intake” (the treatment with lower accumulated intake) than in treatment “High Intake” (F
1,8 = 12.1, p < 0.008; Figure 3A). The details of the supplementary feeding are given in Materials and Methods, but it is important to highlight that the difference in supplementary intake between treatments “High Intake” and “Low Intake” simulated the differences in state that would be consequent on repeated experience of decoys DA and DD, respectively. These results thus show that energetic state per se can directly affect the strength of preference between alternatives.
The stability criteria (see Materials and Methods) were reached by all but one bird. We therefore also conducted the analysis excluding this bird. The results were the same: Preference for FA over FD was significantly higher in treatment “Low Intake” than in treatment “High Intake” (F
1, 7 = 18.9, p = 0.003).
Test of Economic Rationality in the Presence and Absence of Controls for Intake
In this experiment, two groups of six starlings each (group C had intake controlled between treatments, and NC had intake not controlled between treatments) were trained with three options (the two focal options and one of the decoys) and then allowed to choose between either two (binary trials) or three (trinary trials) of those options. The two treatments, “High Intake” and “Low Intake”, differed in which of the two decoys (DA and DD, respectively) was present during training and in the trinary choices. Within each group, every subject experienced both treatments.
Considering the results of the previous experiment, preference for FA over FD should be higher in the treatment with lower accumulated intake (treatment “Low Intake”) for group NC, in which such intake differences were not eliminated. No differences should, however, be observed in group C, in which intake differences were abolished. The results from the binary choice trials (where only the focal options were present) are shown in Figures 3B and 3C. As predicted, for group NC, preference for FA tended to be higher in treatment “Low Intake” (F
1,4 = 7.4, p = 0.06; Figure 3B). In group C, intake differences resulting from the decoys were abolished by supplementary feeding, and no differences in preference between treatments were detected (F
1,4 = 0.2, p = 0.677; Figure 3C). To summarise, differences in the level of preference for the focal options in binary choices are present when intake differs but there are no decoys (Figure 3A) and when decoys are present and their intake consequences are not controlled (Figure 3B), but disappear when the decoys are present but their intake consequences are neutralised (Figure 3C).
We also analysed the temporal aspects of state changes on choice. Because the long-term rate of gain offered by DA was higher than that offered by DD, in group NC the difference in cumulative intake between the “High Intake” and “Low Intake” treatments must have increased over the trials in a session. Accordingly, the rate of increase in the strength of preference for FA over FD along the trials (as measured by the slope of the regression of trial number against average proportion of choices for FA) was significantly higher in the latter than in the former treatment (group NC: F
1,4 = 27.35, p = 0.006). This difference was not observed for the group in which intake was controlled (group C: F
1,4 = 1.29, p = 0.32).
The rational principle of regularity and the constant-ratio rule can be examined by comparing choices between the focal options in binary (only the two focal options present) versus trinary (two focal options and one decoy) trials (see details in Data Analysis). We make two types of comparisons, between-treatments and within-treatments. In the between-treatments comparison we compare the binary trials of one treatment with the trinary trials of the other. For example, we compare the binary trials of treatment “Low Intake” (when training included exposure to FA, FD, and DD, but choices were between FA and FD presented alone) against the trinary trials of treatment “High Intake” (when training included FA, FD, and DA, and choices were between FA, FD, and DA), and vice versa. In the within-treatments comparisons, we compare binary versus trinary trials within the same treatment (e.g., binary versus trinary trials of treatment “Low Intake” and binary versus trinary trials of treatment “High Intake”). Notice that within a given treatment (within-treatment comparisons), accumulated intake was the same in binary and trinary trials for both groups of subjects (C and NC), whereas between treatments (between-treatment comparisons), accumulated intake differed between binary and trinary trials for the group of subjects in which intake differences were not controlled (NC). Therefore, if intake, rather than purely cognitive effects, is the cause of changes in preference for the focal options, apparent violations of regularity and of the constant-ratio rule should be observed only in the between-treatments comparisons for group NC. Conversely, if the presence of the decoys has a cognitive effect upon preferences that is independent of state, such violations should be observed both in the between- and within-treatments comparisons. Table 1 lists the predicted direction of preferences for each of the treatments, considering the hypothesis that differences in intake generated by exposure to the decoys, rather than purely cognitive effects of the decoys, cause the apparent violations of rationality. The directions of preferences were predicted on the basis of the results of the experiment without decoys, which showed that preference for FA was higher in the treatment with lower accumulated intake. Hence, we expect preference for FA to be higher in treatment “Low Intake”; namely, we expect P(FA[“Low Intake”]) > P(FA[“High Intake”]), and consequently, P(FD[“Low Intake”]) < P(FD[“High Intake”]), where P is the strength of preference for the corresponding focal option in the relevant treatment. For simplicity, only the predictions for group NC are shown in Table 1, since under the energetic hypothesis we do not expect differences in preference levels (and therefore violations of rationality) for the group in which intake differences were abolished (group C).
Table 1 Between- and Within-Treatments Comparison of Binary and Trinary Choice Trials for Group NC
All predictions are based on the results of the experiments without decoys, which showed that preference for FA was higher in the treatment with lower accumulated intake, that is, P(FA[Low Intake]) > P(FA[High Intake]), and conversely P(FD[Low Intake]) < P(FD[High Intake]), and assume that differences in intake generated by exposure to the decoys is the sole cause of difference in preferences between treatments
“FA*” indicates preference for FA relative to FD in the trinary choice trial, calculated as shown in equation 1 (see text)
Bin, binary; F(int.), P(F[Intake]); Int, intake; N, no breach, Trin, trinary
Figures 4 and 5 show the results for the between- and within-treatments comparisons, respectively. The left panels in both figures show the results for group NC (Figures 4A, 4C, 5A, and 5C) and the right panels for group C (Figures 4B, 4D, 5B, and 5D). No violations of either regularity or differences in relative choice proportions were observed in group C (repeated-measures ANOVA, all p > 0.1). For group NC, the observed directions of preferences were, in all cases, consistent with all predictions shown in Table 1. In terms of the significance of the observed changes in preference in the between-treatment comparisons (Figure 4A and 4C), one out of the two predicted apparent violations of regularity was statistically significant: There was a significant increase in the absolute proportion of choices for an option (FD) in the trinary with respect to the binary context (F1,4 = 7.8, p = 0.049; Figure 4A). The constant-ratio rule (see Data Analysis in Materials and Methods) was also violated as predicted in Table 1, because the preference for FA relative to FD was significantly higher in the binary than in the trinary context (F1,4 = 9.2, p = 0.039; Figure 4A). Finally, against the hypothesis that the effect of decoys on preferences were caused by purely cognitive processes of comparison, there were no significant differences in preferences in the within-treatments comparisons of binary versus trinary trials (repeated-measures ANOVA, all p > 0.1; Figure 5A and 5C).
Figure 4 Between-Treatments Comparison in Binary and Trinary Choice Trials
The bars show the mean (± s.e.) absolute (FD and FA: leftmost and centre pairs of columns in each panel, respectively) and relative (FA*: rightmost pair of columns in each panel) proportion of choices for each option in binary (white bars) and trinary (black bars) trials when intake rate is not controlled (group NC: A and C; white background) or is controlled (group C: B and D; grey background). Relative preferences were calculated using equation 1 (see text). We compared the preference between the same two focal options between the binary context of one treatment (e.g., treatment “High Intake”) and the trinary of the other (e.g., treatment “Low Intake”). In group C (B and D), none of the differences between binary and trinary contexts were statistically significant. For group NC (A and C), the asterisk (*) indicates a significant violation of either regularity or the constant-ratio rule at p < 0.05.
Figure 5 Within-Treatments Comparison of Binary and Trinary Choice Trials
The bars show the mean (± s.e.) absolute (FD and FA) and relative (FA*) proportion of choices for each option in the binary (white bars) and trinary (black bars) trials for group NC (A and C) or group C (B and D). Relative preferences were calculated using equation 1 (see text). We compared the preference for the same options between the binary and trinary contexts of the same treatment (when there are no differential energetic effects). There were no violations of either regularity or the constant-ratio rule (p > 0.1).
The presence of apparent violations when the effect of the decoys on intake was allowed and their absence when the effect was abolished was again consistent with the hypothesis that these apparent irrationalities were caused by differences in intake brought about by exposure to the decoys. The hypothesis is further confirmed by the absence (both for the group that received supplementary feeding and the one that did not receive it) of violations in the within-treatment comparisons, when the cognitive effect of the decoys was allowed but state was neutralised.
In group NC, all birds reached the stability criteria. In group C, however, one bird did not reach the criteria in treatment “Low Intake”, and another did not reach stability in treatment “High Intake”. We therefore reanalysed the data for this group excluding these two birds. The results were the same, namely, in none of the tests for this group was rationality breached.
Discussion
Our aim in this study was to foster the development of a solid interdisciplinary basis upon which to compare research on economic rationality in humans and nonhumans, and to investigate whether normatively inspired hypotheses of animal behaviour may be systematically misleading, as they implicitly assume rationality. To this end, we examined whether violations of economic rationality in animals that have recently been reported in the literature represent real violations of rationality caused by the use of comparative cognitive mechanisms of choice as proposed for humans or, alternatively, to unwittingly imposed differences in the state of the subjects. To do this requires testing whether one can reproduce the reported breaches of rationality and whether they are abolished when cognitive effects are allowed but state differences are eliminated. A further test requires generating such violations by changes of state alone. We have achieved all of these conditions in our experiments.
Why should preferences be modulated by energetic state? To start dealing with this question, it is necessary to start by considering why choices do not go exclusively to the option with maximum value. From an evolutionary perspective, one possibility is that subjects are adapted to some level of ambiguity (for instance, because the properties of options may change with time), and tracking these properties requires some level of response to each available option (Houston et al. 1982). If partial preferences are taken as a given, the next stage is to model the factors that may affect them quantitatively. Here, it is possible that partial preferences depend on the benefits that could be derived from each of the available options and that these benefits depend on the state of the subject. To capture this possibility, the probability of choosing a suboptimal action (in this case FD, which offers a poorer long-term rate of gain) could be modelled as a function of the difference between the benefit accruing from each option while the subject is in a given state. For example, inspired by the “matching law” from behavioural analysis (Herrnstein 1961), Kacelnik (1984) tested the fit of a model termed “profitability matching” for starlings experiencing the conditions of the “marginal value” foraging model. In the model, each strategy is deployed in proportion to the ratio of its payoff relative to the sum of the payoffs of all available alternatives. Functionally, such a strategy, while failing to maximise rate of return, may often approximate the optimal strategy or at least avoid costly deviations from it. As highlighted by other authors (McNamara and Houston 1987), more frequent deviations from the optimal policy should be expected when their costs are smaller.
In Figure 6, we build on this assumption and on the “state” model proposed by Kacelnik and Marsh (2002; see also Marsh et al. 2004) and extend it to illustrate the putative effects of variations in state. We assume that repeated exposure to treatments offering lower and higher objective intake rates (corresponding to decoys DD and DA, respectively) causes some correlated measure of state to be higher in the latter case (Figure 6B). That is, state is assumed to be a positive function of rate of intake during the period preceding the choice itself. We then consider the improvement in state produced by choosing either of the targets (FA and FD). The difference between the improvement in state (ΔS) caused by choosing FA over FD is the same under both treatments, but the biological consequences may differ in magnitude if benefit is not linearly related to state. For the conditions experienced by the starlings in our experiment (where deprivation was very mild), it is reasonable to assume that biological gains were a decreasing function of their initial state (e.g., the contribution of a food item decreases with increasing reserves; see also McNamara and Houston 1982). Figure 6A illustrates this relationship. The figure shows that the cost of choosing the target option with lower long-term rate of energetic gain (FD) is more severe in treatment “Low Intake” than in “High Intake” (|δDD|>|δDA|). Preference for FA should thus be higher under treatment “Low Intake” if the frequency of choices for the leaner focal option is inversely related to their cost. This model is consistent with the equivalence between the effect of supplementary feeding and that of the decoys.
Figure 6 A Functional Model of How State Can Affect Partial Preferences
(A) Fitness is plotted as a concave function of the organism's state. Exposure to DD leads to a poorer state (SdD) than that reached after exposure to DA (SdA) (see also B). SdD + FD and SdD + FA denote the state reached by subjects under treatment “Low Intake” as a consequence of choosing focal options FD and FA, respectively. Similarly, SdA + FD and SdA + FA represent the state reached by subjects in treatment “High Intake” after choosing FD and FA, respectively.
(B) State is assumed to be a growing, linear function of energy intake. DD and DA represent the average intake rates experienced by subjects that include the decoys with the same names in their diet.
Although choosing FA is always better than choosing FD, and the difference between the states caused by this choice is the same under either treatment (SdD and SdA), the fitness difference between choosing FA and FD is higher under treatment “Low Intake” (δDD) than “High Intake” (δDA). This should lead to a higher level of preference for FA in the former treatment if choices of the low-yielding option were to be reduced in proportion to their cost.
From a mechanistic perspective, it is also possible that, under conditions of higher energetic intake, animals are less motivated to search and work for food. Our data support this possibility. The time subjects took to start working once presented with any option in no-choice trials (i.e., their latency to first peck) was significantly longer in the treatment “High Intake” than in “Low Intake” in the experiment without decoys (F
1,8 = 24.6, p = 0.01) and in the experiment with decoys, but only for the group of subjects for which intake was not controlled (group NC: F
1,4 = 39.3, p = 0.003; compare this result to that for group C: F
1,4 = 3.7, p = 0.13). It is thus possible that these potential differences in motivational state led subjects to pay less attention to the alternatives during a choice opportunity in the treatment “High Intake”, resulting in the observed differences in preference levels.
Within this framework, we now use two examples to consider whether unwittingly induced changes in intake between contexts could also underlie previously reported findings of irrational behaviour.
Energetic State and Rationality in Jays
A recent study tested the effect of background context on the foraging preferences of semi-tame food-hoarding grey jays (Waite 2001a). The jays were initially split into two groups, and each group was given 25 binary choices in only one of two backgrounds: In background context A, the jays had to choose between one and three raisins placed 0.5 m inside separate tubes (where distance into the tubes should correlate with perceived risk); in B, jays chose between two identical options, each offering one raisin 0.5 m inside the tubes. Both groups were subsequently presented with the choice between one raisin 0.3 m into one tube and three raisins 0.7 m into another tube. In violation of IIA, context had an effect: Preference for the option offering more raisins at a greater perceived risk was higher in the group that had experienced context B, a result interpreted as consistent with the existence of cognitive biases leading to departures from value maximisation (Waite 2001a). However, experience with the two background contexts and consequent differences in amount of food hoarded (i.e., level of energy reserves for future use) between groups could also have led to the observed results. Those jays that had been in context A had collected approximately 62 raisins, whereas those in context B had collected an average of 25 raisins. Assuming that the state of the jays was such that fitness increased following a decelerated function of accumulated raisins, it is possible that those jays previously presented with lower food supplies had more to gain by choosing the option yielding the larger amount of food. Equating their hoards with energy reserves, one could say that they were “hungrier” in context B and hence afforded greater risks to pick the maximum reward. This trade-off between energetic state and predation risk has been extensively discussed within the behavioural ecology literature (e.g., Houston and McNamara 1999)
Energetic State and Rationality in Hummingbirds
The comparison between binary and trinary choices sometimes employed in studies designed to test economic rationality in animal behaviour can also lead to changes in state. For example, Bateson et al. (2002) compared preferences of Rufous hummingbirds (Selasphorus rufus) among three flower types differing in volume and concentration of sucrose (target: 15 μl, 40% sucrose; competitor: 45 μl, 30%; decoy: 10 μl, 35%) in binary (target and competitor) and trinary (target, competitor, and decoy) contexts. The birds experienced both contexts consecutively. In each of them, they made repeated choices between the available flower types until a minimum number of 150 choices for the target and competitor had been reached. The strength of preferences for the competitor over the target increased significantly in the presence of the decoy (trinary context), and the authors interpreted these results as being inconsistent with the use of absolute evaluation mechanisms as normally postulated by functional accounts of behaviour. Yet, the resulting differences could have been caused by exposure to energetically different contexts. Net rate of energy intake of the target, competitor, and decoy were, respectively, 81.9, 92.0, and 59.5 J/s. Considering, for instance, an average proportion of choices for the decoy of about 20% (as described in the study), subjects would necessarily experience a higher intake rate in the binary than in the trinary context unless they modified the relative allocation of responses. Therefore, if the interval between consecutive foraging bouts did not differ systematically between contexts, cumulative gain along the 150 choices would have been lower in the trinary context, favouring preference for the option offering the higher net rate of intake, as reported. Further analyses on the extent to which state differed between contexts, testing for differences in inter-bout intervals, variability in choices, and unplanned differences in nutrient balance (i.e., in the actual volumes and concentration of sugar and water; see, for example, Simpson and Raubenheimer 1993) experienced in each context in this and other experiments with hummingbirds (e.g., Bateson et al. 2003) are therefore needed before concluding that the results imply violations of rationality rather than compensations for differences in state generated by the introduction of a decoy.
These examples were provided to illustrate that acknowledging and controlling for the effects that differences between choice sets may produce on an organism's state is paramount when investigating the influence of context on choice behaviour. This is particularly important because it is often difficult to predict how changes in state will affect preferences. For instance, our results showed a higher level of preference for the larger but more delayed reward when the starlings were under a poorer schedule—a result also previously reported by some authors (Christensen-Szalanski et al. 1980; Rechten et al. 1983). Conversely, results showing an effect of state in the opposite direction have also been reported in the literature and interpreted as demonstrating greater impulsivity under hungrier conditions (Snyderman 1983; Lucas et al. 1993). From a functional viewpoint, whether lowering energetic state should shift preference towards bigger and later rewards over smaller and more immediate ones or vice versa depends on the details of the problem. While many authors dealing with the problem of temporal discounting focus on one-shot choices, animal experiments are conducted in repeated trials, where delays mean lost opportunity, and where consideration of variance (“risk”) must come into the picture. For example, when the pressing factor is maximisation of rate of intake, greater ITIs have two effects: They alter the state of the subjects (lowering their energy reserves), and they shift the difference in long-term rates in favour of larger, more delayed rewards (a result that may mistakenly be considered a decrease in impulsivity). On the other hand, when risk is the main factor, it is impossible to make a general prediction, because the consequences of variance in both size and delay to reward are functionally sensitive to the curvature of the fitness versus state function, and this is likely to have one or more inflexion points.
Because the directionality of state effects under biologically rational choice is difficult to predict, to demonstrate the presence of true breaches of rationality or to confirm previous findings as evidence of irrationality using these experimental economics paradigms, it is therefore essential not only to investigate the immediate effects of state on preference, but also to ensure that these violations are reproduced and not altered in any direction when state is controlled. Additionally, the observation that rewards received under higher states of need often lead to faster acquisition (Capaldi and Havancik 1973; Tarpy and Mayer 1978; Balleine 1992) makes it fundamental to control for differences in state whenever subjects have to learn the properties of the rewards.
Conclusion
Following the growing body of claims for irrational choice behaviour by human subjects, recent reports on breaches of rationality in animals may be interpreted as questioning the predictive power of the optimality approach in behavioural ecology, favouring the view that the reported inconsistencies result from rigid rules of evaluation and choice leading to the assignment of context-dependent values to options and devaluing the contribution of functional reasoning. We do not doubt that the precise empirical description of decision rules is important. Indeed, findings of locally irrational behaviour are useful tools for the investigation of the mechanisms underlying choices, often forcing a reinterpretation of existing data and models of optimal decision-making. Additionally, the potential dependence of valuation mechanisms on the context of choice might have direct implications for other biological systems. For example, Shafir et al. (2003) have recently emphasised the role of pollinator perception and choice strategies in mediating the evolution of floral nectar distribution strategies, as well as the potential use of knowledge of cognition-mediated mechanisms of choice on the development of biological control programs. Still, if ideas are going to travel safely between economics and biology, crucial details of the experimental paradigms must be scrutinised, and differences between human and nonhuman research must be acknowledged. Here we emphasise that, due to the need of exposing animals to the contingencies of the choice problem, contextual changes may lead to variations in the state of individuals, which in turn can affect both the amount of knowledge acquired by subjects and the parameters of the decision faced by the individuals, thus calling into question the significance of apparent violations of rational axioms.
We do not claim that state dependence accounts for all reported inconsistencies in animal choice (e.g., Waite 2001b; Shafir et al. 2002), nor are we suggesting that animal choices are based directly upon calculations of optimal state-dependent actions instead of direct psychological mechanisms of choice. Indeed, the notion of “rules of thumb” that perform well in most relevant ecological situations, but may also lead to suboptimal behaviour, has been long accepted in behavioural and evolutionary biology, and may well also comprise some of the comparative mechanisms of choice and “fast and frugal” heuristics previously described for human beings (e.g., Gigerenzer et al. 1999). However, we believe that if evolutionarily inspired normative models of behaviour are to be treated fairly, a deep scrutiny of the causes underlying observations of apparent economic irrationality in animal (and, for that matter, human) choices should be attempted. Economic theory has been and still is a source of inspiration for optimality theorising in biology, and experimental economics may just as well inspire understanding of the predictive failure of some of these models. Conversely, the systematic observation of local cases of irrationality in animals may provide insights into the nature of the mechanisms of choice employed by humans. However, our study highlights that at least some apparent similarities in the expression of “maladaptive” behaviours may be due to oversights in the implementation of experiments testing ideas that originate in other disciplines.
Materials and Methods
Our main experiment consisted of training starlings to choose between either three or two simultaneously presented foraging options. Each option was implemented as a coloured, intermittently flashing key that, when pecked once by the subject, caused the other keys to darken, stopped flashing, and then delivered a certain amount of food following the first peck after a programmed delay. The amount and delay to food determined the features of each option. In total, there were four options in the experiment, two forming a “focal pair” of target options and two that were called “decoys”.
Parameters of the options
The actual reward parameters corresponding to the four options are shown in Figure 1. The two focal options, FA and FD, offered a ratio of amount of reward to delay to reward of 0.5 food units per second, while each of the two decoys, DA and DD, offered a ratio of 0.25 units per second (the slopes of the solid lines in Figure 1). We call these ratios “short-term rates” for consistency with previous literature (viz., Bateson and Kacelnik 1996). Short-term rate is known to be strongly correlated to attractiveness, but it is not a description of objective intake rate, because it does not include times other than the delay between choice and outcome. This difference is important and underlies this study. Functional approaches to foraging behaviour, such as classical optimal foraging theory (Stephens and Krebs 1986), have highlighted that energetic gains are a function of total intake over total time. This relationship is expressed by defining the value of an option by its real rate of returns as given by A/(D + ITI), where A is food amount, D is the delay between choice and outcome, and ITI, is the sum of all other times in the foraging cycle. This expression, known as “long-term rate”, is the slope of the broken lines in Figure 1. Any consideration of the effect of energy state on preferences must consider long-term rate, even if the subjects used only short-term rates to form preferences. Scholars concerned with the mechanisms by which stimuli acquire significance (and hence potential attractiveness) to a learning animal, such as the behavioural analysis and conditioning literatures, have focused on the conditions that make the association between the outcome and the predictive event easier. In this case, the predictive event is the onset of the stimulus marking the delay to food, which coincides with the animal's action of pecking at it (Green et al. 1981; Kacelnik 1984; Mazur 1987; Bateson and Kacelnik 1996).
We programmed the alternatives so that the two focal options were as close as possible to being equally attractive and superior to the two decoys, themselves equated in short-term rate. At the same time, we used the fact that the energetic consequences of the two decoys are very different to manipulate energetic consequences.
The parameters of the decoys were chosen to maximise their putative cognitive effect on preferences between the focal options. Several accounts of the effect of poorer alternatives have proposed that they may have an effect because decision-makers compare each attribute of the options (in our case amount and delay) independently, not integrated into a single expression of value. In the conditions described by Figure 1, two putative mechanisms could cause DA to increase preference for FA. One of them, referred to as the comparative model (Shafir et al. 1989; Wedell 1991; Shafir et al. 1993; Tversky and Simonson 1993), postulates that DA could favour FA by means of its asymmetric relationship of dominance (with dominance used as a synonym for superiority) with the focal options. The overall idea is that an option gains value when it is better than other options in the set along a particular attribute. In the present case, DA is dominated by FA in one attribute (delay) and equal in the other (amount), but it is dominated by FD in one attribute (delay) while it dominates it in the other (amount). Thus, if subjects are influenced by the number of relationships of dominance between attributes, FA could be more attractive than FD for being the only option that dominates DA completely. A second mechanism of interest, known as the “range effect”, says that the same difference in a physical attribute can have a greater effect when embedded in a narrower range of values (Parducci 1965). Therefore, the quantitative advantage of an option along a given attribute decreases as a function of the range of values present. For example, if only FA and FD are present, the range of delays is 6 s, and FD's advantage is 100%. When, say, DA is added, the range increases to 16 s, and FD's advantage over FA is only 37.5% of the total range. Since the range in amounts is not modified by adding DA, this again may favour FA. The same two mechanisms could make DD enhance the attractiveness of FD over FA. Note, however, that although these mechanisms provide a possible direction for which differences in preferences could be observed, changes in preference levels between contexts are usually interpreted as being compatible with context dependence regardless of their direction and the number of attributes describing the options (Hurly and Oseen 1999; Bateson 2002; Bateson et al. 2002).
Subjects.
Subjects were 28 naïve starlings captured in Oxford (English Nature licence # 20020068). After capture, the birds were kept outdoors and, during the experiment, transferred to individual indoor cages (120 cm × 60 cm × 50 cm) that served as housing and testing chambers. Lights were on between 0500 and 1900 h, and temperature ranged from 12 °C to 16 °C. Subjects were visually but not acoustically isolated. The experiments took place between June and October 2002.
Apparatus
Each cage had a panel with a central food hopper and three response keys. Computers running Arachnid language (Paul Fray, Cambridge, UK) served for control and data collection. Rewards were units of Orlux pellets, crushed and sieved to an even size (0.025 ± 0.005 g). Automatic pellet dispensers (Campden Instruments, Leicester, UK) delivered rewards at a rate of 1 unit/s. Each option was signalled by a different key colour (red, green, yellow, blue, white, or pink).
Experimental protocol
Subjects were first trained to peck at keys to obtain rewards until all birds pecked at least 80% of the food opportunities. A discrete trials procedure with two types of trials (no-choice and choice) was then employed. No-choice trials provided the birds information about each alternative, but also contributed to their rate of intake. These trials started with one key blinking. The first peck caused the light to stay steadily on. The first peck after the programmed delay had elapsed triggered the delivery of the programmed amount of food, followed by a fixed ITI of 60 s, during which all keys were off. Since food was delivered following the first peck after the end of the designated delay, experienced delays were slightly longer than those programmed (median [interquartile range] in experiment with decoys: treatment “Low Intake”, delay FD = 4.1 s [0.2 s], delay FA = 10.2 s [2.0 s], delay DD = 4.1 s [0.2 s]; treatment “High Intake”, delay FD = 4.1 s [0.2 s], delay FA = 10.2 s [1.8 s], decoy DD = 20.2 s [0.3 s]).
Choice trials began with two or three keys (depending on whether choice was binary or trinary) simultaneously blinking. The first peck on any of them caused the pecked key to turn steadily on and the others to turn off. After that, the trial continued as in no-choice trials. The order and sides in which the options were presented was randomised. After the sessions, subjects were fed ad libitum with turkey crumbs for at least 2 h, then supplemented with ten mealworms, and then food deprived until the beginning of the experimental sessions on the following morning.
In all experiments (except the calibration for discrimination of amounts) we used a within-subjects design with two treatments. The within-subjects design was preferred owing to the high level of variability between individual starlings' energetic requirements, which would have prevented accurate control of energetic state between groups. We therefore focus our analyses on within-subjects comparisons, since variations in the energetic state of subjects between groups of different subjects would hinder the comparison of their preference levels. The pairing of options with colours was balanced across subjects and changed between treatments. Treatment order was balanced across birds. Subjects were given one resting day with ad libitum food between treatments. Each treatment lasted for 20 sessions. Data from the last five sessions were used for analyses.
Discrimination of amounts
In the discrimination experiment, we used a between-subjects design with six male starlings, split into two groups of three birds each. Group 1 chose between 1 and 2 units of food, and group 2 chose between 2 and 5 units. Rewards were delivered after the first peck on the corresponding key. Subjects experienced two sessions per day, at 0800 and 1300 h. Each session consisted of 84 trials, divided into 21 blocks of four trials each: two no-choice (each option presented once) followed by two choice trials.
Effects of intake rate on choice without decoys
To investigate the potential effect of different intake rates on preference between the target options, we used ten birds (five males and five females) in a within-subjects design with two treatments, which differed with respect to the amount of supplementary food delivered to the starlings. One of the treatments simulated the intake effect (total amount of food consumed) of experience with decoy DA (treatment “High Intake”) on 25% of the foraging opportunities (this proportion was established from the average proportion of trials in which the decoy was experienced in a pilot study). The other treatment simulated the intake effect of experience with decoy DD (treatment “Low Intake”) on 25% of all foraging opportunities. To achieve this we delivered unconditionally the reward corresponding to the appropriate decoy once per experimental block (details below), after the ITI that followed the last trial of that block. Unlike the trials with the pair of focal options, no action was needed on the part of the subjects to receive the unconditional reward, nor was any specific discriminative stimulus associated with it.
There were three daily sessions, at 0600, 1000, and 1400 h. Each session consisted of 36 trials, grouped into 12 blocks of three trials each. Each block started with two no-choice trials (each focal option once), followed by an ITI and an unconditional food delivery in which the amount of the simulated decoy was delivered after the delay corresponding to that decoy had elapsed. The third and last trial of each block was a choice between the two focal options.
Test of economic rationality in the presence and absence of controls for intake
Twelve birds were randomly assigned to two groups (intake controlled, or group C, and intake not controlled, or group NC) of six birds each (three males and three females in each group). All subjects experienced two treatments, one with decoy DA (treatment “High Intake”) and another with DD (treatment “Low Intake”). In group C, the differences in intake rate between treatments caused by the exposure to the energetically different decoys were eliminated with supplementary feeding.
Three daily sessions started at 0500, 0900, and 1400 h. Each session consisted of 63 trials, grouped into seven blocks of nine trials each: three no-choice trials followed by six choice trials in a random order (two trinary choices, two binary choices between the focal options, and two binary choices between each focal and the decoy). To equalise intake between treatments in group C, we adopted the following procedure. We calculated the maximum obtainable amount of food and delay per block for treatment “High Intake” (the treatment offering the higher cumulative delay and amount), and in both treatments delivered supplementary rewards up to this amount and delay twice per block. Thus, in every block of trials we equalised intake and total time to the same value in both treatments. The supplement was delivered after the fourth and the ninth trial of each block, after adding the appropriate delay to the ITI. In both treatments, supplements in the middle of the block were followed by a 5-min no-food interval to prevent satiation. Blocks were separated by 10-min intervals.
Data analysis
According to the principle of IIA, the strength of preference between two options should be independent of the presence of other (less preferred) options. These other options may either form part of the general situational background and be absent at the time of the choice, or form part of an enriched set of options at choice time. To test whether differences in background led to breaches of IIA, we compared choice proportions in binary choices (in which the two target options of the focal pair were paired) between the two treatments by conducting separate tests for each group of subjects. To test the temporal effects of potential state changes over the trials in the experimental sessions (i.e., whether the strength of preference between the focal options changed along a session), we calculated the slope of the regression of trial number against (transformed; see below) proportion of choices for FA over FD and tested whether the group of slopes was different between treatments for both groups of subjects.
We also tested for differences in preference between the focal options across contexts, comparing binary with trinary choice trials. We performed two analyses. First, we tested whether the relative strength of preference between the focal options differed between the binary and trinary contexts. Relative preferences were calculated as
where p(FA,FD;{FA,FD,D}) is the relative preference for FA over FD when the alternatives indicated inside the curly brackets were present, and n(FA;{FA,FD,D}) is the number of choices for FA within the same set of alternatives. D stands for either of the decoys (DA or DD). The second term in the denominator follows the same notation. According to a strong probabilistic version of IIA known as the constant-ratio rule (Luce 1959), relative preferences should be the same between binary and trinary contexts.
Second, we compared absolute strength of preference for each of the targets between the two contexts to test for violations of regularity. Regularity is a weaker form of IIA (Luce and Suppes 1965), which asserts that the absolute proportion of choices for an option cannot increase when a new option is added to the choice set. Again, breaches of regularity are usually taken as strong evidence that the value of an option is assigned in a context-dependent way (see Schuck-Paim and Kacelnik [2002] for an intuitive explanation).
We used repeated-measures ANOVA on square-root arcsine–transformed choice proportions, having treatment and order as within- and between-subjects factors, respectively. In all cases we tested the effect of order and interaction between the factors, but neither was significant. The assumptions of normality and homogeneity of variances were not violated for any of the transformed datasets. The Greenhouse-Geiser correction was applied whenever the assumption of sphericity was violated. Tests were always two-tailed. In all conditions, we additionally tested whether the birds' preferences were already stable when the experimental sessions were interrupted. We considered preferences to be stable when the regression of choice proportions (for the focal choice pair in binary choices, where only FA and FD were available) in five consecutive sessions (against session number) was not significant and the standard deviation of these proportions did not exceed 0.20.
We are grateful to Wladimir J. Alonso, Innes Cuthill, Alasdair Houston, John Krebs, Steven Simpson, Tom Waite and the anonymous referees for helpful comments on the manuscript. The paper also benefited from discussions with Tom Waite and the other members of the workshop on “The Limits of Rationality” held at the institute for advanced studies in Berlin in May 2003. English Nature provided us with the licence (20020068) to keep the starlings. Financial support was received from CAPES (CSP), Clarendon Fund from Oxford University Press (LP), and a BBSRC grant (43/S13483) to AK.
Conflicts of interest. The authors have declared that no conflicts of interest exist.
Author contributions. CSP and AK conceived and designed the experiments. CSP and LP performed the experiments. CSP analyzed the data. CSP, LP, and AK wrote the paper.
Academic Editor: André A. Dhondt, Cornell University
¤Current address: Cognitive Ethology Lab, Department of Experimental Psychology, University of Sao Paulo, Brazil
Citation: Schuck-Paim C, Pompilio L, Kacelnik A (2004) State-dependent decisions cause apparent violations of rationality in animal choice. PLoS Biol 2(12): e402.
Abbreviations
DAdecoy amount
DDdecoy delay
FAfocal amount
FDfocal delay
IIAindependence from irrelevant alternatives
ITIinter-trial interval
s.e.standard error
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| 15550984 | PMC529314 | CC BY | 2021-01-05 08:21:17 | no | PLoS Biol. 2004 Dec 23; 2(12):e402 | utf-8 | PLoS Biol | 2,004 | 10.1371/journal.pbio.0020402 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1555098510.1371/journal.pbio.0020403Research ArticleDevelopmentNeuroscienceMus (Mouse)A Chemoattractant Role for NT-3 in Proprioceptive Axon Guidance NT-3 and Proprioceptive Axon GuidanceGenç Barış
1
Özdinler P. Hande
1
¤Mendoza April E
1
Erzurumlu Reha S [email protected]
1
1Department of Cell Biology and Anatomy, Louisiana State University Health Sciences CenterNew Orleans, LouisianaUnited States of America12 2004 23 11 2004 23 11 2004 2 12 e4033 9 2004 23 9 2004 Copyright: © 2004 Genç et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
A Neuron Survival Protein May Give Directions, Too
Neurotrophin-3 (NT-3) is required for proprioceptive neuron survival. Deletion of the proapoptotic gene Bax in NT-3 knockout mice rescues these neurons and allows for examination of their axon growth in the absence of NT-3 signaling. TrkC-positive peripheral and central axons from dorsal root ganglia follow proper trajectories and arrive in close proximity to their targets but fail to innervate them. Peripherally, muscle spindles are absent and TrkC-positive axons do not enter their target muscles. Centrally, proprioceptive axons branch in ectopic regions of the spinal cord, even crossing the midline. In vitro assays reveal chemoattractant effects of NT-3 on dorsal root ganglion axons. Our results show that survival factor NT-3 acts as a short-distance axon guidance molecule for muscle sensory afferents as they approach their proper targets.
In vivo and in vitro experiments suggest a role for NT-3 in guiding axons from the spinal cord to their muscle targets
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Introduction
Neurotrophin-3 (NT-3) is a key requirement for the development of proprioceptive inputs to motor neurons (Chen and Frank 1999; Chen et al. 2003). Mice deficient in NT-3, its tyrosine kinase receptor, TrkC, or in TrkC-positive neuron-specific transcription factor Runx3 display severe ataxia associated with the absence of muscle spindles, and loss of proprioceptive neurons in dorsal root ganglia (DRGs) or their axons (Ernfors et al. 1994; Klein et al. 1994; Tessarollo et al. 1994; Fariñas et al. 1996; Liebl et al. 1997; Inoue et al. 2002; Levanon et al. 2002). NT-3 is expressed in the ventral spinal cord, in the developing limb buds, and in intrafusal bag fibers of muscle spindles later in development (Copray and Brouwer 1994; Fariñas et al. 1996; Tojo et al. 1996). When sensory axons contact developing myotubes, they induce muscle spindle differentiation, forming ring-like spiral nerve endings around them. In the chicken embryo, limb ablations or anti-NT-3 antibody injections into limb buds lead to elimination of TrkC-positive neurons and decreased innervation of motor neurons (Oakley et al. 1995, 1997). Is NT-3 only a chemotrophic survival factor for muscle sensory afferents, or does it have additional roles in the development of the proprioceptors and the establishment of the monosynaptic reflex arc? Here we provide evidence that NT-3 acts as a chemoattractant for sensory axons during the final phase of their target-directed pathfinding.
Results
TrkC-Positive DRG Neurons Are Rescued in Bax/NT-3 Double Knockout Mice
Mice lacking proapoptotic protein Bax allow for distinguishing survival effects of neurotrophins from other effects. Bax-deficient sensory neurons no longer require neurotrophins for survival (White et al. 1998; Patel et al. 2000), thus they can be used to examine axonal effects. We bred NT-3 heterozygote and Bax knockout (KO) mice to obtain mice with double KO of both NT-3 and Bax genes, and examined proprioceptive axonal projections. All NT-3 and double KOs died within 48 h after birth (Tessarollo et al. 1994). We performed TrkA/TrkC double immunohistochemistry (Huang et al. 1999), enabling detection of both proteins in the same sample. TrkC-positive cells (Figure 1A) and fibers (Figure 1E) were absent in NT-3 KOs at embryonic day (E) 15. Two subsets of DRG cells expressing either TrkA or TrkC were detected in double KOs, similar to wild-type (WT) or Bax KO animals. Surprisingly, at postnatal day (P) 0, a few cells expressed TrkC even in NT-3 KO animals in the Bax
+/+ genetic background, and some cells co-expressed TrkA and TrkC, regardless of the genotype (Figure 1B). In order to quantify our results, we analyzed the ratio of TrkA and TrkC immunopositive cells from four different DRGs of animals of different genotypes. At all ages studied, Bax/NT-3 double null DRGs had TrkA/TrkC ratios similar to those of Bax null DRGs, and higher than those of NT-3 null DRGs (Figure 1D). TrkC-positive neurons rescued by Bax deletion, however, failed to differentiate properly, as evidenced by the lack of expression of the proprioceptive molecular marker Parvalbumin (PV) (Figure 1C).
Figure 1 TrkA/TrkC and PV Immunohistochemistry in DRG and Spinal Cord
Red represents TrkA, green represents TrkC (and PV in [C]), and yellow represents co-expression.
(A) TrkA/TrkC immunostaining in E15 DRG. TrkC-positive neurons normally eliminated in NT-3 KOs are rescued in double KOs.
(B) TrkA/TrkC immunostaining at P0 in DRG.
(C) PV immunostaining in P0 DRG. Rescued TrkC-positive cells fail to express PV.
(D) Ratio of TrkA-immunopositive cells to TrkC-immunopositive cells in E15 and P0 DRGs. Data are presented as percentage of cells with standard deviation. Double KOs always had similar ratios to Bax KOs, and NT-3 KOs had the least amount of TrkC-positive cells, if any.
(E) TrkA/TrkC immunostaining in E15 spinal cord. Arrow points to group Ia fibers. Dorsal is up.
Scale bar: 50 μm (A–C), 1 mm (E).
NT-3 Is Necessary for Proper Innervation of Motor Neurons
TrkA/TrkC-positive fibers in the spinal cord could be detected at E15 (Figure 1E). TrkA-positive fibers were restricted to and terminated in the dorsolateral spinal cord, whereas TrkC-positive fibers entered the cord dorsomedially, and descended into the ventral horns in WT (Ozaki and Snider 1997) and Bax KO embryos. There was no detectable TrkC expression in NT-3 KO spinal cord, indicating complete absence of proprioceptive fibers. In double KO spinal cord, TrkC-positive fibers entered the dorsal spinal cord and descended medially in a manner similar to that seen in WT or Bax KO cases. However, it was not possible to follow TrkC immunolabeled fibers all the way to their terminal zones in any of the cases. Next we examined the central projections of DRG axons with the lipophilic tracer DiI at P0. In WT and Bax KO pups, proprioceptive afferents entered the dorsal spinal cord and followed a medial course towards the ventral horn. They then turned laterally towards motor neurons in the lateral motor column, where they branched and terminated (Figure 2A). DiI labeling was confined to dorsal spinal cord in NT-3 KOs (Figure 2A), as reported earlier, consistent with a complete absence of proprioceptive innervation (Ernfors et al. 1994; Tessarollo et al. 1994). In double KOs, proprioceptive afferents initially followed a trajectory similar to that of WT counterparts, but most of them failed to project all the way to the ventral cord and into the lateral motor column. Instead, they arborized near the ventral midline; some crossed the midline and extended into the contralateral ventral cord (Figure 2A and 2C). In order to distinguish between a role for NT-3 in initiation of motor neuron innervation and a role for maintenance, we repeated the DiI labeling at E17. Innervation patterns of E17 spinal cords (Figure 3) were similar to those at P0 (Figure 2). Dorsal horns of all genotypes were filled with DiI-labeled fibers corresponding to nerve growth factor–dependent nociceptive axons. In WT and Bax KO embryos, proprioceptive fibers extended towards ventral horn motor neurons (Figure 3A and 3B), whereas the ventral horns of the NT-3 KO embryos were devoid of innervation (Figure 3C). In the Bax/NT-3 double KOs, DiI-labeled fibers entered the ventral spinal cord, but extended towards the midline instead of the ventral horn (Figure 3D), in a pattern similar to that observed at P0. Our data point to a complete absence of proprioceptive innervation of the ventral horn of the Bax/NT-3 null spinal cord throughout the developmental stages investigated. As the sensory axons never reach motor neuron dendrites in the ventral horn (Figure S1), the stretch reflex arc circuit is not established. The failure to initiate contact between sensory axons and motor neurons in the absence of NT-3 suggests a requirement for NT-3 for proper axon targeting in addition to a role in sensory axon maintenance.
Figure 2 Axonal Projections in the Spinal Cord after DiI Labeling of DRG at P0
(A) Rescued DRG proprioceptive neurons fail to properly innervate motor neurons in double KOs. Instead, some axons are directed towards the ventral midline; they cross the midline and branch.
(B) Schematic drawing of the monosynaptic reflex arc as it normally develops. Small black dots represent NT-3 released centrally by the motor neurons and peripherally by the muscle spindles.
(C) High-power magnification of the inset in (A). Arrow points to the midline, and arrowheads point to synaptic bouton-like structures.
Scale bar: 1 mm (A), 400 μm (C).
Figure 3 Sensory Axons Labeled with DiI through the DRG at E17
(A) DiI-labeled fibers in WT spinal cord. Notice proprioceptive axons extending towards the motor neurons located in the ventral horn of the spinal cord in cross section.
(B) Bax null spinal cord.
(C) NT-3 null spinal cord. Stained fibers are restricted to the nociceptive axons in the dorsal horn, as evidenced by the complete absence of labeling in the ventral spinal cord.
(D) Bax/NT-3 double null spinal cord. Although fibers extend into the ventral spinal cord, they never grow towards the motor neurons, but are directed towards the midline instead.
Scale bar: 100 μm.
NT-3 Is Required for Proper Peripheral Innervation
In order to study peripheral innervation in double KO animals, we investigated spindle development in the gastrocnemius muscle. Muscle spindles could be identified easily in P0 WT and Bax KO animals by their characteristic morphology (Figure 4A and 4B). Proprioceptive fibers labeled with neurofilament-M (NF-M) antibody formed ring-like spiral endings wrapped around intrafusal bag fibers labeled with S46 antibody, specific for slow developmental myosin heavy chain protein (Miller et al. 1985). As reported earlier, there were no muscle spindles in NT-3 KO animals (Ernfors et al. 1994). On the other hand, Bax KO animals had more spindles than WT, and spindles were in clusters similar to animals over-expressing NT-3 in the muscle (Wright et al. 1997). Although NT-3-dependent cells were rescued, no muscle spindles were detected in the limbs of double KOs (Figure 4A and 4B). Muscle spindles could be observed with TrkC antibody in WT and Bax KO animals, but not in NT-3 or double KOs (Figure 4E). In both NT-3 and double KO animals, NF-M-labeled fibers could be detected in muscles, thus the muscles of these animals were not completely devoid of nerve fibers. Since there were no TrkC-positive fibers in these muscles, we think that these NF-M-labeled fibers correspond to motor axons. Absence of muscle spindles might be due to a failure in initiation of differentiation by proprioceptive axons, or a failure of maturation and maintenance in the absence of NT-3. To distinguish between these two possibilities, we investigated muscle spindle development at E15 and E17 with S46/NF-M immunostaining as well as DiI labeling. No structures with the characteristic muscle spindle shape could be detected in any of the genotypes at E15 (Figure S2). However, numerous developing spindles could be identified at E17 in WT and Bax KOs, but not in NT-3 KO embryos (Figure 4C). Bax/NT-3 double null muscles were devoid of spindles (Figure 4C), except for one spindle-like structure observed in one leg of an embryo (Figure 4C, inset, denoted by the asterisk). DiI labeling through the DRGs at E17 yielded similar results, with muscle spindles identified in parallel sections in WT and Bax null muscles (Figure 4D), but not in NT-3 or double KO animals. Although DiI-labeled fibers could be detected in double null muscles, they never formed ring-like structures characteristic of muscle spindles. We also examined TrkA/TrkC expression at P0 in the tibial nerve, which carries sensory fibers to the gastrocnemius muscle as well as the skin of the lower leg. In NT-3 KOs, TrkA-positive axons could be seen in the tibial nerve but there were no TrkC-positive axons; in contrast, TrkA- and TrkC-labeled axons are both present in WT, Bax KO, and double KO animals (Figure 4F). These results suggest that although proprioceptive axons follow proper trajectories in distal peripheral nerves, they fail to innervate their target muscles in the absence of NT-3.
Figure 4 Muscle Spindles in Gastrocnemius Muscle and TrkA/TrkC Staining in the Tibial Nerve at P0
(A) NF-M (red) and S46 (green) immunostaining in cross section of gastrocnemius muscle at P0. There are no muscle spindles detected in double KOs.
(B) NF-M (red) and S46 (green) immunostaining in parallel sections of gastrocnemius muscle at P0.
(C) NF-M (red) and S46 (green) immunostaining in parallel sections of gastrocnemius muscle at E17. Double null muscles are mostly devoid of muscle spindles, except for one spindle-like structure detected (shown in inset, denoted by the asterisk).
(D) Muscle spindles detected by DiI labeling through the DRG. Gastrocnemius muscle is sectioned at 40 μm thickness in parallel plane to the muscle fibers.
(E) Muscle spindles detected by TrkC staining in cross section of gastrocnemius muscle at P0.
(F) TrkA (red) and TrkC (green) immunostaining in the tibial nerve cross section at P0. TrkC-positive fibers are missing in NT-3 KOs. Red-green overlap (yellow) is due to the thickness of the section and overlapping of red- and green-labeled (TrkA and TrkC) fibers present at different focal depths, rather than co-localization.
Arrows indicate muscle spindles.
Scale bar: 50 μm (A, B, D), 25 μm (C, E), 75 μm (F).
NT-3 Is a Chemoattractant for DRG Axons In Vitro
To test the hypothesis that NT-3 acts as a chemoattractant for sensory axons, we performed a series of in vitro assays. Proprioceptive axons in mice enter the gray matter in the spinal cord and advance ventrally parallel to the midline by E13 and reach the motor neurons by E15 (Ozaki and Snider 1994). We co-cultured collagen-embedded E13 WT DRG explants with NT-3-soaked sepharose beads (n = 26). Control cultures were set up using bovine serum albumin (BSA)– or phosphate buffer saline (PBS)–soaked beads (n = 12). DRG axons began extending towards the localized NT-3 source by the end of the first day and consistently displayed a strong chemoattraction by 3 d in vitro, whereas they did not show such preference for BSA-loaded control beads (Figure 5A and 5B). This attraction was not due to survival support of NT-3 because Bax null ganglia displayed the same chemoattraction (Figure 5C; n = 16). NT-3 may act through either TrkC, or the p75NTR. We repeated the co-culture experiments with DRG explants derived from p75NTR KO mice (n = 18). Axons of these ganglia also showed strong chemoattraction towards the NT-3 beads (Figure 5D). Finally, we used diffusible TrkC receptors conjugated to IgG constant regions (TrkC-Fc) added to the medium (n = 6) to deplete soluble NT-3 from the collagen gels (Figure 5E). In the presence of TrkC-Fc the chemoattraction was completely blocked, demonstrating that the effect we see is specific for NT-3. In order to investigate the active range of our beads, we have repeated the cultures with WT E13 DRGs by placing the beads at increasing distances from the ganglia (n = 4 each) (Figure 5F). There was still preferred growth towards the bead at 1,200μm, the longest distance studied, although the number of axons and the extent of growth were not as robust. Next, we set up E13 DRG spinal cord explant cultures using NT-3-loaded beads placed at the midline at mid-spinal cord level as an ectopic NT-3 source (n = 15). Control cultures were set using PBS-loaded beads (n = 6). DiI labeling through the DRGs revealed numerous fibers entering the spinal cord at ectopic regions and growing towards the NT-3 beads (Figure 6A), surrounding the beads and forming bundles around them (Figure 6C–6E). In control cultures, all labeled fibers were directed towards the dorsal spinal cord and terminated there, where they normally enter the gray matter at E13 (Figure 6B). No axons were observed around the PBS-loaded control beads (Figure 6F). Axons that normally enter the gray matter through dorsal spinal cord grow towards the midline when presented with a localized NT-3 source, and new axon growth towards the NT-3 bead is initiated from DRGs, entering the spinal cord at ectopic loci at lateral mid-spinal cord (Figure 6G). NT-3 is therefore capable of acting as a chemoattractant for DRG axons.
Figure 5 Chemoattraction of E13 DRG Axons to Local NT-3 Observed by In Vitro Co-Culture Assays
(A) WT DRG with NT-3-loaded bead.
(B) WT DRG with BSA-loaded bead.
(C) Bax null DRG with NT-3-loaded bead.
(D) p75 null DRG with NT-3-loaded bead.
(E) WT DRG with NT-3-loaded bead and TrkC-Fc in the medium.
(F) WT DRG with NT-3 loaded beads placed at increasing distances away from the ganglia (range, 500–1,200 μm).
Scale bar: 150 μm (A–E), 350 μm (F).
Figure 6 Chemoattraction Towards NT-3 Beads Placed in E13 Spinal Cord DRG Explant Co-Cultures
(A) NT-3 bead placed in the midline of E13 WT spinal cord. Notice axons labeled through the DRGs (circled with black dashed lines) growing towards the bead (circled with white dashed lines) enter the spinal cord at ectopic loci instead of dorsal spinal cord.
(B) PBS-loaded bead in E13 spinal cord. All labeled axons extend along the dorsal spinal cord, where they terminate.
(C) High-power image of the bead in (A). Notice labeled axons surrounding the bead.
(D) High-power image of an NT-3-loaded bead. Notice axons bundled around the bead.
(E) High-power image of an NT-3-loaded bead. Notice the axons approaching the bead via the dorsal spinal cord.
(F) High-power image of a PBS-loaded bead. No labeled fibers were observed around control beads.
(G) Summary of our observations from E13 spinal cord DRG organotypic cultures. In control cultures fibers extend along the dorsal spinal cord, where they normally enter the gray matter at E13. In the presence of an ectopic NT-3 source localized at the midline, these axons grow towards the NT-3 bead. NT-3 also initiates axon growth from the DRGs, entering the spinal cord at ectopic lateral loci, growing towards the bead, surrounding the bead, forming nerve bundles, and branching around it.
Scale bar, 175 μm (A and B), 100 μm (C–F).
Discussion
Relatively few studies have implicated NT-3 as a chemoattractant agent for sensory and motor axons. Previously noted chemoattractant action of the embryonic mouse maxillary process on trigeminal ganglion neurons (Lumsden and Davies 1986) is now attributed to NT-3 and brain-derived neurotrophic factor (BDNF) (O'Connor et al. 1999). Recently, Tucker et al. (2001) showed that developing sensory and motor axons in limb slice cultures preferentially grow towards neurotrophin-soaked beads instead of following their normal trajectories. Conversely, beads soaked with neurotrophin function-blocking antibodies led to reduction of sensory and motor axon growth towards the limb. In transgenic mice, which over-express NT-3 under the nestin promoter in the central nervous system, the course of the proprioceptive afferents are altered and directed towards the regions with high levels of ectopic NT-3 expression in the spinal cord (Ringstedt et al. 1997). Ringstedt et al. considered the possibility that NT-3 may play a chemoattractant role during the innervation of ventral horns by proprioceptive afferents. However, earlier findings of normal proprioceptive afferent trajectories in chicken embryos despite injection of function-blocking NT-3 antibody into the spinal cord (Oakley et al. 1995) led them to discount this possibility. In that study, though, assays on the effectiveness of antibody perturbation on NT-3-dependent cell survival showed that effectiveness was significant (approximately 90%) but not complete (Oakley et al. 1995). Ringstedt et al. (1997) argue that while central sensory axons may still navigate properly in the absence of NT-3, ectopic expression of NT-3 disrupts their targeting. Recently, another member of the neurotrophin family, BDNF, has been suggested to act as a chemoattractant for sensory axons innervating ear (Tessarollo et al. 2004). In a gene replacement strategy in which BDNF expression was driven by the NT-3 promoter, vestibular axons rerouted towards ectopic sources of BDNF in the cochlea that normally expressed NT-3 and were not innervated by these axons. Ectopic NT-3 supplied using osmotic pumps and adenovirus-mediated expression induce sensory axon growth during regeneration (Zhang et al. 1998, Bradbury et al. 1999, Oudega et al. 1999, Ramer et al. 2002), and induce axonal plasticity of corticospinal axons in injured adult spinal cord (Zhou et al. 2003), where the sprouting axons from the intact site cross the midline towards the NT-3 source on the lesion side of the spinal cord. Our present results are in agreement with these observations, and provide further evidence that NT-3 acts as a chemoattractant for sensory afferents.
Previously, normal motor neuron innervation was rescued in NT-3 KO animals by over-expressing NT-3 selectively in their muscles (Wright et al. 1997). It appears that peripheral NT-3 alone is sufficient for rescuing proprioceptive neurons in NT-3 KO animals and proper axonal pathfinding in the spinal cord. However, it is unclear whether NT-3 is absent from the ventral spinal cords of these animals. The possibility has been raised that motor neurons may retrogradely transport NT-3 in muscle to the spinal cord (Chen and Frank 1999). Assays to determine whether any NT-3 is present in the ventral horns of these mice would be informative.
Recently, Patel et al. (2003) reported their observations on Bax/NT-3 double KO mice they bred. Their results are significantly different from ours. They see no proprioceptive afferents in the periphery of the double knockouts and note that central proprioceptive afferents terminate in the intermediate spinal cord without extending ventrally. Their observations are based on PV immunostaining and DiI labeling of the peripheral nerves. We noted that PV immunolabeling is diminished in our Bax/NT-3 double KOs. In various manipulations of the neurotrophins it has been noted that molecular markers for proprioceptive axons such as PV or calcitonin gene-related peptide for nerve growth factor–responsive axons are compromised (Ringstedt et al. 1997; Patel et al. 2000), thus PV immunostaining in Bax/NT-3 double KOs cannot reveal the extent of proprioceptive axons in the periphery.
Seventy-three percent of retrogradely labeled gastrocnemius muscle afferents were reported to be expressing TrkC RNA in the adult rat DRG, although some of these (about 10%) may represent cutaneous innervation (McMahon et al. 1994). Presently, it is not clear whether TrkC protein is also made by cutaneous afferents and if so at what stage in development this receptor is expressed by cutaneous axons. Based on the available evidence showing that all proprioceptive neurons are eliminated in NT-3 or TrkC null mice, we think that TrkC staining in the tibial nerve mostly represents proprioceptive axons in the vicinity of their peripheral target. Altogether, our findings suggest a role for NT-3 in initiation of muscle innervation and spindle differentiation by the proprioceptive axons.
Patel et al. (2003) examined DiI-labeled central DRG axons in the spinal cord of three E17 and two P0 cases, and report that central proprioceptive axons stop in the intermediate laminae, never entering the ventral cord. We have examined nine Bax/NT-3 double KO cases, and often incomplete DiI labeling gives the impression that there are no axons reaching the ventral spinal cord. We have also seen cases similar to theirs (n = 4), but at higher magnification these axons did not have terminal boutons and were not completely labeled. However, with complete fills (n = 5), it was possible to trace these axons into the ventral midline and across to the contralateral side, and visualize terminal boutons at their tips (see Figure 3C). Previously, Arber et al. (2000) reported that members of the Ets family of transcription factors, Er81 and Pea3, are expressed by DRG neurons as well as motor neurons and their target muscle fibers. They found that in the spinal cord of Er81 KO mice, ventral projections of proprioceptive axons were mostly absent, and very few axons made it to the ventral cord. Patel et al. (2003) note that the phenotype they observed in their Bax/NT-3 double KO mice is quite similar to that of Er81 KO mice. They provide evidence that NT-3 induces Er81 expression in DRG explants in vitro. Patel et al. (2003) report that Er81 mRNA expression is diminished (but not abolished) in both NT-3 KO and Bax/NT-3 double KO mice, while their immunohistochemistry shows much less protein expression in the double KO mice. It is highly possible that a small but considerable number of DRG cells express the transcription factor Er81 and that their axons grow beyond the intermediate levels of the spinal cord in Bax/NT-3 double KO mice. While the phenotype of Er81 KO mice is quite dramatic, and most proprioceptive axons stop within the intermediate spinal cord, it is important to note that a few axons still find their way to the ventral spinal cord and target properly to the motor neurons (Arber et al. 2000).
Patel et al. (2003) also present observations from islet2DTA mice, which lack a significant portion of the motor neurons in the ventral cord. In these mice PV immunostaining shows axons in the ventral horns. Patel et al. argue that since motor neurons are absent in these mice, NT-3 secreted by them could not be a signal for proprioceptive axons to enter the lateral motor columns. However, there is no evidence showing that NT-3 mRNA or protein expressed in the ventral spinal cord is exclusively from motor neurons, and there are no available data indicating that in islet2DTA mice, NT-3 expression in the ventral spinal cord is abolished (Yang et al. 2001; Pun et al. 2002). Studies in embryonic mice reported NT-3 mRNA in the ventral horns of the spinal cord, but it is not definitive that both mRNA and protein are expressed solely by motor neurons. In the adult spinal cord, while motor neurons express high levels of NT-3, other cells, including glia, also express it (Zhou and Rush 1994; Dreyfus et al. 1999; Buck et al. 2000). Our present results, along with those from transgenic mice with NT-3 over-expression in ectopic regions of the spinal cord (Ringstedt et al. 1997), argue for a role of NT-3 in chemoattractant axon guidance of proprioceptive axons in the spinal cord.
Finally, in culture assays, we see a strong chemoattraction of DRG neurons to localized sources of NT-3. This response is seen in WT, Bax null and in p75 null DRG explants, and in the absence of any other neurotrophins or target-derived axon guidance molecules. Furthermore, in vitro, sensory axon response to NT-3 does not appear to be dose-dependent (Ringstedt et al. 1997; Tucker et al. 2001). NT-3 is capable of attracting axons along distances of up to 1 mm in collagen gel matrix, covering the physiological range it needs to attract axons during development. Previous studies with exogenous or local applications of NT-3 to developing primary sensory axons have indicated that this neurotrophin can attract and induce axonal branching (Ulupınar et al. 2000; Özdinler and Erzurumlu 2001, Özdinler et al. 2004).
Along the monosynaptic stretch reflex pathway, only Wnt-3 has been implicated as an axon arborization factor in the spinal cord (Krylova et al. 2002). Another molecule, Slit2, expressed in the midline and by motor neurons (Wang et al. 1999) is capable of inducing axonal branching (Nguyen Ba-Charvet et al. 2001; Özdinler and Erzurumlu 2002). DRG neurons express Robo receptors, which bind to Slits, and proprioceptive axons are therefore capable of responding to Slit signals (Wang et al. 1999). Slit2 does not cause repulsion of NT-3-responsive DRG axons in vitro (Nguyen Ba-Charvet et al. 2001), but causes ectopic branching and arborization of trigeminal axons in the brainstem (Özdinler and Erzurumlu 2002). Thus, Slit2 might also be involved in terminal branching of propioceptive axons in the ventral cord and in the midline branching observed in our double KOs.
Lack of PV expression in Bax/NT-3 KO mice suggests that PV expression might be responsible for proper axon targeting and muscle spindle differentiation. Presently we cannot completely rule out this possibility. However, no defects in axon pathfinding along the monosynaptic reflex arc or in muscle spindle differentiation have been noted in PV KO mice, which develop normally and show no apparent changes in their behavior or physical activity (Schwaller et al. 1999). These observations suggest that axonal targeting defects in Bax/NT-3 double KO mice cannot be simply due to lack of PV expression in proprioceptive cells.
Our present results suggest that NT-3 acts as a short-range axon guidance cue for proprioceptive axons centrally and peripherally, as they navigate to their targets using other axon guidance cues. In its absence, these axons terminate in inappropriate loci. However, NT-3 may not be the only molecule that plays a role in targeting and terminal branching of sensory axons in the ventral spinal cord. NT-3 most likely acts cooperatively with other axon guidance molecules or by regulating expression of yet to be identified proprioceptive neuron-specific receptors/ligands for numerous axon guidance cues.
Materials and Methods
Generation of double KOs.
We crossed Bax KO females on a C57BL/6 background (Jackson Laboratory, Bar Harbor, Maine, United States) to NT-3 heterozygote males on a 129 Sv background to generate double heterozygote animals. Progeny was genotyped with PCR, and animals heterozygous for both genes were bred to obtain the double KOs. Primers used for the Bax locus were R661, GTT GAC CAG AGT GGC GTA GG; R662, CCG CTT CCA TTG CTC AGC GG; and R663, GAG CTG ATC AGA ACC ATC ATG. Primers used for the NT-3 locus were NT3A, CGT GGT GAG GTT CTA TTG GCT AC; NT3B, CAG AGC ACC CTG CCC AAA GCA GAG; NT3R, CCT TGA CAA TAC TGA ATG CC; and NEOF, GGG AAC TTC CTG ACT AGG. WT, Bax KO, and NT-3 KO littermates were used as controls. A total of 11 Bax/NT-3 double KO mice were analyzed, of these nine were P0 pups. The p75 colony on a 129 S1 background was received from Jackson Laboratory. We used tail DNA to genotype animals using the primers IMR0013, CTT GGG TGG AGA GGC TAT TC; IMR0014, AGG TGA GAT GAC AGG AGA TC (generic neo primers); IMR0710, TGT TAC GTT CTC TGA CGT GGT GAG; and IMR0711, TCA GCC CAG GGT GTG CAC TC (p75 locus). For embryonic experiments, day of plug positivity was considered E0. All of the protocols used in this study were approved by the Louisiana State University Health Sciences Center Institutional Animal Care and Use Committee and conformed to the National Institutes of Health guidelines for use of experimental animals.
TrkA/TrkC immunohistochemistry.
Frozen spinal cord sections (10 μm thick) were blocked and incubated in a cocktail of rabbit anti-TrkA and goat anti-TrkC antibodies (gift of Dr. Reichardt; Huang et al. 1999), followed by a cocktail of CY3 conjugated donkey anti-rabbit and FITC conjugated donkey anti-goat antibodies (Chemicon, Temecula, California, United States) in the presence of 0.3% TritonX-100 and 10% normal donkey serum. For PV immunohistochemistry, sections were reacted with monoclonal mouse anti-PV antibody (Sigma, St. Louis, Missouri, United States), and developed by Vector fluorescein mouse on mouse kit (Vector Laboratories, Burlingame, California, United States). For quantification purposes, TrkA- and TrkC-labeled cells from four different DRGs of each genotype and age studied were counted, and ratios plotted.
Muscle spindle detection.
Gastrocnemius muscle was dissected out in P0 pups and sectioned longitudinally, or, in some cases, the whole leg at the level of gastrocnemius muscle with tibial nerve was sectioned in cross section. Sections were incubated with monoclonal S46 antibody (gift of Dr. Stockdale) reactive to the spindle-specific slow-tonic myosin heavy chain isoform and developed by Vector mouse on mouse kit as described above, followed by rabbit anti-NF-M antibody (Chemicon) and CY3 conjugated goat anti-rabbit antibody (Chemicon) in a sequential double-labeling protocol. In another method, DiI crystals were placed in DRGs of E17 embryos from different genotypes (n = 2), and the gastrocnemius muscle was isolated and sectioned into 40-μm-thick slices on a vibratome.
DiI labeling.
Spinal cords were dissected out with the DRGs attached, motor root was cut to prevent backfilling of motor neurons, and crystals of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI; Molecular Probes, Eugene, Oregon, United States) were inserted in DRGs. Labeled spinal cords were incubated at 37 °C for 8 d and sectioned on a vibrotome at 100 μm. Sections were observed under the fluorescent microscope and photographed, and images were transferred to Adobe Photoshop, inverted, and adjusted for brightness and contrast. Photoconverted in 0.15% DAB (3,3′-diaminobenzidine; Sigma) in 0.1M Tris buffer (pH 8.2).
In vitro co-culture assay.
DRG explants were derived from Swiss Webster, Bax null, and p75 null mouse embryos (E13). For collagen matrix assays, DRGs were dissected out under a stereomicroscope using tungsten needles. Collagen matrix was prepared with 430 μl of collagen (3 mg/ml dissolved in 0.1M acetic acid, Sigma), 50 μl of 10X DMEM medium, and 2.5 μl of 0.8 M NaCa2, and the pH was adjusted to 7.5. Individual ganglion explants were placed in 24-well plates and covered with freshly prepared collagen. Sepharose beads with an average diameter of 150 μm were used. Beads were washed twice with PBS, air dried, and loaded with 10, 20, 50, or 100 ng/μl NT-3 (Collaborative Research, Chemicon) at 4 °C overnight with constant shaking. For negative control, beads were loaded with BSA (10–100 ng/μl) or PBS. Either a single neurotrophin-loaded bead or a single BSA (or PBS)–loaded bead was implanted about 200–1,200 μm away from the ganglion explant. Collagen-embedded cultures were then placed at 33 °C for 15 min for the matrix to harden. Serum-free culture medium was then added to each well. In cultures with WT DRG and control beads, 10% serum was added to ensure viability of the explants. TrkC-Fc (Regeneron Pharmaceuticals, Tarrytown, New York, United States) was added (20 μg/ml) into the culture medium.
Explant co-cultures
Spinal cords with attached DRGs were dissected out from Swiss Webster mouse embryos at E13, and sectioned into 300-μm-thick slices. Tissue slices were placed on Millicell Tissue Culture Inserts (Millipore, Billrica, Massachusetts, United States). NT-3-loaded (n = 15) or PBS-loaded (n = 6) sepharose beads were prepared as described above, and placed in the midline at mid-spinal cord level. Inserts were placed in six-well plates with serum-free medium at the bottom of the wells, and kept at 33 °C for 3 d in the presence of 5% CO2. Cultures were then fixed with 4% paraformaldehyde in PBS, and small crystals of DiI were placed into the DRGs. Samples were incubated in a 37-°C incubator, allowing the dye to diffuse, and photographed under a Nikon (Tokyo, Japan) inverted epifluoresence microscope.
Supporting Information
Figure S1 Motor Neuron Innervation Initiated in WT E17 Embryos Cannot Be Detected in Bax/NT-3 Double KOs
In some of the samples, motor neurons were labeled by backfilling through the ventral root in addition to the sensory axons labeled through the DRGs.
(A) WT embryo showing proprioceptive axons contacting motor neuron dendrites in the ventral horn, forming synapses.
(B) Bax/NT-3 double null spinal cord. Although labeled fibers enter the ventral spinal cord, they extend towards the midline instead of the ventral horn and never contact the motor neuron dendrites.
(C) High-power image of the inset in (A). Arrows point to the proprioceptive fibers contacting motor neurons (asterisk).
(D) High-power image of the inset in (B). Notice that there are no sensory axons contacting labeled motor neurons (asterisk).
Scale bar: 100 μm (A and B), 50 μm (C and D).
(24 MB TIF).
Click here for additional data file.
Figure S2 S46/NF-M Immunohistochemistry at E15 Gastrocnemius Muscle
Although numerous muscle and nerve fibers were labeled, no muscle spindles could be identified because the characteristic morphology of sensory nerve ending wrapped around muscle bag fiber had not begun to develop in any of the genotypes yet. Scale bar: 25 μm.
(13 MB TIF).
Click here for additional data file.
We thank L. Tessarollo for providing NT-3 KO animals, L. Reichardt for TrkA and TrkC antibodies, F. Stockdale for the S46 antibody, K. Muneoka for sepharose beads, and B. King for maintenance of animals used in this study. We particularly thank E. Frank for many helpful comments and discussions about this study and for sharing unpublished data from his laboratory. Supported by National Institutes of Health/National Institute of Dental and Craniofacial Research grant P01 DE07734.
Conflicts of interest. The authors have declared that no conflicts of interest exist.
Author contributions. BG and RE conceived and designed the experiments. BG, AEM, and PHO performed the experiments. BG, PHO, and RE analyzed the data. BG and RE wrote the paper.
Academic Editor: Joshua R. Sanes, Harvard University
¤Current address: Massachusetts General Hospital, Harvard Medical School Center for Nervous System Repair, Boston, Massachusetts, United States of America
Citation: Genç B, Özdinler PH, Mendoza AE, Erzurumlu RS (2004) A chemoattractant role for NT-3 in proprioceptive axon guidance. PLoS Biol 2(12): e403.
Abbreviations
BDNFbrain-derived neurotrophic factor
BSAbovine serum albumin
DRGdorsal root ganglion
Eembryonic day
KOknockout
NF-Mneurofilament-M
NT-3neurotrophin-3
Ppostnatal day
PBSphosphate buffer saline
PVparvalbumin
TrkC-Fcdiffusible TrkC receptors conjugated to IgG constant regions
WTwild-type
==== Refs
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| 15550985 | PMC529315 | CC BY | 2021-01-05 08:21:21 | no | PLoS Biol. 2004 Dec 23; 2(12):e403 | utf-8 | PLoS Biol | 2,004 | 10.1371/journal.pbio.0020403 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1555098610.1371/journal.pbio.0020405Research ArticleBioinformatics/Computational BiologyGenetics/Genomics/Gene TherapyImmunologyMolecular Biology/Structural BiologyHomo (Human)DNA Methylation Profiling of the Human Major Histocompatibility Complex: A Pilot Study for the Human Epigenome Project Human Epigenome ProjectRakyan Vardhman K
1
Hildmann Thomas
2
Novik Karen L
1
¤Lewin Jörn
2
Tost Jörg
3
Cox Antony V
1
Andrews T. Dan
1
Howe Kevin L
1
Otto Thomas
2
Olek Alexander
2
Fischer Judith
3
Gut Ivo G
3
Berlin Kurt
2
Beck Stephan [email protected]
1
1The Wellcome Trust Sanger Institute, HinxtonCambridgeUnited Kingdom2Epigenomics AGBerlinGermany3Centre National de GénotypageEvry CedexFrance12 2004 23 11 2004 23 11 2004 2 12 e4056 6 2004 23 9 2004 Copyright: © 2004 Rakyan et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
All HEPped Up about Methylation
The Human Epigenome Project aims to identify, catalogue, and interpret genome-wide DNA methylation phenomena. Occurring naturally on cytosine bases at cytosine–guanine dinucleotides, DNA methylation is intimately involved in diverse biological processes and the aetiology of many diseases. Differentially methylated cytosines give rise to distinct profiles, thought to be specific for gene activity, tissue type, and disease state. The identification of such methylation variable positions will significantly improve our understanding of genome biology and our ability to diagnose disease. Here, we report the results of the pilot study for the Human Epigenome Project entailing the methylation analysis of the human major histocompatibility complex. This study involved the development of an integrated pipeline for high-throughput methylation analysis using bisulphite DNA sequencing, discovery of methylation variable positions, epigenotyping by matrix-assisted laser desorption/ionisation mass spectrometry, and development of an integrated public database available at http://www.epigenome.org. Our analysis of DNA methylation levels within the major histocompatibility complex, including regulatory exonic and intronic regions associated with 90 genes in multiple tissues and individuals, reveals a bimodal distribution of methylation profiles (i.e., the vast majority of the analysed regions were either hypo- or hypermethylated), tissue specificity, inter-individual variation, and correlation with independent gene expression data.
DNA is frequently modified by methylation, which can affect its function. The Human Epigenome Project aims to identify, catalog, and interpret DNA methylation throughout the genome
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Introduction
DNA methylation is indispensable for vertebrate genome function. It is involved in diverse genomic processes such as gene regulation, chromosomal stability, and parental imprinting (Bird 2002), and interest in the function of DNA methylation is further heightened by the various human diseases associated with epigenetic dysfunction, a notable example being cancer (Laird 2003). However, the DNA methylation profile of the human genome is still largely a mystery.
The sequencing of the human genome (IHGSC 2001) and creation of a whole-genome map of single nucleotide polymorphisms (SNPs) (Sachidanandam et al. 2001) laid the foundation for the Human Epigenome Project (HEP). For the HEP, we aim to analyse DNA methylation in the regulatory regions of all known genes in most major cell types and their diseased variants, along with producing high-density snapshots of non-genic regions spread evenly across the human genome. Although genome-wide DNA methylation analyses have been performed previously (Costello et al. 2000; Strichman-Almashanu et al. 2002), the HEP is the first systematic whole-genome study of DNA methylation at the sequence level.
As a prelude to the HEP, here we report the results of the HEP pilot study: DNA methylation profiling of the human major histocompatibility complex (MHC). The MHC, located on Chromosome 6 (6p21.3), is one of the most gene-dense regions in the human genome, containing genes with a high diversity of function, many of which are involved in the innate and adaptive immune systems. We chose to analyse the MHC for the pilot HEP study for three main reasons. (i) The MHC is associated with more diseases than any other region of the human genome, and therefore the generated data will be of interest to researchers with diverse biomedical interests. (ii) It is also the most polymorphic region in the genome, and therefore the data will allow study of the potential effects of the loss or gain of cytosine–guanine dinucleotide (CpG) methylation sites (due to SNPs) on gene expression and possibly other phenotypes. (iii) At the time when the HEP pilot study was initiated in 1999 (Beck et al. 1999), the MHC was one of the few regions within the human genome for which finished sequence and annotation were readily available (MHC Sequencing Consortium 1999).
Using an integrated pipeline involving high-throughput bisulphite DNA sequencing, we have determined the DNA methylation levels within the vicinity of the promoter and other relevant regions, such as CpG islands and first exons and introns of 90 genes within the 3.8-Mb MHC region in multiple tissues and individuals. Our analysis reveals a bimodal distribution of methylation levels, tissue specificity, and inter-individual variation. We have also developed matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) assays for high-throughput epigenotyping of the analysed regions. Finally, we have established a publicly available database for the HEP data (http://www.epigenome.org), which integrates, for the first time, epigenetic information with the existing genome annotation.
Results/Discussion
For the DNA methylation profiling of the human MHC, the following regions of interest (ROIs) were chosen: (i) a potential regulatory region for each gene and (ii) the most CpG-dense region of each gene. It is well established that epigenetic modifications at regulatory regions, in particular promoters, correlate with the transcriptional state of the cognate gene (reviewed in Bird 2002). Because the precise locations of promoters within the human MHC were unknown at the time this study was initiated, we surmised that analysing a region from 2 kb upstream to 500 bp downstream of the annotated start codon would, in many cases, include the promoter region. Such regions were designated as “upstream” ROIs. ROIs representing the most CpG-dense region within each gene were defined for the region from 500 bp downstream of the annotated start codon to the end of the gene and did not exceed a total length of 2.5 kb. These ROIs were named “intragenic”. For longer genes, more than one intragenic ROI was chosen. Within each ROI, we used the amplicon with the highest CpG density that could be successfully amplified. Other amplicons, if used, were chosen based on the ranking of their CpG density. Wherever possible, CpG islands associated with genes were included. (CpG islands have been defined by Bird [1986] as a contiguous window of DNA of at least 200 bp in which the G + C content is at least 50% and the ratio of observed over expected CpG frequency is greater than 0.6. We used a slightly stricter definition: regions of at least 400 bp in which the G + C content is at least 50% and the ratio of observed over expected CpG frequency is greater than 0.6.) All known repeat sequences were avoided during amplicon design. Methylation was analysed in seven human tissues—adipose, brain, breast, liver, lung, muscle, and prostate—with multiple samples from different individuals for all tissues (except adipose) (see Table S1).
Figure 1 shows the locations and the coverage provided by the bisulphite PCR amplicons across the 3.8-Mb human MHC in the context of annotated genes, CpG content, CpG islands, and SNPs extracted from the SNP database (http://www.ncbi.nlm.nih.gov/SNP). A total of 253 unique amplicons were successfully analysed (Table 1). On average, the amplicons were 438 bp in length (which is close to the optimum amplicon length for the bisulphite PCR), were relatively GC-rich (average G + C > 50%), and had a high density of CpGs (approximately 1 CpG/31 bp). Ninety genes (i.e., more than 70% of all expressed genes within the MHC) were represented by at least one amplicon. Of the analysed CpG sites, 80% displayed methylation levels that varied (i.e., by more than 20%) either between individuals and/or tissues, suggesting that the potential information content of the selected amplicons was relatively high.
Figure 1 Map of the Human MHC Showing Coverage and Locations of the Bisulphite PCR Amplicons for Which Methylation Data Have Been Generated
Tracks from top to bottom are as follows. (1) CpG content—the proportion of CpGs in 8-kb windows. The expected proportion of CpG dinucletides is 0.04 based on the background base composition of Chromosome 6 (Mungall et al. 2003). (2) Random SNP density in 1,000-bp windows. (3) Location of predicted CpG islands. (4) Bisulphite PCR amplicons. (5) Location of annotated gene structures. Right and left arrows indicate gene structures on the sense and antisense strand, respectively. Official gene symbols are used where available.
Table 1 Amplicon Statistics
a Total number of CpG sites for which methylation levels were determined from forward and reverse sequencing
b Proportion of unique CpG positions analysed that showed a difference of 50% or greater between the minimum and maximum methylation levels observed at that site across all samples
c Proportion of unique CpG positions analysed that showed a difference of 20% or greater between the minimum and maximum methylation levels observed at that site across all samples
d Proportion of unique CpG positions analysed that showed a difference of 20% or greater between the minimum and maximum average methylation levels observed at that site across different tissue types. The mean methylation for a unique CpG in a tissue was calculated by averaging the measurements of the individual samples from that tissue type
Quantification of DNA Methylation by Direct Sequencing of Bisulphite PCR Products
We analysed DNA methylation using bisulphite sequencing (Olek et al. 1996). In the presence of sodium bisulphite, unmethylated cytosines are converted to uracil, whereas methylated cytosines are unreactive under the same conditions. After bisulphite treatment the DNA is subjected to PCR and sequencing. Methylated cytosines are detected as cytosines in the sequencing reaction, whereas all unmethylated cytosines appear as thymidines. Traditionally, bisulphite PCR analysis involves sequencing multiple sub-clones of the bisulphite PCR product. This approach is time-consuming, and there have also been reports of bias (Grunau et al. 2001) and hetero-duplex amplification (Sandovici et al. 2003) during sub-cloning of bisulphite PCR products. We sequenced the bisulphite PCR products directly with the same primers used in the PCR and developed software, called ESME (Lewin et al. 2004), to determine the DNA methylation levels from the sequence trace files. Briefly, ESME performs quality control, normalises signals, corrects for incomplete bisulphite conversion, and maps positions in the trace file to CpGs in the reference sequence. The program calculates methylation levels by comparing the C to T peaks at CpG sites, with the ability to discriminate levels of methylation that differ by as little as 20%. Methylation estimation by ESME at any given CpG site is the average from all the copies generated during PCR and is, therefore, compared to sub-cloning, a more accurate representation of the methylation level. Furthermore, we reanalysed the methylation levels of 77 amplicons by MALDI-MS, which allows for discrimination of methylation levels that differ by as little as 5% (Tost et al. 2003). Figure 2 shows the comparison of the two methods, demonstrating a concordance rate of 88% between ESME and MALDI-MS.
Figure 2 Comparison of Methylation Measurements Obtained Using MALDI-MS with Those from ESME Analysis of Directly Sequenced Bisulphite PCR Products
(A) Comparison of methylation measurements obtained by MALDI-MS (x-axis) with ESME-processed data from sequencing (y-axis). Methylation rates at CpGs from forward and reverse sequencing were binned into ten intervals from zero to one using corresponding MALDI-MS measurements at the same CpGs and in the same tissue samples.
(B) Comparison of methylation measurements obtained from ESME-processed data (x-axis) with measurements from MALDI-MS (y-axis). Methylation rates from MALDI are binned as in (A), using the corresponding methylation values from sequencing. Red lines show the means of the binned rates; bars show the standard deviations. The overall correlation of the data is 0.887. Data points that are not around a methylation rate of zero or one are covered by few measurements because of the bimodal distribution of methylation measurements.
The HEP Database
To make the data generated in this study a publicly available resource, we have designed a Web-based, ENSEMBL-like genome browser (http://www.epigenome.org) that allows easy access to the data from the pilot HEP study (Figure 3A and 3B). Methylation levels calculated by ESME are displayed in a colour-coded matrix. Rows represent the averages of forward and reverse sequences for various tissues while columns represent individual CpG sites. Each matrix square therefore represents the average methylation level at a given CpG site for a given tissue. Multiple data rows are available for all tissues (except adipose). Clicking on a square in the genome browser reveals the level of methylation observed at that particular CpG site (the average of the forward and reverse sequence) and information about the tissue source. Additional annotation includes chromosome coordinates, CpG islands, SNPs, ENSEMBL and high-quality, manually curated Vertebrate Genome Annotation database transcripts, the ROIs, and amplicon and primer sequences. The browser provides a zoom function to view the genomic sequence (Figure 3B), and a link to ENSEMBL facilitates access to additional information and the ENSEMBL search engines. The data from the full-scale HEP will be made available via the same browser, providing a novel public resource for the research community.
Figure 3 The HEP Database
(A) We have created a Web-based, ENSEMBL-like genome browser for displaying HEP data that is publicly available at http://www.epigenome.org. The methylation levels calculated by the ESME software are displayed in the form of a matrix. Each matrix contains the data obtained from all the samples of one amplicon. Each colour-coded square (yellow represents 0% methylation, blue represents 100% methylation, and green represents intermediate levels) within the matrix represents one CpG site. Clicking on a square reveals the tissue source of the sample and the level of methylation observed at that particular CpG site. Grey squares indicate CpG sites for which methylation levels could not be determined. Each row of squares represents all the CpG sites for one sample of a particular amplicon, and the samples are grouped by tissue type. The red bar indicates the genomic region analysed. Also shown are chromosome coordinates, CpG islands, SNPs, and ENSEMBL and high-quality, manually curated VEGA transcript information. The HEP database links to the Ensembl genome browser, providing additional information about the region of interest. The example shows amplicons within the SynGAP 1 gene that correspond to regions that were determined to be hypomethylated (second amplicon from the left), hypermethylated (first and fifth amplicons), and heterogeneously methylated (fourth amplicon). Insufficient data were obtained for the third amplicon.
(B) By using the zoom function, the user can view the complete DNA sequence for the analysed amplicon.
Methylation Profile Characteristics of the MHC
The methylation profile of the human MHC region appears to be strongly bimodal, with over 90% of the amplicons being either relatively hypomethylated (i.e., median methylation of amplicon 30% or less) or relatively hypermethylated (i.e., median methylation of amplicon 70% or greater) (Figures 3A and 4). Re-analysis of a subset of the data by MALDI-MS confirmed the bimodality of the methylation profile (Figure 4). Extensive bimodality of genomic methylation profiles has been observed by several authors (reviewed in Bird 2002). Furthermore, the experiments of Lorincz et al. (2002) suggest that the extremes of methylation profiles may in fact be the most stable states within the genome. Lorincz et al. showed that a high density of methylation at a proviral construct is stably propagated in vivo, whereas a low density of proviral methylation is inherently unstable, with daughter cells harbouring proviral cassettes that are demethylated or de novo methylated. It must be noted that even though the amplicons displayed hypo- or hypermethylated profiles, small variations in the levels of methylation at individual CpG sites within an amplicon were also frequently observed. Although there may be technical reasons for this heterogeneity, numerous studies (using a variety of techniques) have shown that the methylation profile of a given region in vivo is rarely homogenous (Costello et al. 2000; Kondo et al. 2000; Grunau et al. 2001; Cui et al. 2003). The functional outcome of these small variations, particularly when they exist between tissues or individuals, remains to be elucidated.
Figure 4 Bimodal Distribution of DNA Methylation within the Human MHC
(A) Determined by direct sequencing/ESME analysis (based on 86,374 single CpGs in different tissue samples building the median for measurement repetitions).
(B) Determined by MALDI-MS (based on 1,019 MALDI measurements).
Comparison of the methylation values for upstream amplicons (median methylation of 10%) versus intragenic amplicons (median methylation of 86%) revealed that upstream amplicons were more likely to be hypomethylated (p < 0.0001). Interestingly, within the upstream category we found that CpG sites located within the 5′ UTR were less likely to be methylated (median methylation of 7%) than the CpG sites located within 2 kb of the first start codon but not within the 5′ UTR (median methylation of 14%) (p < 0.0001). Within the intragenic category, we found that CpG sites located within introns (median methylation of 84%) were less likely to be methylated than CpG sites located within exons (median methylation of 89%) (p < 0.0001). Whether these significant but small differences reflect any bias for the presence of regulatory elements close to the transcriptional start site or within introns, or some other functional consequence, is currently hard to assess.
Analysis of Heterogeneously Methylated Regions
Fourteen amplicons displayed significant heterogeneous methylation profiles (i.e., median methylation between 30% and 70%) (see Figure 3A). These might represent differentially methylated regions at which parental alleles display reciprocal methylation profiles that are determined by the parent-of-origin of the allele, or regions that were heterogeneously methylated on both alleles. Our sequencing method could not discriminate between these two possibilities, and none of these regions corresponded to known imprinted sites within the human genome. We therefore sub-cloned the PCR products and sequenced individual sub-clones of ten different heterogeneously methylated amplicons and used polymorphisms to discriminate between the parental alleles. The overall methylation profiles determined by sequencing individual sub-clones were consistent with those obtained by direct sequencing of the bisulphite PCR products. None of the six amplicons for which polymorphisms were found showed allele-specific methylation (data not shown). This is consistent with the fact that so far there have been no reports of imprinted regions within the human MHC. Since these amplicons were heterogeneously methylated to a similar extent in samples from various tissues and individuals, they might represent regions where maintenance of a specific epigenetic state is not essential. It is also possible that these regions are located at the boundaries of hypermethylated regions, and, consequently, the methylation levels are “trailing off”. However, both these possibilities contradict models that suggest that the genome prefers to maintain methylation profiles in bimodal states.
Interestingly, a few regions were heterogeneously methylated in some tissues only, suggesting that the tissue sampled was a mosaic of several sub-types among which the methylation profile at certain genes varied, or that the region displays tissue-specific parental imprinting similar to the insulin-like growth factor 2 (IGF2) gene, which is imprinted in all tissues except brain (Pham et al. 1997).
Direct sequencing of the heterogeneously methylated amplicons was unsuccessful in a small proportion of cases. Possible reasons include incomplete bisulphite conversion and genetic polymorphisms within the primer binding site. We also noticed a mobility shift of the sequence in a few cases. This occurs because a population of bisulphite PCR products generated from a heterogeneously methylated region contains a mixture of molecules, some with cytosines at certain CpG sites (i.e., initially methylated) and others with thymidines at those CpG sites (i.e., initially unmethylated). When these PCR products are sequenced directly, the cumulative effect of the molecular weight difference between cytosines and thymidines is that some molecules migrate faster than others during capillary electrophoresis. The sequence trace therefore contains two traces that do not perfectly overlap, resulting in erroneous estimation of methylation levels. Such sequences were excluded from further analyses.
Analysis of CpG Islands
CpG islands are GC-rich regions that contain a high density of CpGs and are positioned at the 5′ ends of many human genes (reviewed in Bird 2002). Although most CpG islands remain hypomethylated throughout development in all tissues (Antequera and Bird 1993), regardless of expression state, a small proportion become hypermethylated during development (reviewed in Bird 2002), and this correlates with transcriptional silencing of the associated gene. In our study, 27 amplicons overlapped CpG islands, and 22 of these (i.e., 80%) were hypomethylated in all tissues examined. Interestingly, this proportion of hypomethylated CpG islands is similar to that reported by Yamada et al. (2004), who analysed the methylation status of CpG islands on human Chromosome 21q and found that 103 out of 149 CpG islands (i.e., 70%) were hypomethylated.
In our study, CpG island amplicons situated in the upstream ROIs were always hypomethylated, whereas hypermethylated CpG island amplicons were found only in the intragenic regions. Among the intragenic CpG island amplicons, those situated at the 5′ end of the gene (i.e., overlapping exon 1, intron 1, or exon 2) were always hypomethylated. A tissue-specific methylation profile was observed for the CpG island situated within exon 3 of the tenascin-XB (TNXB) gene, which was hypomethylated in muscle samples only. This hypomethylation correlates with the temporally regulated and tissue-specific expression of TNXB, which is abundantly expressed in connective tissues. It has been suggested that TNXB has a role in limb, muscle, and heart development (Burch et al. 1995), and, therefore, epigenetic modifications at the TNXB CpG island may have an important regulatory role (tissue specificity of methylation profiles is discussed in more detail below). Interestingly, the CpG island amplicon located within exon 3 of the HLA-G gene spanned a methylation boundary, being hypomethylated at the 5′ end with a sharp transition to a hypermethylated profile at the 3′ end. Overall, the results are consistent with the prevailing model of CpG islands being regions of the genome that are hypomethylated, especially when they occur upstream or within the 5′ end of the gene.
Tissue Specificity of the Methylation Profiles
DNA methylation profiles are complex and dynamic, and can vary with developmental stage, tissue type, age, the alleles' parent-of-origin, and also phenotype or disease state (reviewed in Bird 2002). In particular, the role of DNA methylation in setting up and maintaining tissue-specific expression patterns has received a lot of attention. However, the extent of tissue specificity of DNA methylation profiles is relatively unknown. The HEP pilot study involved the analysis of 32 samples (from different individuals) comprising seven tissues: adipose, brain, breast, liver, lung, muscle, and prostate.
Upon comparison of the amplicon profiles, we found that 10% of all amplicons displayed differential methylation between the tissue types (examples are shown in Figure 5). Of these amplicons, 31% were located in the upstream regions, a proportion that is in the same range as the total number of upstream amplicons relative to intragenic amplicons analysed in this study (see Table 1). We scanned the literature and publicly available gene expression databases to determine whether the cognate genes displayed tissue-specific expression. An example is the complement protein C2 mRNA, which normally has a long 5′ upstream region; in the liver, an additional transcript with a much shorter 5′ upstream region is expressed (Horiuchi et al. 1990). In our study we found that a region that overlaps intron 2 and exon 2 of the C2 gene was hypomethylated in liver samples only (however, this region is downstream of the transcriptional start sites of both forms of C2 mRNA). Another example is DOM3Z, which is ubiquitously expressed but occurs only at very low levels in the lung (Yang et al.1998), and this correlates with a region overlapping exons 4 and 5 of DOM3Z that is hypermethylated in lung (and brain) but hypomethylated in the other tissues examined. It has also been demonstrated that the murine complement factor B utilises differential tissue-specific start sites (Garnier et al. 1995), and in our analysis the human homologue is hypomethylated at a region overlapping exons 3 and 4 only in liver. However, the majority of the genes that were associated with tissue-specific methylation profiles in our study did not show corresponding tissue-specific expression profiles in a previously reported whole human genome expression microarray analysis (Su et al. 2002). Some of these genes are known to be associated with various mRNA isoforms, but detection of such alternative transcripts is quite difficult with conventional microarray analysis and usually requires more detailed analysis. It is also possible that the tissue-specific methylation profiles we observed in adult tissue may hint at tissue-specific expression profiles that existed during early development, or they may be associated with as yet unknown transcripts, e.g., non-coding RNAs. Alternatively, there may be only a modest proportion of genes in which tissue specificity of gene expression is affected by methylation.
Figure 5 Example of METHANE Output Showing Regions That Display Tissue-Specific Methylation Profiles
The top colour-scale bar refers to the degree of methylation (percent). The bottom colour-scale bar refers to the absolute difference in the methylation level observed between tissues at a given CpG site, and is therefore a measure of the confidence level for a CpG site to be defined as a MVP.
(A) The upper matrix represents an amplicon that contains 18 CpG sites within a 386-bp region overlapping exon 3, intron 3, and exon 4 of the complement factor B gene. It is hypomethylated in liver (median methylation is 17%) and hypermethylated in all other tissues examined (median methylation is 100%). The lower matrix shows pairwise comparisons of the methylation values for each CpG site between tissues.
(B) The upper matrix represents an amplicon that contains 19 CpG sites within a 550-bp region overlapping exon 3 and intron 3 of the DAXX gene. It is relatively hypomethylated in breast (median methylation is 64%) compared with the other tissues examined (median methylation is 100%). The lower matrix shows pairwise comparisons of the methylation values for each CpG site between tissues.
Inter-Individual Variation of Methylation Profiles
There is increasing evidence that an individual's epigenetic profile can influence phenotype and susceptibility to various diseases such as cancer, an example of such evidence being a recent report linking the loss of imprinting at the IGF2 locus with an increased risk of developing colorectal cancer (Cui et al. 2003). In our study, nearly all loci displayed some degree of heterogeneity, which probably has no bearing on the differences in genome function among individuals. However, considerable differences in methylation profiles between individual samples within a tissue were observed for a number of amplicons. We calculated a median methylation value for each individual sample and then compared these values within each tissue type for each amplicon. A total of 118 amplicons displayed a difference of greater than 50% between the lowest and highest median methylation values in at least one tissue. Of these amplicons, 76% were intragenic, which is a similar proportion to the overall number of intragenic amplicons (71%; 181 out of 253 amplicons) analysed in the study. This proportion is also similar to the overall proportion of amplicons that showed tissue-specific methylation profiles and were classified as intragenic (69%). Although inter-individual variation for a given amplicon was not observed in every tissue, there was no apparent tissue-specific enrichment for inter-individual variability of methylation profiles.
Examples of amplicons that displayed significant inter-individual variation in methylation profiles include a region overlapping the last exon in CYP21A2 that showed considerable inter-individual variation in prostate (Figure 6A), and a 5′ upstream region of tumour necrosis factor (LocusID 7124) that varied significantly between individuals in liver (Figure 6B). Although the differences could be attributable to the technical variability inherent in our approach or the fact that we did not control for age or sex of the tissue donors, it is also possible that certain genotypes are associated with unique epigenotypes. In a recent study, Van Laere et al. (2003) mapped a porcine quantitative trait locus that affects muscle growth, fat deposition, and heart size to an evolutionarily conserved CpG island within the imprinted Igf2 gene. Pigs inheriting the mutation from their sire had a 3-fold increase in Igf2 expression in postnatal muscle (i.e., the quantitative trait locus is paternally expressed). Furthermore, the mutation abrogated in vitro interaction with a nuclear factor, and this effect was phenocopied following in vitro DNA methylation of the region. Evidence for an interaction between genotype and epigenotype at the IGF2 gene in humans has also recently been reported (Murrell et al. 2004). Of the 3,273 unique CpG sites we analysed, 101 overlapped with known SNPs (relatively evenly distributed over all amplicons), all representing sites at which the CpG was lost (see Figure 1; Table 1). The SNPs were extracted from dbSNP (http://www.ncbi.nlm.nih.gov/SNP) and are annotated in the HEP database. One could postulate that the gain or loss of one or more critical CpG sites may affect the overall methylation profile of a locus and, consequently, promoter activity. Alternatively, non-CpG SNPs located within an epigenetically sensitive regulatory element could also influence the epigenetic makeup of that region. Therefore, mutations in regulatory sequences could influence epigenetic profiles, resulting in altered phenotypes.
Figure 6 Example of METHANE Output Showing Regions That Display Inter-Individual Variation of Methylation Profiles
(A) Example of a region that displays significant inter-individual variation, especially in prostate. The matrix represents an amplicon that contains 27 CpG sites within a 527-bp region overlapping the last exon of the CYP21A2 gene.
(B) Another example of a region that displays significant inter-individual variation. The matrix represents an amplicon that contains 13 CpG sites within a 453-bp region overlapping the 5′ UTR and exon 1 of the tumour necrosis factor gene.
Analysis of Methylation Variable Positions by MALDI-MS
A major aim of the HEP is to identify genomic regions at which DNA methylation profiles display statistically significant variation due to biological or environmental influences. Therefore, based on the tissue-specific and inter-individual variation in methylation profiles discussed above, we were interested in establishing high-throughput assays for epigenotyping. This involved the identification (manually or using the METHylation ANalysis Engine [METHANE]) of methylation variable positions (MVPs), which we define as CpG sites that have statistical power to discriminate between different biological samples or states. In other words, by assaying the methylation state of just a few select CpG sites within a given region, information can be inferred about the tissue source or disease state. Such a high-throughout MVP epigenotyping method was recently developed based on the GOOD assay (Tost et al. 2003). This recently developed epigenotyping assay allows for accurate discrimination of methylation levels that differ by 5% or more. Furthermore, MALDI-MS is a relatively inexpensive method that offers a high degree of automation and integration and that has no requirement for sample purification. Assays for 231 MVPs in 77 amplicons, including all those that displayed differential methylation profiles between different tissue types or inter-individual variability, were designed and analysed in a triplex format (i.e., methylation levels at three independent CpG sites are analysed in one assay). A subset of 11 MALDI-MS assays is shown in Figure 7.
Figure 7 Comparison of Methylation Values Measured in Five Tissues and Eleven Amplicons Using MALDI-MS and ESME Analysis of Directly Sequenced PCR Products
Each column is a tissue sample, each row a CpG site. Data are ordered in blocks by tissue type and amplicons. Positions of measurements for MALDI-MS (A) correspond to those for ESME analysis (B). The methylation values are colour coded from 0% methylation (yellow) to 100% methylation (blue), with intermediate methylation levels represented by shades of green. White indicates missing measurement values.
Comparison of Methylation Profiles with Independent Gene Expression Data
The primary function of epigenetic modifications is to modulate gene expression: a specific combination of epigenetic modifications at regulatory elements, notably promoters and enhancers, influences the transcriptional state of a gene (reviewed in Bird 2002). In many cancers, aberrant epigenetic modifications occur within CpG islands that overlap promoters (some of which are candidate tumour suppressors), which is thought to result in aberrant transcription of the cognate gene, thus contributing to tumour progression.
We compared the amplicon methylation profiles with the human genome expression patterns available from the Genomics Institute of the Novartis Research Foundation Gene Expression Atlas database (http://expression.gnf.org). This publicly available database contains whole-genome mRNA expression data obtained by Su et al. (2002) using human U95A Affymetrix microarray chips. We calculated a median methylation value for each amplicon (see Materials and Methods). As mentioned above, the methylation profiles displayed a bimodal distribution, with more than 90% of the amplicons being either hypomethylated (median methylation of 30% or less) or hypermethylated (median methylation of 70% or greater). Therefore, to perform the analyses we divided the amplicons into two categories: hypomethylated (methylation less than 50%) and hypermethylated (methylation greater than 50%) (see Materials and Methods). We then compared the range of expression values associated with hypomethylated amplicons with those of hypermethylated amplicons. Most genes on the U95 microarray are represented by multiple probes, and, in a few cases, contradictory expression values were obtained for the same gene, in which case the gene was excluded from our analyses. Analyses were performed for liver, lung, and prostate samples only (Figure 8), since appropriate Gene Expression Atlas data were unavailable for the other tissues. For prostate and liver, a significant difference was found between expression levels associated with hypomethylated versus hypermethylated upstream amplicons: hypomethylated upstream amplicons correlated with a wide range of expression levels whereas hypermethylated upstream amplicons correlated with a lack of expression (p < 0.0001 for prostate and p < 0.01 for liver). The intragenic amplicons did not show any correlation between methylation and expression levels (p > 0.3 for both prostate and liver). A list of all upstream amplicons included in the analysis is given in Table S2.
Figure 8 Comparison of DNA Methylation with Gene Expression
Amplicons generated from prostate (yellow), lung (blue), and liver (green) samples were divided into two categories: “upstream” and “intragenic”. The median methylation values for the amplicons were calculated as described in the text, and these were then classified as hypomethylated (median methylation less than 50%) or hypermethylated (median methylation greater than 50%), and plotted against the cDNA microarray expression data available at http://expression.gnf.org (Su et al. 2002). The expression values are expressed as average difference values (ADVs) for each gene. The average difference value is computed using Affymetrix software and is proportional to mRNA content in the sample, with a value of 200 being a conservative cut-off below which a gene can be classified as being not expressed. The average difference values are the mean of 2 or 3 independent experiments. For prostate and liver, the expression levels associated with the hypermethylated upstream amplicons were significantly lower than the expression levels associated with the hypomethylated upstream amplicons (p < 0.0001 for prostate and p < 0.01 for liver). For lung, there was no significant difference between the expression levels associated with the hypermethylated upstream amplicons and those of the hypomethylated upstream amplicons (p > 0.3). There was no correlation between expression and methylation for the intragenic amplicons for any of the three tissues (p > 0.3). The width of the bars is indicative of the number of amplicons in each category: prostate upstream, hypermethylated (n = 9); prostate upstream, hypomethylated (n = 15); prostate intragenic, hypermethylated (n = 109); prostate intragenic, hypomethylated (n = 53); liver upstream, hypermethylated (n = 9); liver upstream, hypomethylated (n = 14); liver intragenic, hypermethylated (n = 115); liver intragenic, hypomethylated (n = 45); lung upstream, hypermethylated (n = 9); lung upstream, hypomethylated (n = 13); lung intragenic, hypermethylated (n = 112); and lung intragenic, hypomethylated (n = 57).
For the lung samples there was no significant correlation between expression and methylation state for amplicons within the upstream or intragenic categories (p > 0.3 for both categories). Although the lung data show the same trend as the prostate and liver data, the lung hypomethylated data contained a number of outlier data points representing very high expression values (as shown in Figure 8 by the unfilled circles). The overall trend of the data suggests that these data may be artefactual, but there is nothing that indicates these data points are not real. These data points were enough to influence the analysis such that we could not find a significant difference in the expression of between hypo- and hypermethylated lung genes. If the data points are real, the lack of correlation for the lung samples may be due to inconsistencies within the expression or methylation datasets for lung. Alternatively, there may be additional regulatory elements that influence the expression state of the analysed genes in the lung.
Overall, the findings are consistent with a model in which the DNA methylation profile of the upstream region of the gene is an informative indicator of the expression of the cognate gene, specifically, in which hypermethylation within the upstream region is associated with transcriptional silencing. Furthermore, the data also suggest that epigenetic modifications within the upstream regions influence the transcriptional state of a significant number of the genes within the MHC. This is supported by the study of Jackson-Grusby et al. (2001) in which they employed homogeneous cultures of primary mouse embryonic fibroblasts and used the Cre-loxP system to conditionally inactivate Dnmt1, an enzyme that methylates DNA. They found that in the absence of Dnmt1, several mouse MHC class I genes showed altered expression profiles.
Concluding Remarks
One of the principal challenges in the post-genomic era is to provide a holistic view of genome function, a challenge which is currently being addressed by several large-scale studies of the transcriptome, proteome, metabolic networks, and haplotype maps. The HEP is therefore timely, since DNA methylation is an indispensable part of the genome's regulatory mechanisms. Here we have described the pilot study for the HEP—DNA methylation profiling of the MHC region—which is the first systematic large-scale study of methylation profiles at the sequence level within a multi-megabase region of the human genome. For this project, we developed an integrated pipeline for high-throughput methylation analysis using bisulphite DNA sequencing, MVP discovery, and epigenotyping by MALDI-MS, and created an integrated database (http://www.epigenome.org) for public access to the data generated by the study. The results from the pilot study demonstrate that a significant proportion of the analysed loci within the MHC show tissue-specific methylation profiles, and inter-individual methylation differences are common. Furthermore, the tissue-specific differences in DNA methylation suggest that epigenetic mechanisms are involved in the use of alternative transcriptional start sites. We have also shown that the generated methylation data allow the identification of MVPs that can be typed with high quantitative resolution and sensitivity using MALDI-MS, providing a tool for large population-based studies and for diagnosing diseases in the future.
The study reported here lays the foundation for the HEP, which aims to analyse the methylation state of the regulatory regions of all annotated genes in most major cell types and their diseased variants. In the first phase, which is well underway, we are analysing the DNA methylation profiles of over 5,000 amplicons (representing a 20-fold scale-up relative to the pilot HEP study reported here) associated with nearly all the annotated genes (approximately 3,000) on human Chromosomes 6, 13, 20, and 22. The excellent genomic annotation available for these four chromosomes, e.g., high-quality transcript information and location of SNPs, will enable us to perform comprehensive analyses linking the epigenetic information gained from the HEP with the underlying genetic information. Samples from over 40 different individuals representing 20 tissues will be used in the study.
The resulting data will generate a map that complements other large-scale efforts that are linking our knowledge of gene sequence and cellular phenotypes: studies involving DNA sequencing, SNPs, histone modifications, and transcriptome and proteomic analyses. The epigenome map will be invaluable for understanding gene regulation and the interactions between genes in normal and disease states. It will offer new explanations in well-studied areas such as cancer research, and will also provide a basis for novel approaches to research on environmental effects, nutrition, and ageing (Eckhardt et al. 2004). The HEP also promises to provide DNA methylation markers for disease states, and new targets for drug development and diagnostic applications based on DNA methylation research are already emerging (Cairns et al. 2001). Current efforts to target the epigenomic machinery of cells with drugs have global effects (Besterman and McLeod 2000; Lubbert 2000; Munster et al. 2001), and more refined approaches will become possible with accumulating knowledge in the new field of epigenomics.
Materials and Methods
Tissue samples.
Human tissue samples were obtained from the National Disease Research Interchange (Philadelphia, Pennsylvania, United States) and consisted of tissue material from healthy individuals. Tissue samples included seven different tissue types (adipose, brain, breast, lung, liver, prostate, and muscle) from 32 different individuals (Table S1). DNA was extracted using standard protocols (Sambrook et al. 1989).
Bisulphite conversion.
Bisulphite treatment of genomic DNA was performed with minor modifications to a method described previously (Olek et al. 1996).
Amplicon design and PCR.
Primers were designed to be at least 21 bases (G + C ≥ 30%) and to contain at least two bases complementary to bisulphite-converted sequence to increase the specificity. To ensure that the primers were not biased for either hypomethylated or hypermethylated sequences, controls were performed using bisulphite-converted unmethylated or in vitro methylated target sequences. PCRs were performed in 96-well plates on MJ Research (Waltham, Massachusetts, United States) thermocyclers in a final volume of 25 μl containing 250 μM dNTPs, 1X PCR Buffer (Qiagen, Valencia, California, United States), 10 pmol each of forward and reverse primer, 1 U Taq polymerase (Qiagen), and 8 ng of bisulphite-treated genomic DNA. Water-only controls were also included in each 96-well PCR plate. The cycling conditions were 95 °C for 15 min followed by 40 cycles of 95 °C for 60 s, 55 °C for 45 s, and 72 °C for 90 s, and a final extension step of 10 min at 72 °C. The PCR amplicons for some of the genomic regions that displayed heterogeneous levels of CpG methylation were sub-cloned using the pGEM-T Easy Vector System according to the manufacturer's instructions (Promega, Madison, Wisconsin, United States). The clones were sequenced on ABI 3700 capillary sequencers (Applied Biosystems, Foster City, California, United States) using ABI Prism Big Dye terminator V 3.1 sequencing chemistry.
Sequencing.
PCR amplicons were purified using MultiScreen PCR plates (Millipore, Billerica, Massachusetts, United States) and sequenced directly in forward and reverse directions with the same primers used in the PCR. Sequencing was performed on ABI 3700 capillary sequencers using ABI Prism Big Dye terminator V 3.1 sequencing chemistry.
Analysis and database generation.
Quantitative methylation rates were estimated from sequence traces using the ESME software (Lewin et al. 2004). This involved appropriate quality control, normalisation of signals, correction for incomplete bisulphite conversion, and mapping of positions in the trace file to CpGs in reference sequences as described in Lewin et al. (2004). Amplicons were mapped to the human genome assembly (NCBI34) using BLAST (Altschul et al. 1990) and CrossMatch (http://www.genome.washington.edu/UWGC/analysistools/Swat.cfm). Using these offsets the positions of CpG dinucleotides were determined in genomic coordinates. Positional data were then loaded into an LDAS database (http://www.biodas.org) suitable for serving to applications using the distributed annotation system (DAS) XML format (Dowell et al. 2001). HEP methylation data converted to DAS format could then be incorporated dynamically into any third-party applications (such as ENSEMBL) capable of understanding DAS. A Web-based, ENSEMBL-like genome browser, driven entirely from DAS data, was created for displaying HEP data and is publicly available at http://www.epigenome.org. The data reported in the pilot HEP study are subject to the HEP's data release policy (http://www.sanger.ac.uk/PostGenomics/epigenome/drp.shtml).
Kolmogorov-Smirnov tests confirmed that the methylation data did not follow Gaussian distribution (p <0.0001 in all cases). We therefore used the Mann-Whitney U test (a non-parametric test that compares the medians of two unpaired groups of samples that do not follow a Gaussian distribution) to perform three different comparative analyses for methylation profiles: (i) upstream versus intragenic amplicons, (ii) within the upstream amplicon category, CpG sites located within the 5′ UTR versus CpG sites not located within the 5′ UTR but still within 2 kb of the first start codon, and (iii) within the intragenic amplicon category, intronic versus exonic CpG sites.
We designed a software package, METHANE, to identify and generate graphical views of MVPs (see Figures 5 and 6). This tool uses the same DAS data source as the Web browser to generate graphical views but adds additional analysis facilities to compare and display relative methylation level differences in pairwise comparisons. METHANE provides options to compare CpG methylation level differences calculated from either simple averages or medians per tissue or per site. METHANE can export data as tabular text output or as images in SVG, PDF, or postscript formats and is available on request from the authors.
Epigenotyping by mass spectrometry.
Assays, based on the GOOD assay for DNA methylation analysis (Tost et al. 2003), were established for amplicons displaying differential methylation between tissue types or inter-individual variability in the sequencing effort. In most cases, three MVPs within an amplicon were simultaneously queried and analysed. Assay volumes started at 3 μl for PCR, with 2-μl additions for shrimp alkaline phosphatase treatment, primer extension, and 5′-phosphodiesterase digest, and the addition of 10 μl of alkylation mix in the respective reaction steps. After dilution with acetonitrile, 0.5-μl samples were transferred to a matrix coated MALDI target plate. All liquid handling was carried out with automated liquid-handling robotics (BasePlate, The Automation Partnership, Royston, United Kingdom). All MALDI-MS analyses were performed with positive ion mode detection on Bruker Autoflex MALDI mass spectrometers (Bruker Daltonics, Billerica, California, United States), equipped with target-plate-changing robots.
To ensure accurate quantification and to compensate for the common preferential amplification in bisulphite-treated DNA, triplicate calibration standards from 0% to 100% methylation in 25% increments were included into the analysis. Statistical parameters were defined to compensate for factors complicating quantification by MALDI-MS, such as bad reproducibility of the crystallisation, different rates of ionisation of analytes, and signal-to-signal interactions. Quantitative results were obtained with high accuracy when 200 laser shots were accumulated on a sample spot, and eight preparations accounted for differences in the sample preparations. Thus, in total, a quantitative data point is obtained from the average of 1,600 individual spectra and calibrated with the help of mixtures with a known degree of methylation. Success rates are above 97%, and standard deviation for data points is less than 2%.
Comparison of DNA methylation with mRNA expression levels.
The methylation data were compared with data in the Gene Expression Atlas database (Su et al. 2002) that contains human transcript data based on the U95 build of Unigene, Affymetrix U95A chip. We calculated methylation values for each amplicon using the median of the methylation values for each CpG site within that amplicon. A quantile plot indicated a strongly non-normal distribution of the data, specifically, platykurtosis (one of two different kinds of kurtosis) demonstrating a bimodal distribution. Therefore, for the purposes of simplified statistical analysis, amplicons were classified as either hypermethylated or hypomethylated dependent on whether their median methylation value was either greater than or less than 50%, respectively. We performed methylation versus expression comparisons after removing the data that corresponded to multiple probes that gave contradictory expression patterns for the same gene on the U95 microarray. Equality of mean expression levels between hypermethylated and hypomethylated datasets was tested using a Welch two-sample (unpaired) t-test.
Supporting Information
Table S1 Tissues Used in the HEP Pilot Study
(63 KB DOC).
Click here for additional data file.
Table S2 Upstream Amplicons Included in the Comparative Analysis of DNA Methylation with mRNA Expression
(32 KB DOC).
Click here for additional data file.
Accession Numbers
The LocusLink (http://www.ncbi.nlm.nih.gov/projects/LocusLink/) accession numbers for the genes and gene products discussed in this paper are C2 (LocusID 717), CYP21A2 (LocusID 1589), DOM3Z (LocusID 1797), HLA-G (LocusID 3135), IGF2 (LocusID 3481), TNXB (LocusID 7148), and tumour necrosis factor (LocusID 7124).
This work was supported by a grant of the European Union (EU-FP5 QLRI-CT-2000–00417). VKR was supported by a CJ Martin Fellowship from the National Health and Medical Research Council of Australia. AVC, TDA, KLH, and SB were supported by the Wellcome Trust. JT and IGG were supported by the French Ministry of Research. We thank Paul Bevan for help with the http://www.epigenome.org Web site, Rob Davies for implementing ESME software at the Sanger Institute, Matthew Francis and Mark Griffiths for providing data analysis scripts, Sarah Lindsay for sequencing, and Lianne Stanford for assistance with statistical analysis. We also thank the four anonymous reviewers for helpful suggestions.
Conflicts of interest. JL, KB, TH, TO, and AO are employees of Epigenomics AG. IGG and SB are members of the scientific advisory board of Epigenomics AG, but do not benefit financially from their involvement in this study. Epigenomics AG has filed for patents based on some of the work described here. Epigenomics AG was a scientific collaborator in the study. The work described here was funded by a grant from the European Union Framework 5 Programme.
Author contributions. VKR, TH, KLN, AO, SB, KB, and IGG conceived and designed the experiments. VKR, KLN, JL, and JT performed the experiments. VKR, TH, KLN, JL, DA, KLH, AVC, SB, JT, KB, and IGG analyzed the data. VKR, JL, TO, DA, KH, and AVC contributed reagents/materials/analysis tools. VKR and SB wrote the paper.
Academic Editor: Peter B. Becker, University of Munich
¤Current address: Cancer Genetics Group, Genome Sciences Centre, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada
Citation: Rakyan VK, Hildmann T, Novik KL, Lewin J, Tost J, et al. (2004) DNA methylation profiling of the human major histocompatibility complex: A pilot study for the human epigenome project. PLoS Biol 2(12): e405.
Abbreviations
CpGcytosine–guanine dinucleotide
DASdistributed annotation system
HEPHuman Epigenome Project
IGF2
insulin-like growth factor 2
MHCmajor histocompatibility complex
MALDI-MSmatrix-assisted laser desorption/ionisation mass spectrometry
METHANEMETHylation ANalysis Engine
MVPmethylation variable position
ROIregion of interest
SNPsingle nucleotide polymorphism
TNXB
tenascin-XB
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| 15550986 | PMC529316 | CC BY | 2021-01-05 08:21:17 | no | PLoS Biol. 2004 Dec 23; 2(12):e405 | utf-8 | PLoS Biol | 2,004 | 10.1371/journal.pbio.0020405 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1555098710.1371/journal.pbio.0020408Research ArticleGenetics/Genomics/Gene TherapyNeuroscienceDrosophila
Lmo Mutants Reveal a Novel Role for Circadian Pacemaker Neurons in Cocaine-Induced Behaviors PDF Neurons and Drosophila Cocaine BehaviorsTsai Linus T.-Y
1
Bainton Roland J
2
Blau Justin
3
Heberlein Ulrike [email protected]
4
1Department of Anatomy, Program in Neuroscienceand Medical Science Training Program, University of California, San Francisco, CaliforniaUnited States of America2Department of Anesthesia, University of CaliforniaSan Francisco, CaliforniaUnited States of America3Department of Biology, New York UniversityNew York, New YorkUnited States of America4Department of Anatomy, Programs in Neuroscience and Developmental BiologyUniversity of California, San Francisco, CaliforniaUnited States of America12 2004 23 11 2004 23 11 2004 2 12 e4087 5 2004 24 9 2004 Copyright: © 2004 Tsai et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
This Is Your Fly's Brain on Drugs
Drosophila has been developed recently as a model system to investigate the molecular and neural mechanisms underlying responses to drugs of abuse. Genetic screens for mutants with altered drug-induced behaviors thus provide an unbiased approach to define novel molecules involved in the process. We identified mutations in the Drosophila LIM-only (LMO) gene, encoding a regulator of LIM-homeodomain proteins, in a genetic screen for mutants with altered cocaine sensitivity. Reduced Lmo function increases behavioral responses to cocaine, while Lmo overexpression causes the opposite effect, reduced cocaine responsiveness. Expression of Lmo in the principal Drosophila circadian pacemaker cells, the PDF-expressing ventral lateral neurons (LNvs), is sufficient to confer normal cocaine sensitivity. Consistent with a role for Lmo in LNv function, Lmo mutants also show defects in circadian rhythms of behavior. However, the role for LNvs in modulating cocaine responses is separable from their role as pacemaker neurons: ablation or functional silencing of the LNvs reduces cocaine sensitivity, while loss of the principal circadian neurotransmitter PDF has no effect. Together, these results reveal a novel role for Lmo in modulating acute cocaine sensitivity and circadian locomotor rhythmicity, and add to growing evidence that these behaviors are regulated by shared molecular mechanisms. The finding that the degree of cocaine responsiveness is controlled by the Drosophila pacemaker neurons provides a neuroanatomical basis for this overlap. We propose that Lmo controls the responsiveness of LNvs to cocaine, which in turn regulate the flies' behavioral sensitivity to the drug.
Expression of the Drosophila LIM-only (LMO) gene in circadian pacemaker cells is required for the normal behavioral sensitivity of these animals to cocaine
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Introduction
Cocaine, a naturally occurring plant alkaloid, is the prototype addictive psychomotor stimulant. It elicits a variety of acute behavioral changes ranging from mood elevation, disinhibition, and motor activation at low doses to compulsive stereotypies and psychosis at higher doses (Gawin 1991). Long-term cocaine use generally results in tolerance to many of its subjective effects, an increased craving towards the drug, and, eventually, drug abuse and addiction.
Cocaine's primary mechanism of action is to bind and inhibit plasma membrane monoamine transporters, thereby increasing synaptic monoamine neurotransmitter levels and potentiating their actions. Cocaine's direct role in increasing dopamine (DA) in the nucleus accumbens via inhibition of the DA transporter (DAT) has contributed to the prevalent DA hypothesis of drug addiction, which posits that the shared ability of drugs of abuse to increase DA in the nucleus accumbens underlies their reinforcing properties (Wise and Bozarth 1985; Kuhar et al. 1991). More recent animal studies, however, have shown that cocaine still elicits a robust conditioned place preference in mice with genetic deletions of DAT, suggesting that the rewarding properties of cocaine are not solely mediated by its action on DAT (Sora et al. 1998). Additional studies on mice in which the serotonin transporter or norepinephrine transporter has been deleted, together with studies using selective serotonin transporter or norepinephrine transporter inhibitors, have suggested roles for both serotonin and norepinephrine systems in mediating cocaine's rewarding properties (Uhl et al. 2002). These data show that the molecular bases for cocaine's psychostimulant and reinforcing properties are more complicated than once thought, and that a combination of actions at multiple sites may mediate its effects. Indeed, multiple genes and signaling pathways have been implicated in the stimulant and rewarding properties of cocaine in mice (Laakso et al. 2002).
The fruit fly Drosophila melanogaster has been advanced as a useful model system for identifying novel genes that regulate behavioral responses to drugs of abuse including cocaine (Wolf and Heberlein 2003). Several of cocaine's most characteristic properties have been recapitulated in flies. First, cocaine induces motor behaviors in flies (see below) that are remarkably similar to those observed in mammals (McClung and Hirsh 1998; Bainton et al. 2000). Second, repeated cocaine administration induces behavioral sensitization (McClung and Hirsh 1998), a form of behavioral plasticity believed to underlie certain aspects of addiction (Robinson and Berridge 1993; Schenk and Partridge 1997). Finally, a key role for dopaminergic systems in mediating cocaine's effects has been demonstrated through both pharmacologic and genetic methods (Bainton et al. 2000; Li et al. 2000). More importantly, Drosophila studies have identified genes and pathways whose role in cocaine responsiveness had not been anticipated (Hirsh 2001; Rothenfluh and Heberlein 2002). For instance, the Drosophila circadian gene period was identified as a necessary mediator of cocaine sensitization (Andretic et al. 1999). Subsequently, mice carrying various period mutations were found to have altered cocaine sensitization and conditioned place preference (Abarca et al. 2002).
In order to identify novel molecules and pathways involved in behavioral responses to cocaine, we carried out a genetic screen for Drosophila mutants with altered acute responses to cocaine. Here, we report the phenotypic and molecular characterization of mutations in the Drosophila LIM-only gene, Lmo (also called Beadex [Bx]), isolated due to their increased sensitivity to cocaine-induced loss of negative geotaxis. The products of Drosophila and mammalian Lmo genes modulate the function of LIM-homeodomain (LIM-HD) proteins (Milan et al. 1998; Retaux and Bachy 2002), which in turn regulate various aspects of nervous system development, including the specification of neural identity (Thor and Thomas 1997; Hobert and Westphal 2000; Shirasaki and Pfaff 2002; Tsalik et al. 2003). We find that cocaine sensitivity is inversely related to the levels of Lmo function: reduced function causes increased sensitivity, while increased function causes resistance. This bidirectional regulation of cocaine responsiveness is mediated by Lmo function in a small set of neurons, the ventral lateral neurons (LNvs), which are the primary circadian pacemaker cells in Drosophila (Helfrich-Förster 1997; Renn et al. 1999). Like other mutants that affect LNv function, Lmo mutants show altered circadian locomotor behaviors. However, the roles of the LNvs in regulating cocaine sensitivity and circadian behavioral rhythms are genetically separable, as mutants lacking the neuropeptide PDF—the only known functional output of these neurons—show normal cocaine sensitivity. Thus, Lmo defines a novel role for the circadian pacemaker cells in regulating behavioral responses to cocaine.
Results
Loss- and Gain-of-Function Mutations in the Lmo Locus Show Altered Cocaine Sensitivity
To identify novel molecules involved in cocaine-related behaviors, we carried out a genetic screen for mutants with altered acute responses to volatilized freebase cocaine using the crackometer, a simple assay that measures cocaine-induced loss of negative geotaxis (Bainton et al. 2000). Screening of 400 first chromosome P-element insertions from the EP collection (Rørth 1996) led to the identification of five mutants with reduced cocaine sensitivity and seven with increased sensitivity. Two of the mutants conferring increased cocaine sensitivity, EP1306 and EP1383 (Figure 1A), carry a P-element insertion in the promoter region of the Drosophila Lmo locus (Milan et al. 1998; Zeng et al. 1998). Drosophila LMO protein has been shown to inhibit the activity of the LIM-HD transcription factor apterous through its interactions with the LIM-HD activator Chip (Milan and Cohen 1999; Weihe et al. 2001).
Figure 1
Lmo Loss-of-Function Mutants Show Increased Sensitivity to Cocaine
(A) Cocaine phenotypes of various Lmo mutants. Male flies hemizygous for the indicated Lmo alleles (and their appropriate genetic controls) were exposed to 150 μg of cocaine and tested in the crackometer as described in Materials and Methods. Compared to their control (Ctl-1), EP1383 (p < 0.02) and EP1306 (p < 0.001) flies show significantly increased sensitivity to cocaine. Similarly, compared to their respective controls, pdrm and hdp flies are significantly more sensitive to cocaine (p < 0.001). Asterisks denote significant differences from controls (Student's paired t-test assuming equal variance); n = 20 experiments.
(B) Cocaine dose–response. EP1306 flies (filled squares) and pdrm flies (filled circles) and their respective controls were exposed to the indicated doses of cocaine. At each dose, the responses of EP1306 and pdrm flies are significantly higher than their controls (p < 0.001, n = 16–20 experiments).
(C) EP1306 flies show alterations in cocaine-induced locomotor patterns of activity. Flies were exposed to 0, 75, or 100 μg of cocaine, as indicated, for 1 min. Representative traces shown correspond to 30 s of recorded activity of about ten flies starting 1 min after the end of cocaine exposure (n ≥ 4). Top panels show response of control flies to indicated amounts of cocaine; bottom panels show activity of EP1306 flies after cocaine administration. Ctl-1 is EP1631, Ctl-2 is P[GAL4] line 8.142, and Ctl-3 is w1118.
Independently, we isolated a P[GAL4] insertion in the Lmo locus, dubbed pipedream (pdrm), that also showed increased cocaine sensitivity (Figure 1A). Finally, heldup (hdp) mutant flies, which carry well-characterized loss-of-function mutations in Lmo (Milan et al. 1998), displayed increased sensitivity to cocaine that is similar in magnitude to that of the EP1306 line (Figure 1A). This increased sensitivity was observed at all cocaine doses tested (Figure 1B and 1C; data not shown). The EP1306 and hdp alleles consistently demonstrated stronger phenotypes than EP1383 and pdrm. All mutants tested performed within the normal range in task-specific baseline behaviors (see Materials and Methods). Finally, we were able to revert the cocaine phenotype of EP1306 by excision of the P element (data not shown). Taken together, these data demonstrate that loss-of-function mutations in Lmo result in increased sensitivity to cocaine.
The dominant Beadex (Bx) mutations were first described in 1925 by Morgan, Bridges, and Sturtevant, and named for their characteristic beaded wing margin (Morgan et al. 1925). More recently, Bx mutants were shown to be overexpression alleles of Lmo (Milan et al. 1998; Shoresh et al. 1998; Zeng et al. 1998). Overexpression results from the insertion of naturally occurring transposable elements into the 3′ UTR of Lmo, which leads to transcript stabilization (Shoresh et al. 1998). The resultant overexpression of Lmo causes decreased apterous activity in the dorsal compartment of the wing and, consequently, disorganization of the wing margin (Milan and Cohen 1999, 2000).
We tested multiple available Bx alleles, and found that they all caused resistance to the acute effects of cocaine at every dose tested (Figure 2A and 2B). The strength of the cocaine resistance phenotype correlated well with the severity of the beaded wing-margin phenotype, BxJ > Bx1 = Bx2 = Bx3 (Figure 2A and 2B; data not shown). Because in Bx mutants Lmo transcripts are stabilized in their normal spatiotemporal pattern (Shoresh et al. 1998), the observed cocaine phenotypes likely result from overexpression, rather than from misexpression of Lmo. Taken together with the loss-of-function effects described above (see Figure 1A and 1B), these data reveal a graded behavioral response to cocaine that is inversely related to Lmo levels.
Figure 2
Lmo Gain-of-Function Bx Alleles Show Reduced Sensitivity to Cocaine
(A) Cocaine phenotypes of Bx mutants. Male flies hemizygous for Bx alleles Bx1 or BxJ show significant reductions in sensitivity to cocaine compared to control (Ctl) flies (p < 0.001, n = 12 experiments). Asterisks denote significant differences from control (Student's paired t-test assuming equal variance).
(B) Dose–response. BxJ flies (filled circles) show reduced sensitivity compared to Ctl flies (open circles) at all doses tested (p < 0.001, n = 16–20 for all doses except for 250 μg, where p = 0.0015, n = 8). Two additional Bx alleles (Bx2 and Bx3) had similar phenotypes to Bx1 (not shown).
(C) BxJ flies show alterations in cocaine-induced locomotor patterns of activity. Flies were exposed to 0, 100, or 125 μg of cocaine, as indicated, for 1 min. Representative traces shown are 30 s of recorded activity of about ten flies starting 30 or 60 s after the end of cocaine exposure (n ≥ 3). Top panels show response of control flies to indicated amounts of cocaine; bottom panels show activity of BxJ flies after cocaine administration. Ctl flies are w1118.
In order to study Lmo mutant behavior in more detail, we recorded the cocaine-induced patterns of locomotor activity of control and Lmo flies using a movement tracking system (Bainton et al. 2000; Wolf et al. 2002). Upon mock exposure, control flies showed robust locomotor activity, generally walking in straight lines (Figure 1C, top left panel). At relatively low doses of cocaine (75 and 100 μg), flies engaged in stereotyped circling patterns of activity (Figure 1C, top right panels). At higher doses (125–200 μg), flies began to show spasmodic movements (seen as zigzag patterns in movement) and severe hypokinesis (data not shown; Bainton et al. 2000). EP1306 flies, while showing a reduced baseline speed, showed normal patterns of activity upon mock exposure (Figure 1C, bottom left panel). Upon exposure to cocaine, these mutant flies showed a shift in the dose–response relationship. At low doses (75 μg), EP1306 flies showed a marked increase in stereotyped circling behavior compared to controls (Figure 1C, bottom middle panel), while at higher doses (100 μg), the mutant flies were more likely to be spasmodic or akinetic (Figure 1C, bottom right panel). Thus, despite the reduced speed observed in the mock exposures, increases in specific cocaine-induced locomotor behaviors demonstrate that EP1306 flies have a shifted dose–response to cocaine: mutant flies exposed to 75 μg of cocaine behaved similarly to control flies exposed to 100 μg of the drug.
BxJ flies also showed changes in walking patterns that are consistent with a shift in the cocaine dose–response relationship. After exposure to 100 μg of drug, most control flies showed slow circling behavior and some were akinetic (Figure 2C, top middle panel). BxJ flies were much less affected, showing increased locomotion (mostly in straight lines), decreased slow circling, and almost no akinesia (Figure 2C, bottom middle panel). At 125 μg, control flies showed very little movement (Figure 2C, top right panel), while most BxJ flies continued to show circling behaviors seen in control flies at lower doses (Figure 2C, bottom right panel). BxJ flies, like EP1306 flies, showed reduced activity upon mock exposure (Figure 2C, bottom left panel). Despite this reduced activity, these two mutants showed very different sensitivities to cocaine: EP1306 flies were more affected and BxJ flies were less affected, suggesting that the reduced activity upon mock exposure is unrelated to the cocaine response. Long-term activity recordings revealed no differences between BxJ, EP1306, and control lines (data not shown; see below).
In summary, using two different behavioral assays we show that flies carrying loss-of-function mutations in Lmo display increased sensitivity to the effects of cocaine on locomotor behaviors, while gain-of-function mutations show the converse effect, a reduced response to the drug. This inverse relationship between Lmo gene activity and drug responsiveness suggests that Lmo may regulate the expression of genes that might play a direct role in controlling cocaine responses (see Discussion).
Molecular Characterization of Lmo Mutants
The Lmo locus produces at least three transcripts by differential promoter use (Figure 3A). The RA and RC transcripts differ only in their 5′ UTRs and are predicted to encode identical proteins of 313 amino acids; the RB transcript is predicted to utilize an alternative translational start site in its first exon, which would result in an addition of 71 N-terminal amino acids. The insertions that produce increased cocaine sensitivity all lie within the putative promoter region for the RA transcript, 25–90 bp upstream of its transcriptional start site (Figure 3A). In order to identify molecular changes caused by these insertions, we assessed Lmo transcript levels by quantitative RT-PCR in wild-type flies and Lmo mutants. In wild-type flies the RA transcript was about 4-fold more abundant in heads as compared to bodies, suggesting that this transcript is enriched in the nervous system (Figure 3B; see also Figure 4). Importantly, the RA transcript was reduced by greater than 50% in the heads, but not the bodies, of EP1306 flies (Figure 3B); a 40% increase in the RA transcript was observed in the heads of BxJ flies (data not shown). Thus, as predicted by the location of the P-element insertion, the EP1306 mutation causes a reduction in Lmo transcript levels, a finding that is consistent with the observation that the cocaine sensitivity of EP1306 flies is similar to that seen with known loss-of-function alleles of Lmo (the hdp alleles) and opposite to that seen with gain-of-function Bx alleles. In addition, the finding that transcript levels are specifically reduced in the heads of EP1306 flies, and not their bodies, suggests that this mutation affects a nervous-system-enriched (or -specific) Lmo transcript (see below). Finally, none of the P-element insertions in the promoter of the RA transcript leads to a held-up wing phenotype, which is characteristic of loss-of-function hdp alleles (Shoresh et al. 1998; Milan and Cohen 1999). This suggests that the P-element insertions isolated in our genetic screen cause either less severe or more spatially restricted changes in Lmo gene expression.
Figure 3 Molecular Structure of the Lmo Locus
(A) A genomic map of the Lmo locus. Three different first exons can be utilized, forming the basis for three alternative transcripts. Exon RA-1 is separated from the alternative start sites RB-1 and RC-1 by a large (∼30 kb) intron. EP1306, EP1383, and pdrm carry insertions 25, 73, and 91 bp, respectively, upstream of the exon RA-1 transcriptional start site. Arrows within the EP elements refer to the orientation of the insertion and the expected direction of inducible expression via UAS sites contained within the EP element. Bx alleles are insertions of natural transposons into the 3′ UTR of the Lmo gene that have been shown to stabilize Lmo transcript (Shoresh et al. 1998). Protein-coding exons are shaded.
(B) Expression of the Lmo RA transcript is enriched in Drosophila heads, and is reduced in the EP1306 mutant. RNA was isolated from heads and bodies; after cDNA synthesis, quantitative RT-PCR was performed using primers specific to the RA transcript of Lmo in addition to primers to a reference transcript, the ribosomal protein rp49. Relative abundance is expressed as fold increase over control (EP1631) body mRNA. No detectable amplification was seen in RNase-treated controls (data not shown). Error bars represent standard error of the mean. Asterisk denotes significant difference from control (Student's paired t-test assuming equal variance; p < 0.001, n = 3).
Figure 4
pdrm's GAL4 Expression Is Sufficient to Drive Lmo-Transgene-Mediated Rescue of Cocaine Sensitivity
(A) pdrm's GAL4 expression pattern. UAS-GFP reveals GAL4 expression pattern of the pdrm enhancer-trap insertion in MB lobes and calyces, ALs, the large cell bodies of the peptidergic LNvs, and neurons of the pars intercerebralis (PI).
(B) Lmo expression restores wild-type cocaine responses. In the absence of a UAS transgene (“no UAS” columns), hemizygous male pdrm flies (hatched bar) are more sensitive than controls (Ctl GAL4 is line 8.142, solid bar), as shown before in Figure 1A. Male flies hemizygous for pdrm and heterozygous for either of two UAS-Lmo transgenes (UAS-Lmo1 and UAS-Lmo2; hatched black bars), show normal cocaine sensitivity when compared to either UAS-Lmo transgene alone (white bars) or UAS-Lmo transgenes in the presence of a control GAL4 line (Ctl GAL4, line 8.142; black bars). To control for non-specific effects of transgene overexpression, UAS-GFP and UAS-lacZ transgenes were also driven by pdrm GAL4. Male flies were hemizygous for pdrm (or heterozygous for the control GAL4 insertion) and heterozygous for the specific UAS transgene. One-way ANOVA revealed a significant effect of genotype in the UAS-lacZ (F = 17.4, p < 0.001), UAS-GFP (F = 19.47, p < 0.001), or no UAS transgene (F = 4.1, p < 0.001) groups, but not in either of the UAS-Lmo transgene groups (F = 1.58, p = 0.22 and F = 1.21, p = 0.31 for UAS-Lmo1 and UAS-LMO2, respectively); thus, UAS-Lmo expression specifically restores normal cocaine sensitivity to pdrm flies. Post hoc pairwise planned comparisons, with the critical p-value adjusted to 0.025, revealed significant differences between the “non-rescued” pdrm/UAS-GFP flies and the appropriate controls (UAS-GFP/+ or 8.142/UAS-GFP, p < 0.002); similarly, pdrm/UAS-lacZ flies are significantly different from their controls (UAS-lacZ/+ and 8.142/UAS-lacZ, p < 0.002). Pairwise comparisons revealed no significant differences between “rescued” pdrm/UAS-Lmo1 flies and their “normal” controls (8.142/UAS-Lmo1 and UAS-Lmo1/+, p = 0.99 and p = 0.14, respectively) or pdrm/UAS-Lmo2 flies and their controls (8.142/UAS-Lmo2/+ and UAS-Lmo2/+, p = 0.09 and p = 0.99, respectively), indicating full rescue of pdrm cocaine sensitivity. For all genotypes, n = 16–20 experiments.
Restricted Expression of Lmo in the Nervous System Is Sufficient for Wild-Type Cocaine Sensitivity
The pdrm P[GAL4] insertion, which acts as a promoter/enhancer detector, is expected to reproduce, at least in part, the expression pattern of the Lmo gene. In adult flies, the pdrm line showed extensive GAL4 expression in the brain as visualized with the UAS–green fluorescent protein (GFP) transgene (Figure 4A). Prominent expression was observed in the antennal lobes (ALs) and the Kenyon cells of the mushroom bodies (MBs), which are major brain centers involved in olfaction and olfactory conditioning, respectively (Stocker 1994; Zars 2000). In addition, GAL4 was expressed in the LNvs, which are the major pacemaker cells regulating circadian locomotor rhythmicity in flies (Renn et al. 1999). Finally, GAL4 expression was seen in neurosecretory cells located in the pars intercerebralis, in glial cells surrounding the optic lobes, and in scattered cells throughout the ventral nerve cord (VNC) (Figure 4A; data not shown). In pdrm larvae and adult flies, GAL4 expression was restricted to the nervous system. GAL4 expression was not detected in larval imaginal discs, such as the wing disc (data not shown), where Lmo expression has been shown to play an important patterning role (Milan and Cohen 1999). Thus, pdrm traps a specific subset of Lmo regulatory elements, possibly those controlling nervous-system-restricted expression of the RA transcript.
We hypothesized that the expression pattern of the pdrm enhancer trap may identify the cells in which Lmo expression is disrupted in the P-element insertion lines, causing increased cocaine sensitivity. If this is the case, pdrm-driven expression of UAS-Lmo transgenes is expected to restore normal cocaine sensitivity to pdrm flies. Indeed, we found that two UAS-Lmo transgenes rescued the cocaine-sensitivity defect of pdrm mutant flies (Figure 4B). This effect was specific to Lmo, as pdrm-driven expression of UAS-tauGFP or UAS-lacZ failed to restore normal behavior. Finally, Lmo mutants that did not contain a GAL4 enhancer trap, such as EP1306, failed to be rescued by the presence of UAS-Lmo transgenes (data not shown). These data demonstrate that the cocaine-sensitivity defect of pdrm flies is due to the loss of Lmo function, and that nervous-system-specific expression of Lmo in cells dictated by the pdrm GAL4 line is sufficient to confer normal cocaine responses.
Lmo Expression in PDF Neurons Is Sufficient to Confer Normal Cocaine Responses
In order to refine further the spatial requirements for Lmo function, we attempted to rescue the EP1306 phenotype by expression of Lmo in specific brain regions. EP lines carry a P-element insertion containing multiple UAS sites to which GAL4 can bind to drive expression of adjacent genomic sequences (Rørth 1996). Therefore, mutagenic EP insertions, if oriented appropriately, can be used to drive expression of the disrupted gene. It was demonstrated previously that GAL4-driven Lmo expression can be mediated by the EP1306 and EP1383 insertions (Milan et al. 1998; Zeng et al. 1998). Consistent with this, we were able to rescue the EP1306 phenotype in the presence of the heat-inducible hs-GAL4 transgene, which drives low levels of ubiquitous GAL4 expression even in the absence of heat shock (data not shown).
We then used GAL4 enhancer-trap lines with expression in specific brain regions to drive Lmo expression from the EP1306 insertion. We focused specifically on GAL4 lines that drive expression in the MBs, ALs, pars intercerebralis neurons, LNvs, and glia, sites of expression revealed by the pdrm line (Figure 4A). GAL4 lines that drive expression specifically in the MBs and the ALs failed to rescue the cocaine-sensitivity phenotype of EP1306 (data not shown). Similar negative results were obtained with the glial-specific repo-GAL4 driver (Xiong et al. 1994). However, expression of Lmo in the LNvs using the pdf-GAL4 driver, which drives expression in cells that contain the neuropeptide PDF (Renn et al. 1999), restored nearly wild-type cocaine responsiveness to EP1306 flies (Figure 5A and 5C). pdf-GAL4 did not rescue the cocaine phenotype of hdp and pdrm flies, demonstrating dependence on induced Lmo expression provided by the EP1306 insertion. These results show that Lmo expression in the PDF-expressing neurons alone is sufficient to rescue wild-type cocaine responses, implicating these cells in the increased cocaine responses observed in Lmo loss-of-function mutants.
Figure 5
Lmo Expression in PDF Neurons Regulates Cocaine Responses
(A) pdf-GAL4-driven expression of Lmo using the EP1306 element rescues the EP1306 insertional phenotype. Lmo mutants EP1306, hdp, and pdrm, as well as a control EP line (Ctl-1 = EP1413), were tested in the absence (−pdf-GAL4; white bars) and presence (+
pdf-GAL4; black bars) of pdf-GAL4. All male flies are hemizygous for the Lmo mutation (or Ctl-1) and heterozygous for pdf-GAL4 (when carrying the transgene). One-way ANOVAs with post hoc planned comparisons (critical p-value adjusted to 0.0125) confirmed that EP1306, pdrm, and hdp flies (in the absence of pdf-GAL4) had significantly increased sensitivity to cocaine compared to control flies (Ctl-1 = EP1413) (p ≤ 0.003, n = 15–26 experiments). One-way ANOVA with post hoc planned comparisons (critical p-value adjusted to 0.01) revealed a significant difference between pdrm/pdf-GAL4 and hdp/pdf-GAL4 flies and their controls (EP1413/pdf-GAL4, pdf-GAL4/+, or EP1413/+, p < 0.003, n = 24–27 experiments), showing that the presence of pdf-GAL4 does not rescue the cocaine sensitivity of pdrm or hdp flies. In contrast, similar comparisons for “rescued” EP1306/pdf-GAL4 flies revealed no significant differences from their “normal” controls (p ≥ 0.026, n = 27–36). Furthermore, within-group comparisons (+/− pdf-GAL4) using t-tests indicate a significant difference only in the EP1306 group (p = 0.002). Asterisk denotes significant difference between −pdf-GAL4 and +
pdf-GAL4 phenotype.
(B) Flies overexpressing Lmo in PDF cells show decreased sensitivity to cocaine. Flies heterozygous for both pdf-GAL4 and either one of two UAS-Lmo transgenes (black bars) were compared to flies carrying UAS-Lmo (white bars) or pdf-GAL4 (gray bar) alone. One-way ANOVA revealed a significant effect of genotype for both UAS-Lmo transgene groups. Post-test planned comparisons, with the critical p-value adjusted to 0.025, showed significant differences between the pdf-GAL4/UAS-Lmo flies and either pdf-GAL4/+ (p < 0.02) or UAS-Lmo/+ controls (p < 0.005). Asterisks denote significant differences, n = 16 experiments.
(C) Confocal images demonstrate overlap between pdrm and PDF expression in the LNvs. In the left panel, UAS-mCD8GFP reveals the pdrm-driven GAL4 expression pattern (green) in the adult brain, and α-PAP staining (magenta) reveals PDF-expressing LNvs. Right panels are close-ups of the cell bodies of the LNvs; white areas correspond to regions of overlap between GFP (green) and PAP (magenta) expression.
In order to test whether LNvs are also the locus for the resistance to cocaine observed in Bx mutants, we overexpressed Lmo in wild-type flies using the pdf-GAL4 driver and UAS-Lmo transgenes. We found that these flies showed a significant decrease in cocaine sensitivity (Figure 5B). The magnitude of the induced resistance was lower than that observed in the BxJ strain, but similar to that of weaker Bx alleles. It is possible that overexpression of Lmo with the pdf-GAL4 driver may not be as high as that caused by the BxJ mutation, or, alternatively, that overexpression of Lmo in cells other than the LNvs mediates the remaining resistance to cocaine observed in BxJ. Nonetheless, this experiment supports a role for LNvs as a site where Lmo regulates sensitivity to cocaine.
In the adult fly, pdf-GAL4 drives expression in small and large LNvs (s-LNvs and l-LNvs) and in a group of neuroendocrine cells located at the very tip of the VNC (J. H. Park et al. 2000; Figure 5C). The LNvs express many central clock genes and have been demonstrated, through genetic ablations and electrical silencing studies, to play a central role in maintaining circadian locomotor rhythmicity (Renn et al. 1999; Nitabach et al. 2002; Peng et al. 2003); the function of the pdf-GAL4-expressing cells in the VNC is unknown. The pdrm line drives expression in both the s-LNvs and l-LNvs, as demonstrated by immunohistochemical analysis of pdrm/UAS-GFP flies with antibodies directed against the PDF precursor PAP (Figure 5C). pdrm does not, however, drive expression in the PDF-expressing cells located in the VNC (data not shown). This overlap in expression of pdrm and pdf-GAL4 in the LNvs implicates these few neurons as mediators of the altered cocaine sensitivity observed in Lmo mutants.
Because of the known role of LMOs as regulators of developmentally important transcription factors (Hobert and Westphal 2000), we asked whether the development of PDF-expressing LNvs is disrupted in Lmo mutants. Projections of the s-LNvs and l-LNvs can be visualized with an antibody that recognizes the PDF precursor PAP (Renn et al. 1999). L-LNvs make an elaborate network of varicosities on the surface of the optic medulla, and project across the midline to the contralateral LNvs, while s-LNvs make a very specific projection to the dorsal central brain (Figure 5C). Both sets of LNv neurons also make extensive arborizations in the accessory medulla (reviewed in Hellfrich-Förster 2003). In the EP1306 and pdrm mutants, anti-PAP staining revealed that the number and detailed morphology of LNv neurons were completely normal (data not shown). Thus, Lmo does not appear to play a role in the development of the LNvs, although subtle developmental defects could have been missed.
Lmo Is Required for Robust Circadian Locomotor Rhythms
The increased sensitivity to cocaine of Lmo mutants suggested that these mutations alter LNv function without grossly affecting LNv development. In order to determine whether Lmo mutants have a more general dysfunction in the LNvs, we tested Lmo mutants in locomotor rhythm assays. The LNvs are required for the maintenance of circadian locomotor rhythms in constant darkness, and are thus the pacemaker neurons of the fly (Stanewsky 2003). Lmo mutants EP1306, EP1383, hdpR26, and hdprev83 had less robust locomotor rhythms in constant darkness than their control lines (Figure 6A; data not shown), and all alleles had a higher tendency for arrhythmicity (Figure 6B). We used the power of the rhythm as an estimate of the degree of rhythmicity and found that EP1306 flies had weaker behavioral rhythms than EP1383 flies, which, in turn, were less robustly rhythmic than their control line (Ctl-2; Figure 6C); similarly, hdpR26 and hdprev83 flies had significantly weaker rhythms than their control line (Ctl-1; Figure 6C). However, there were rhythmic flies in almost all genotypes assayed (Figure 6B), and the period lengths of the rhythms were very similar across the genotypes (see legend to Figure 6 for details). Together, these data demonstrate that disruption of Lmo results in weak circadian rhythms of behavior and suggest a generalized dysfunction of the LNvs in Lmo mutants.
Figure 6 Wild-Type Lmo Is Required for Robust Circadian Rhythms of Locomotor Activity
Locomotor activity of control (Ctl-1 and Ctl-2) and Lmo mutant (EP1383, EP1306, hdpR26, and hdprev83) flies was recorded in constant darkness as previously described (Nitabach et al. 2002).
(A) Representative actograms of control and Lmo mutants. Control flies show robust circadian rhythms with clear distinctions between activity during the subjective day and inactivity during the subjective night. The pattern of the EP1306 and hdprev83 mutants was more stochastic.
(B) Graph showing the proportion of strongly rhythmic (white), weakly rhythmic (gray), and arrhythmic (black) flies for each genotype. Most control flies had strong rhythms (28/30 for Ctl-1 and 27/29 for Ctl-2) while Lmo mutants formed a series with an increasing fraction of the flies arrhythmic. For example, 13/26 hdprev83 flies were arrhythmic, with the other 13 all having weak rhythms. The power of the rhythm was used to estimate the strength of the activity rhythm, with a power of 300 or more classed as a strong rhythm and a power between 300 and 170 classed as a weak rhythm; arrhythmics were given a power of 170 (for analysis below). Between 24 and 30 flies were assayed for each genotype. There were no major differences in the period length of the rhythmic flies in each genotype (Ctl-1, 23.6 ± 0.3; Ctl-2, 23.5 ± 0.5; EP1383, 23.2 ± 0.4; EP1306, 23.4 ± 0.3; hdpR26, 24.2 ± 0.5; and hdprev83, 23.4 ± 0.3).
(C) Quantitation of the average power of the rhythm with error bars showing standard error of the mean. One-way ANOVA revealed significant differences between genotypes (p < 0.0001). Post-hoc t-tests using a Bonferroni correction revealed that the power of the rhythm was significantly different between control flies and the Lmo mutants hdpR26, hdprev83, and EP1306 (p < 0.01). EP1383 flies had a significantly weaker rhythm than Ctl-2 flies (p < 0.05). Ctl-1, w1118, is the appropriate genetic control for the hdp alleles (black columns); Ctl-2, EP1631, is the appropriate control for EP1306 and EP1383 (gray columns). All flies tested are in the same genetic background, that of the w1118 flies.
(D) Quantitation of average activity (beam crossings per minute) with error bars showing standard error of the mean. ANOVA did not reveal significant differences between genotypes at the 0.01 level. Ctl-1, w1118, is the appropriate genetic control for the hdp alleles (black columns); Ctl-2, EP1631, is the appropriate control for EP1306 and EP1383 (gray columns). All flies tested are in the same genetic background, that of the w1118 flies.
LNvs Regulate Circadian Rhythmicity and Cocaine Sensitivity Independently
The observation that Lmo functions in the PDF neurons to regulate cocaine sensitivity, together with the finding that Lmo mutants show weak circadian locomotor rhythms, suggested that the pathways regulating cocaine sensitivity interact with the circadian clock. Alternatively, Lmo could regulate these two behaviors independently. We addressed these possibilities in two sets of experiments. First, we determined whether cocaine sensitivity was regulated by the circadian clock. For this purpose we entrained flies to light–dark (LD) cycles and then tested them for cocaine sensitivity during the light and dark phases at 3-h intervals over 24 h (see Materials and Methods). We found that cocaine sensitivity was essentially the same at all times tested (Figure 7A), demonstrating that cocaine responsiveness is not a behavioral output of the circadian clock.
Figure 7 Cocaine Responses Are Not a Circadian Output and pdf Mutants Show Wild-Type Cocaine Sensitivity
(A) Cocaine responses do not vary with the circadian clock. Control (EP1631) flies were raised under LD conditions and assayed for cocaine phenotypes in the crackometer at the indicated Zeitgeber (ZT) times. One-way ANOVA revealed no significant effect of time of day (F = 0.53, p = 0.82, n = 32).
(B) Flies lacking the neuropeptide PDF (pdf01) display normal cocaine sensitivity. pdf01 homozygotes (pdf01/pdf01) and pdf01 hemizygotes (pdf01/Df) showed wild-type responses to cocaine in the crackometer. Individual pairwise comparisons using Student's t-tests revealed no significant differences between control (+/+ and +/Df) and pdf mutant genotypes (p = 0.69, p = 0.97, n = 6–8 experiments)
Second, we asked whether PDF, the only known functional output of the LNvs in the context of circadian rhythms, is involved in regulating cocaine sensitivity. pdf mutant flies show a circadian phenotype that has been localized to the LNvs. We found that the pdf01 mutant flies, which completely lack PDF (Renn et al. 1999), showed normal cocaine responses compared to the control strain (Figure 7B). To eliminate the possibility that the absence of a phenotype was caused by genetic modifiers in the background of the pdf01 strain, we also tested flies carrying the pdf01 chromosome over a deficiency for the locus (see Materials and Methods). These pdf01 hemizygous flies also displayed normal cocaine sensitivity (Figure 7B). Taken together, these results show that the altered cocaine sensitivity of Lmo flies is not secondary to their abnormal circadian rhythms. Moreover, our data show that cocaine sensitivity and circadian behaviors, although both localized to the LNvs, are genetically separable.
LNvs and Their Synaptic Activity Regulate Acute Cocaine Responsivity
The hypersensitivity of Lmo mutants to cocaine could result from either the disruption of an LNv output that acts normally to dampen cocaine sensitivity or from an increase in an output of LNvs that normally enhances cocaine sensitivity. In order to differentiate between these two possibilities, we tested flies that lacked LNvs, generated by targeted expression of the cell death gene head involution defective (hid) using pdf-GAL4. This approach was previously used to demonstrate that LNvs are the Drosophila pacemaker neurons responsible for rhythmic locomotor activity (Renn et al. 1999). When tested in the crackometer, flies with LNv ablations showed reduced sensitivity to cocaine when compared to controls (Figure 8). These results show that LNvs normally act to increase cocaine responsiveness and that this effect is antagonized by Lmo function in these cells (see Discussion).
Figure 8 Silencing or Ablating PDF Cells Induces Resistance to Cocaine
Ablation of PDF cells with pdf-GAL4 and UAS-hid reduced sensitivity to cocaine compared to parental lines (pdf-GAL4/+ and UAS-hid/+). Electrical (UAS-Kir2.18 or UAS-Kir2.17) or synaptic (UAS-TeTx) silencing of PDF cells with pdf-GAL4 phenocopied PDF cell ablations. One-way ANOVAs with post hoc planned comparisons revealed a significant effect of genotype for the UAS-hid (p < 0.003, n = 20), UAS-Kir2.17 (p < 0.008, n = 32), UAS-Kir2.18 (p < 0.002, n = 28), and UAS-TeTx (p < 0.001, n = 28) groups, but not for UAS-TeTxin (p > 0.045, n = 28) (n corresponds to the number of experiments). Critical p-value was adjusted to p = 0.025. Asterisks denote significant differences in both planned comparisons (pdf-GAL4/+ and UAS-transgene/+ versus pdf-GAL4/UAS-transgene). Variations in phenotype of pdf-GAL4 flies for each set of experiments is caused by day-to-day variability.
To confirm that the behavioral resistance observed in LNv-ablated animals results from loss of neuronal signaling from these cells, rather than from developmental compensations induced by their ablation, we functionally silenced the LNvs either electrically, by targeted expression of the mammalian inward rectifying K+ channel (Kir2.1), or synaptically, by expression of tetanus toxin light chain (TeTx) (Sweeney et al. 1995; Baines et al. 2001). These manipulations do not affect LNv survival or the normal projection patterns of LNvs (Kaneko et al. 2000; Nitabach et al. 2002). Expression of either Kir2.1 or TeTx resulted in reduced cocaine sensitivity similar to that seen with LNv ablations (Figure 8). Importantly, expression of an inactive TeTx (TeTxin) did not significantly alter cocaine responses, confirming that the actions of these transgenes were specific to their ability to silence or ablate the LNvs (Figure 8). These data further demonstrate that circadian phenotype does not predict cocaine phenotype, as targeted expression of either hid or Kir2.1 in LNvs causes arrhythmia, while expression of TetTx does not (Renn et al. 1999; Kaneko et al. 2000; Nitabach et al. 2002). In summary, these results confirm a novel role for LNv activity and synaptic output in increasing behavioral responses to cocaine in a manner independent from its role in regulating circadian locomotor rhythmicity.
Discussion
In a genetic screen for Drosophila mutants with altered acute responses to cocaine, we isolated multiple mutations in the Lmo locus. Behavioral characterization of gain- and loss-of-function alleles of Lmo demonstrates an inverse correlation between Lmo expression levels and cocaine sensitivity. Through targeted expression of Lmo, we show that these altered cocaine responses are caused by differential Lmo expression in the circadian pacemaker neurons, the LNvs. Consistent with a dysfunction of these pacemaker neurons in Lmo mutants is our finding that the mutant flies also show altered circadian locomotor rhythms. However, using a variety of genetic methods to ablate or functionally silence these neurons, we reveal a novel role for the LNvs in modulating cocaine's acute locomotor responses that is independent of their pacemaker function. These findings add to mounting data in Drosophila and mice supporting a role for circadian genes in cocaine-related behaviors (Andretic et al. 1999; S. K. Park et al. 2000; Abarca et al. 2002; Rothenfluh and Heberlein 2002). Our discovery that cocaine actions are modulated by neurons critical for normal circadian locomotor rhythmicity suggests a basis for this overlap.
Lmo Functions in PDF Neurons to Regulate Cocaine Sensitivity
We provide several lines of evidence that levels of Lmo expression in PDF-expressing LNvs regulate acute sensitivity to volatilized cocaine in Drosophila. First, loss-of-function mutations in Lmo show increased cocaine sensitivity, a defect that can be reversed by induced expression of Lmo in the LNvs. Second, Bx mutants, in which Lmo is overexpressed, show reduced cocaine sensitivity; this resistance can be mimicked by overexpression of Lmo in the LNvs. The LNvs are a group of 8–10 neurons in each brain hemisphere that express the neuropeptide PDF. These neurons have previously been identified as the circadian pacemaker neurons of adult Drosophila and are involved in modulating rhythmic locomotor behavior (Renn et al. 1999; Blanchardon et al. 2001). Interestingly, expression of a mouse homolog of Lmo, Lmo4, is highly enriched in the suprachiasmatic nucleus (SCN) (A. Lasek, D. Kapfhamer, and U. H., unpublished data), the mammalian central pacemaker. Furthermore, microarray analysis revealed that in many tissues, Lmo4 expression varies with circadian time (Panda et al. 2002). These data suggest an evolutionarily conserved role for Lmo in clock neuron function.
LMOs are known to act as regulators of LIM-HD protein activity and stability (Retaux and Bachy 2002). LIM-HD proteins are transcription factors involved in many stages of nervous system development, from neuronal generation and axon guidance to determination of neuronal subtype identity (Hobert and Westphal 2000). In addition, expression of LIM-HD proteins in postmitotic neurons suggests a role in maintaining the differentiated state of these neurons (Hobert and Westphal 2000). The development and structure of the PDF-expressing LNvs, the neurons to which we localize Lmo action, have been studied in detail (Helfrich-Förster 1997). The LNvs can be divided into two groups of 4–5 neurons based on cell body size, projection pattern, and time of development. The s-LNvs, which arise in early larval development, project to the dorsal central brain, terminating near two sets of dorsal neurons that express clock genes at high levels. The l-LNvs arise during pupal stages and project onto the surface of the optic medulla, as well as to the contralateral LNvs through fibers running in the posterior optic tract. Both s- and l-LNvs have dense arborizations in the accessory medulla, a neuropil proposed to be a circadian pacemaker center in cockroaches and crickets (Helfrich-Förster 1998). An assessment of s-LNv and l-LNv numbers and detailed projection patterns revealed no differences between wild-type flies and Lmo mutants. Furthermore, by both quantitative RT-PCR and immunohistochemistry, we determined that PDF levels are normal in Lmo mutants (data not shown). Expression of PDF, the only known LNv output, is restricted to the LNvs and a few tritocerebral neurons. As PDF expression is a specific marker for the differentiated state of the LNvs, it is unlikely that in Lmo mutants these neurons are grossly abnormal in their terminal differentiation.
The absence of obvious structural abnormalities of LNvs suggests that Lmo may play an active role in regulating cocaine responses. In fact, evidence is mounting that the expression and activity of LMOs are dynamically regulated within the nervous system. For instance, expression of the murine Lmo homologs Lmo1, Lmo2, and Lmo3 is differentially regulated by seizure activity in specific regions of the hippocampus and forebrain of adult mice (Hinks et al. 1997). In addition, gene array experiments have revealed that expression of mammalian Lmo homologs is under circadian regulation in the SCN and is increased in the cerebral cortex during sleep deprivation (Cirelli and Tononi 2000; Panda et al. 2002). Furthermore, Lmo3 was isolated as a transcript upregulated by DA administration in cultured astrocytes (Shi et al. 2001). Lastly, Lmo2 and Lmo4 were recently isolated in a screen for calcium-regulated activators of transcription (Aizawa et al. 2004), suggesting a role for LMOs in regulating gene expression changes induced by neural activity. These data are intriguing in light of the many functional changes that occur in reward pathways in the addicted state. Whether LMO activity and/or expression are regulated by acute cocaine exposure remains to be studied.
Separable Roles of LNvs in Regulating Cocaine and Circadian Behaviors
We provide evidence that the LNvs regulate cocaine-induced behaviors, in addition to their well-known role in controlling circadian locomotor behaviors. Flies with LNv ablations show reduced sensitivity to cocaine, establishing that these cells normally increase behavioral responses to cocaine. Our experiments also indicate that LNvs drive circadian locomotor and cocaine behaviors in distinct ways. First, we found that sensitivity to cocaine is not modulated in a circadian manner, showing that the cocaine response is not simply an output of the central clock. Second, we showed that Lmo mutants, pdf mutants, and flies in which LNvs have been electrically silenced or ablated—all known to display similar locomotor rhythm deficits—show completely uncorrelated cocaine sensitivities: increased, unchanged, and reduced sensitivity, respectively. Lastly, we provide evidence that the LNv outputs that mediate cocaine and circadian behaviors are divergent. PDF, the only known LNv neurotransmitter, is required for normal locomotor rhythms (Renn et al. 1999). pdf null mutants, however, show normal cocaine responses. Furthermore, while synaptic silencing of PDF neurons does not disrupt circadian locomotor activity (Kaneko et al. 2000), we show here that the same manipulation reduces cocaine responses to the same extent as neuronal ablation. Together, these results imply the existence of an alternate, TeTx-sensitive functional output that mediates LNv modulation of cocaine responses. Interestingly, Blau and colleagues have also hypothesized a PDF-independent LNv output that regulates another rapid behavioral response, larval photophobicity (E. Mazzoni, C. Desplan, and J. Blau, unpublished data).
How might LNvs modulate circadian rhythmicity and cocaine sensitivity independently? It is possible that these cells use distinct output mechanisms, PDF to regulate circadian locomotor rhythms and another unknown signal to regulate cocaine sensitivity. Alternatively, these behaviors could be regulated by distinct subsets of LNvs. For example, several recent findings suggest that l-LNvs may play a lesser role in regulating circadian rhythmicity (Helfrich-Förster 2003). First, l-LNvs do not project to the dorsal brain, an area implicated in locomotor rhythmicity (Helfrich-Förster 1997). Second, unlike in s-LNvs, molecular clock cycling is not sustained for long in these cells during free-running conditions (Kaneko et al. 2000; Yang and Sehgal 2001; Shafer et al. 2002). Lastly, PDF expression and release is modulated by the molecular clock in s-LNvs, but not in l-LNvs (Blau and Young 1999; J. H. Park et al. 2000). However, another study found that normal rhythmicity can be obtained in flies lacking s-LNvs (Helfrich-Förster 1998). Moreover, the projections of l-LNvs connect the l- and s-LNvs from both brain hemispheres through projections in the posterior optic tract, suggesting a functional link between the two groups of cells. Whether Lmo functions in the s- and/or l-LNvs to regulate cocaine sensitivity and circadian rhythmicity cannot be established with currently available tools.
There is growing evidence for a functional link between circadian neurons and the modulation of cocaine-related behaviors. In mammals, the peptidergic neurons of the SCN have been shown by a variety of studies to be the central pacemakers controlling circadian rhythms (van Esseveldt et al. 2000). Interestingly, the fetal mammalian SCN contains DA D1 receptors through which cocaine can influence entrainment of fetal biological rhythms (Simonik et al. 1994; Viswanathan et al. 1994; Bender et al. 1997). In addition, disruption of another major component of the mammalian circadian system, the pineal gland, or its secretory product melatonin, results in altered cocaine responses (Uz et al. 2002, 2003; Zhdanova and Giorgetti 2002). Hirsh and colleagues showed that mutants in the Drosophila clock genes period, clock, and cycle, but not timeless fail to sensitize to repeated cocaine exposures (Andretic et al. 1999). These genes, which are expressed at high levels in LNvs, have been to shown to act in these cells to modulate circadian behavior (Kaneko et al. 1997). It is not known whether circadian gene function in LNvs also regulates behavioral sensitization to cocaine.
How Might LNvs Modulate Cocaine Responses
We have demonstrated that LNv electrical activity and synaptic output contribute to cocaine-induced behavioral responses, raising a number of questions regarding the interaction of cocaine with these neurons and their output. We propose a simple model whereby the activity of some or all LNvs directly increases upon cocaine administration, which in turn results in cocaine-induced changes in locomotion (Figure 9). Interestingly, a recent report showed that cultured LNv neurons can respond to either DA or acetylcholine, but not to glutamate, serotonin, octopamine, or histamine (Wegener et al. 2004). The inferred presence of DA receptors on a subset of LNvs provides a potential mechanism by which LNv activity could be directly increased by cocaine administration, as cocaine's primary mechanism of action is to inhibit the reuptake of DA by DAT. In this model, when the LNvs are ablated or silenced, one site of cocaine action would be eliminated, thus reducing cocaine's effect (Figure 9B).
Figure 9 A Model for LNv and LMO Regulation of Cocaine Sensitivity
(A) In wild type, LNvs modulate locomotor responses via electrical activity and synaptic transmission. We propose a model in which cocaine acts to directly increase LNv activity. Upon cocaine administration, synaptic DA concentrations are increased (via cocaine's inhibition of the plasma membrane DA transporter). Activation of presumed DA receptors on the LNv (dark arrowheads) stimulates electrical activity and subsequent synaptic output. This activity contributes to the behavioral response of the fly to cocaine.
(B) LNv ablations eliminate LNv contribution to the cocaine response, reducing cocaine sensitivity.
(C) In our model, Lmo loss-of-function mutants (LmoLOF), which have increased cocaine sensitivity, have increased activity/output during the cocaine response. This increased activity may be mediated by increases in receptor content on the LNv or by recruitment of other LNvs that normally do not participate in the cocaine response.
(D) Lmo gain-of-function mutants (LmoGOF) mutants have reduced LNv output and reduced cocaine sensitivity. This could also result from a reduction in receptor density.
How would Lmo fit into this model? We propose that in Lmo loss-of-function mutants, LNv output is boosted, possibly because of increased expression of DA receptors (Figure 9C), leading to enhanced cocaine sensitivity. Conversely, increased Lmo expression (in Bx mutants or in flies specifically overexpressing Lmo in PDF cells) would result in reduced receptor content and, consequently, in dampened cocaine sensitivity (Figure 9D). Further studies are needed to identify the putative DA receptor (or other molecules) that functions in LNvs to regulate cocaine-induced behaviors.
LMO-induced changes in receptor expression are not inconceivable given LMO's interaction with LIM-HD proteins. Changes in LIM-HD protein function have been shown to affect aspects of neuronal subtype identity, including neurotransmitter and receptor expression profiles. For instance, mutations in the Drosophila LIM-HD gene islet cause a loss of DA and serotonin synthesis, while ectopic expression leads to ectopic expression of tyrosine hydroxylase, an enzyme required for DA biosynthesis (Thor and Thomas 1997). In addition, expression of a Drosophila DA receptor in larval neurons requires the function of the LIM-HD gene apterous (Park et al. 2004). The observation that DA receptor expression is also altered in various Caenorhabditis elegans LIM-HD mutants (Tsalik et al. 2003) suggests an evolutionarily conserved role of LIM-HD proteins and possibly LMOs in the regulation of neurotransmitter identity and responsiveness. Consistent with this idea is the finding that Lmo homologs are highly expressed in the mesolimbic DA system of mice (A. Lasek, D. Kapfhamer, and U. H., unpublished observations)—a neural pathway involved in the acute stimulant and rewarding properties of abused drugs. We posit that LMOs are ideally suited to modulate in a subtle manner the neurochemical identity and sensitivity of the nervous system to various stimuli, including drugs of abuse.
Materials and Methods
Drosophila culture and strains.
All flies were maintained on standard cornmeal molasses agar at 25 °C and 70% humidity under constant weak light. The Rørth EP collection was obtained from G. M. Rubin (University of California, Berkeley, California, United States) (Rørth 1996). The pdrm allele was originally isolated in a genetic screen of P[GAL4] insertions (carrying the GawB element) for altered response to the locomotor-activating effects of ethanol (F. Wolf, unpublished data) and consequently tested for cocaine sensitivity. The location of the insertion was determined by inverse PCR (http://www.fruitfly.org/about/methods/inverse.pcr.html). The hdpR590 and hdpR26 loss-of-function alleles were provided by S. Cohen (European Molecular Biology Laboratory, Heidelberg, Germany) (Milan et al. 1998). UAS-Lmo lines were provided by C. Zeng (University of Wisconsin, Milwaukee, Wisconsin, United States) (Zeng et al. 1998). The hdprev83 strain was generated by N. Justice by imprecise excision of the EP1306 line, screening for the hdp phenotype (unpublished data). Bx alleles and pdf deficiencies (Df(3R)T1-X and Df(3R)T1-P) were obtained from the Drosophila Stock Center (Bloomington, Indiana, United States). w33, pdf01 and pdf-GAL4 flies were obtained from P. Taghert (Washington University, St. Louis, Missouri, United States) (Renn et al. 1999). UAS-Kir2.1 lines were obtained from S. Sweeney (University of California, San Francisco, California, United States) (Baines et al. 2001). UAS-TeTx lines were generated as previously described (Sweeney et al. 1995; Scholz et al. 2000). UAS-hid flies were provided by R. S. Stowers (NASA Ames Research Center, Moffett Field, California, United States) (Zhou et al. 1997). UAS-tauGFP and UAS-lacZ lines were obtained from Y.-N. Jan (University of California, San Francisco). All lines used for behavioral experiments, unless noted below, were out-crossed for five generations to a w1118 stock isogenic for Chromosomes II and III. Because pdf01 and its background control strain (w33) are unmarked, only Chromosomes I and II were replaced via crosses to balancer stocks in the w1118 genetic background.
Genetic screen and selection of controls
The approximately 400 X-linked EP lines were initially screened as hemizygotes crossed to a w, X^X/Y tester strain in the crackometer as described below (n = 6–9). These lines distributed normally, and 30 lines with phenotypes greater than 1.5 standard deviation from the mean were out-crossed into our w1118 background and retested. An additional 70 lines were selected to be out-crossed in order to provide a population distribution from which to select control lines. These 100 out-crossed lines were rescreened for acute responses to cocaine. A group of five control EP lines were chosen based on having a normal acute response (near the mode of the distribution). In all behavioral experiments involving EP lines, at least three of these control lines were tested to ensure that the control shown was representative of the EP population. The specific control EP line shown for each experiment is noted in the figure legends. As a control for the pdrm line we used a P[GAL4] insertion (line 8.142) that shows a normal response to multiple drugs, including cocaine (A. Rothenfluh, D. Guarnieri, A. Rodan, and F. Wolf, unpublished data). For data shown in Figures 1 and 3, all lines were crossed to a w, X^X/Y tester strain to reduce possible effects of recessive autosomal modifiers. In addition to the genotypes shown, we tested three P-element lines in the Lmo locus that did not produce cocaine phenotypes: MS1096, which lies 20 bp downstream of the first RA transcript exon, and two additional EP lines, EP0443 and EP1394, inserted 5′ to EP1383; the EP lines have normal wings. MS1096 has been shown to have a very weak wing venation phenotype, but has otherwise not been phenotypically or molecularly characterized (Milan et al. 1998).
Behavioral assays and statistics
For cocaine-sensitivity assays, for all behavioral experiments, 15 male flies were collected under CO2 anesthesia 0–2 d post eclosion (day 12), and tested 2–3 d later. Flies were equilibrated to room temperature for at least 1 h before being exposed to cocaine. Cocaine exposures were performed as described previously (McClung and Hirsh 1998; Bainton et al. 2000), and startle-induced negative geotaxis was assayed in a glass cylinder as described previously (Bainton et al. 2000). A drug effect score was determined as the average (measured every minute over 5 min) number of flies that remained on the bottom of the cylinder, expressed as percent of the total number of flies. Significance was established for each experiment as described in figure legends. Generally, either Student's paired t-tests assuming equal variance or one-way ANOVAs with post hoc Bonferroni planned comparisons or Tukey-Kramer multiple comparisons were performed in GraphPad Prism 4 (GraphPad, San Diego, California, United States). Error bars in all experiments represent the standard error of the mean. To maintain an experiment-wide error rate of α = 0.05, the adjusted error rates were p = 0.05/n for the n subsequent planned pairwise comparisons in each experiment. To observe cocaine locomotor activity patterns, ten flies were exposed to volatilized cocaine for 1 min, transferred to a 7.5 cm × 10 cm × 0.5 cm acrylic box, and images were captured on an Apple G4 computer (Apple, Cupertino, California, United States) using Adobe Premiere (Adobe Systems, San Jose, California, United States) at 10 frames/s for 5 min. Fly locomotion was tracked using the dynamic image analysis system software (Solltech, Oakdale, Iowa, United States). All genotypes were tested at each dose multiple times (n ≥ 3), and representative data were selected for the figures. Baseline negative geotaxis was measured by mock exposures of flies and subsequent assay in the crackometer as above; all genotypes displayed behavioral scores of less than 10%, with no significant differences between genotypes (n > 8).
For circadian experiments, locomotor activity of individual flies was measured using the TriKinetics (Waltham, Massachusetts, United States) infrared beam-crossing system recording total crosses in 10-min bins. Raw activity histograms were analyzed for circadian rhythms using Actimetrics (Wilmette, Illinois, United States) Clocklab software. Chi-square periodograms were constructed according to Sokolove and Bushell (1978), and significant circadian rhythmicity was defined as presence of a peak in periodogram power that extends above the 0.01 Chi-prosquare significance line. Since this line is equal to a power of approximately 175 at a period of 24 h, flies with no periodogram peak crossing the significance line were assigned a circadian power of 170. This would tend to overestimate the circadian power of these flies, and thus is conservative with regard to assessing statistical differences in power between genotypes exhibiting frequent arrhythmicity and those that are predominately rhythmic. For the circadian cocaine-sensitivity experiment, flies were set up and raised in LD, collected under CO2 anesthesia during the light phase 1–2 d after eclosion, and placed back in LD for 2–3 d. At the indicated Zeitgeber times, flies were tested in the crackometer, under lights, within 5 min of being removed from LD conditions.
Histology
Expression pattern of the pdrm P[GAL4] insertion was examined by crossing to UAS-GFPT2 and UAS-mCD8GFP. All adult and larval preparations were dissected in PBS, fixed in 4% formaldehyde/PEM for 40 min, washed in PBS, and dehydrated in 50% glycerol/PBS for 1 h. Tissue was mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, California, United States) and analyzed with a Bio-Rad confocal microscope with Bio-Rad Lasersharp 2000 software (Bio-Rad, Hercules, California, United States). PAP antibody staining (provided by P. Taghert) was performed on CNS preparations that were dissected, fixed, and washed as above. Specimens were incubated in 1:2,000 dilution of anti-PAP in PBT, and with a Texas Red–coupled goat anti–guinea pig secondary antibody, diluted 1:200 (Jackson Laboratory, Bar Harbor, Maine, United States).
Real-time quantitative RT-PCR
Flies 2- to 4-d-old were collected and frozen immediately at −80 °C. Heads were removed from bodies by vortexing, and separated in a sieve. RNA was extracted from heads and bodies by homogenizing the flies in hot phenol and NTES. Complementary DNA (cDNA) was prepared using TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, California, United States) according to the manufacturer's specifications, with the addition of random sequence hexamers to 2.5 μM. cDNA was analyzed by quantitative, real-time PCR using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems). The following probes and primers were designed using ABI PrimerExpress software: LmoRA-For, GAAGAGAAACAACAGCAGCAACA; LmoRA-Rev, ATTTGCATATTTCGCACTTGTTTAGCT; and LmoRA-Probe, CTGCTGCCGTTGCTG. rp49 probe and primers (CT 6405) were obtained from Applied Biosystems. TaqMan PCR reactions consisted of 50 ng of cDNA, 0.9 μM each diagnostic primer, 0.25 μM diagnostic probe, and 1x final of TaqMan Universal PCR Mastermix (Applied Biosystems) in a reaction volume of 25 μl. The TaqMan PCR conditions used were as described in TaqMan guidelines. Each sample was analyzed in triplicate. As negative controls, we used both no-template and DNase-treated non-reverse-transcribed mRNA samples; no significant amplification was observed in these samples. rp49 transcript levels were used as an endogenous normalization control for RNA samples, and relative mRNA abundance was calculated using the comparative delta-Ct method. Reference mRNA is noted in figure legends. Quantitative RT-PCR analysis was performed on at least two independent RNA preparations, with similar results.
We thank Cori Bargmann, Grae Davis, Lily Jan, William Cho, Douglas Guarnieri, Aylin Rodan, Adrian Rothenfluh, and Fred Wolf for interesting discussions and/or comments on the manuscript; Nick Justice, Fred Wolf, Douglas Guarnieri, Ammon Corl, and Adrian Rothenfluh for providing unpublished reagents; Melissa Andres and Rollie Mancilla for help with dissections and immunohistochemistry; and Julie Gesch for performing the qPCR. This work was supported by grants from the National Institutes of Health to UH (R21 DA14809, R01 AA 10035, and RO1 AA 13105), RB (KO8 DA 444906–33821), and LT (Medical Scientist Training Program).
Conflicts of interest. The authors have declared that no conflicts of interest exist.
Author contributions. LTYT and UH conceived and designed the experiments. LTYT and JB performed the experiments. LTYT and JB analyzed the data. RJB participated in the genetic screen that led to the isolation of EP1306 and EP1383. LTYT and UH wrote the paper.
Academic Editor: Eric Nestler, University of Texas Southwestern Medical Center
Citation: Tsai LTY, Bainton RJ, Blau J, Heberlein U (2004) Lmo mutants reveal a novel role for circadian pacemaker neurons in cocaine-induced behaviors. PLoS Biol 2(12): e408.
Abbreviations
ALantennal lobe
Bx
Beadex
cDNAcomplementary DNA
DAdopamine
DATdopamine transporter
GFPgreen fluorescent protein
hdp
heldup
hid
head involution defective
l-LNvlarge ventral lateral neuron
LDlight–dark
LIM-HDLIM-homeodomain
LMOLIM-only
LNvventral lateral neuron
MBmushroom body
pdrm
pipedream
s-LNvsmall ventral lateral neuron
SCNsuprachiasmatic nucleus
TeTxtetanus toxin light chain
TeTxininactive tetanus toxin light chain
VNCventral nerve cord
==== Refs
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| 15550987 | PMC529317 | CC BY | 2021-01-05 08:28:08 | no | PLoS Biol. 2004 Dec 23; 2(12):e408 | utf-8 | PLoS Biol | 2,004 | 10.1371/journal.pbio.0020408 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1555098810.1371/journal.pbio.0020416Research ArticleAnimal BehaviorEcologyEvolutionGenetics/Genomics/Gene TherapyZoologyDrosophilaInsectsThe Genetics of Speciation by Reinforcement Genetics of ReinforcementOrtiz-Barrientos Daniel [email protected]
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Counterman Brian A
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Noor Mohamed A. F 1Department of Biological Sciences, Louisiana State UniversityBaton Rouge, LouisianaUnited States of America12 2004 23 11 2004 23 11 2004 2 12 e41610 6 2004 4 10 2004 Copyright: © 2004 Ortiz-Barrientos et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
The What and Why of Research on Reinforcement
Getting a Whiff of Speciation by Reinforcement
Reinforcement occurs when natural selection strengthens behavioral discrimination to prevent costly interspecies matings, such as when matings produce sterile hybrids. This evolutionary process can complete speciation, thereby providing a direct link between Darwin's theory of natural selection and the origin of new species. Here, by examining a case of speciation by reinforcement in Drosophila, we present the first high-resolution genetic study of variation within species for female mating discrimination that is enhanced by natural selection. We show that reinforced mating discrimination is inherited as a dominant trait, exhibits variability within species, and may be influenced by a known set of candidate genes involved in olfaction. Our results show that the genetics of reinforced mating discrimination is different from the genetics of mating discrimination between species, suggesting that overall mating discrimination might be a composite phenomenon, which in Drosophila could involve both auditory and olfactory cues. Examining the genetics of reinforcement provides a unique opportunity for both understanding the origin of new species in the face of gene flow and identifying the genetic basis of adaptive female species preferences, two major gaps in our understanding of speciation.
Mating discrimination between two species of Drosophila is more pronounced where they hybridize and genes related to odor appear responsible for this "reinforcement," thus providing insights into the genetics of speciation
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Introduction
During reinforcement, mating discrimination is strengthened by natural selection in response to maladaptive hybridization between closely related taxa (Dobzhansky 1940; Fisher 1958). Although reinforcement was a contentious issue in the past (Butlin 1989; Howard 1993; Noor 1999), recent theoretical work has identified the most favorable conditions for its existence (Liou and Price 1994; Kelly and Noor 1996; Servedio and Kirkpatrick 1997; Kirkpatrick and Servedio 1999; Servedio 2000), and empirical data have provided potential examples of its occurrence in nature (Noor 1995; Sæaetre et al. 1997; Rundle and Schluter 1998; Nosil et al. 2003).
Theoretical work on reinforcement shows that reproductive isolation may be strengthened when either the same (one) or different (two) alleles conferring mating discrimination spread in the emerging species (Felsenstein 1981). In two-allele models, alleles conferring mating discrimination spread if they become genetically correlated with alleles reducing hybrid fitness. However, the evolution of such a correlation is opposed by recombination because alleles conferring discrimination in a given species do not confer discrimination in the other species. Consequently, two-allele models require either very strong selection, or tight linkage (e.g., physical or via chromosomal rearrangements) between alleles conferring mating discrimination and alleles reducing hybrid fitness (Kirkpatrick and Ravigné 2002). In contrast, one-allele models are not opposed by recombination because alleles conferring mating discrimination reduce hybridization in the genetic background of both species (Kelly and Noor 1996; Servedio 2000) and so may be more conducive to reinforcement. Unfortunately, empirical data concerning these two models of speciation are lacking (see Ortíz-Barrientos et al. 2002; Servedio and Noor 2003).
In addition to providing fundamental information for theoretical models, discerning the genetics of reinforcement will also develop our understanding of both the physiological basis of and forces governing changes in female preference. Furthermore, because the strengthening of female preference is driven by natural selection, the genetics of reinforcement will provide unique insights into the genetics of adaptation, another unsettled issue in evolutionary biology. Finally, high-resolution genetic studies of reinforcement can identify candidate speciation genes with effects on mating discrimination, information almost nonexistent in speciation studies (but see, Ritchie and Noor 2004). Here, we address these many fundamental issues in speciation by examining a case of reinforcement in Drosophila and present for the first time a high-resolution genetic study of variation within species in female mating discrimination, including a set of candidate reinforcement genes and a discussion of the evolutionary implications of our findings.
The North American fruitflies Drosophila pseudoobscura and D. persimilis hybridize in nature and produce sterile male hybrids. While D. pseudoobscura occurs alone in non-coastal western United States and Central America, the two species co-occur in California and the Pacific Northwest. Males court females from both species indiscriminately (Noor 1996), but females mate preferentially with individuals from the same species. The strength of this discrimination is not homogeneous across the species' geographic range: in a previous study, Noor (1995) showed that D. pseudoobscura females derived from populations where D. persimilis was absent exhibited weak mating discrimination (hereafter, “basal mating discrimination”) while females derived from populations where D. persimilis is present exhibited strong mating discrimination (hereafter “reinforced mating discrimination”). This difference in mating discrimination is likely the evolutionary consequence of maladaptive hybridization where the two species coexist: reinforcement has strengthened mating discrimination in the D. pseudoobscura populations co-occurring with D. persimilis. These observations and the recent completion of the genome sequence of D. pseudoobscura (BCM-HGSC 2004) make these species an ideal system to genetically dissect the enhancement of mating discrimination in sympatry.
Although the genetics of reinforcement has not been studied in D. pseudoobscura, or in any system, the genetic basis of other traits contributing to the species' reproductive isolation (i.e., hybrid sterility and basal mating discrimination) is known in detail. All traits contributing to reproductive isolation between D. pseudoobscura and D. persimilis, including traits for basal discrimination, map primarily or exclusively to regions bearing fixed chromosomal inversion differences between the species (Noor et al. 2001a, 2001b). This result is consistent with a two-allele model of speciation in which the reduction in recombination between alleles for hybrid unfitness (i.e., hybrid sterility) and mating discrimination creates the necessary genetic correlations to advance divergence in the presence of gene flow. However, we do not know whether the genetic basis of reinforced mating discrimination corresponds to this picture, and specifically, whether chromosomal inversions are fundamental to this process. Comparing these genetic architectures will provide the most comprehensive view yet on the genetics of mating discrimination contributing to the formation of new species in the face of interspecies gene flow.
Results
Female Discrimination Is Dominant and Reinforced in Sympatry
Table 1 shows that D. pseudoobscura females derived from sympatry (with D. persimilis) exhibited stronger mating discrimination against D. persimilis males than did D. pseudoobscura females derived from allopatry. This pattern holds for both inbred and outbred lines. Also, our data show that both sympatric-derived lines and allopatric-derived lines vary considerably in their degree of discrimination (p < 0.001 for sympatric inbred lines, and p = 0.0006 for allopatric inbred lines), suggesting some within-population variation in female mating discrimination, both basal and reinforced.
Table 1 Matings of D. persimilis Males to D. pseudoobscura Females Derived Sympatry or Allopatry
Each comparison involves either a sympatric versus an allopatric line of D. pseudoobscura, or F1 females (allopatric × sympatric) versus sympatric lines. Probability values were derived from Fisher's exact tests using geography (allopatric versus sympatric) and copulation occurrence (yes versus no) as variables
O, outbred lines; I, inbred lines; F1, first generation offspring from crossing the D. pseudoobscura sympatric and allopatric line
10.1371/journal.pbio.0020416.t001
Apparent reinforced mating discrimination could result from behavioral differences in D. persimilis males when exposed to sympatric or allopatric D. pseudoobscura females. To exclude this possibility, we measured the copulation latency and number of attempted copulations by D. persimilis males towards D. pseudoobscura females derived from sympatry or allopatry, and found no significant differences between groups (copulation latency, p = 0. 736, n = 138; attempted copulations, p = 0. 937, n = 110). Finally, we investigated the mode of inheritance of the phenotype and observed that F1 females from crosses between sympatric and allopatric flies discriminated as strongly as their sympatric parent, suggesting that reinforced female mating discrimination is inherited as a dominant trait in both inbred and outbred lines (see Table 1). This F1 female mating discrimination is restricted to pairings with D. persimilis males, as F1 females mate readily with conspecifics (data not shown). Taken together, these results suggest that reinforced mating discrimination in D. pseudoobscura is exclusive to females derived from areas of sympatry with D. persimilis, is inherited as a dominant trait, and is not markedly affected by inbreeding.
Within-Species Variation in Reinforced Female Mating Discrimination
We investigated within-species variability in reinforced discrimination by estimating the chromosomal contributions to mating discrimination between two pairs of D. pseudoobscura populations. In each case, we performed a male-parent backcross in which a mixture of whole chromosomes from sympatry and allopatry (F1 genome) was substituted into an allopatric background (F2 backcross genome) (see Figure 1, left panel). Each male-parent backcross was also replicated with the reciprocal F1 cross between parental strains, thus ruling out any maternal effects and providing insight into the effect of the X chromosome on mating discrimination.
Figure 1 Experimental Design to Substitute Chromosomes or Chromosomal Regions Derived from Sympatry into an Allopatric Background and Measure Their Effect on Mating Discrimination
F1 male-parent backcrosses (A) allow measurements of whole chromosome effects, while F1 female-parent backcrosses (B) measure specific chromosomal region effects. Curved arrow represents the reciprocal backcross of the one shown.
Our two backcrosses identified different chromosomes as affecting reinforced mating discrimination (binomial test of proportions for effects of all chromosomes, p < 0.01). For example, sympatric X and fourth chromosomes derived from Mather, California (male-parent backcross 1), contribute significantly to reinforced mating discrimination (p < 0.0001 for X chromosome, p < 0.005 for fourth chromosome, n of approximately 1,000 for all markers), while the same chromosomes show no detectable effect on reinforced mating discrimination when derived from Mt. St. Helena, California (male-parent backcross 2, p = 0.2297, n = 600 for all markers) (see Figure 2A and 2B). In contrast, the second chromosome shows the opposite relationship between the two backcrosses. The third chromosome shows marked effects on reinforced mating discrimination in both backcrosses, although at this level of resolution, it is impossible to tell whether this chromosome carries the same alleles in both sympatric populations. Figure 2C shows the genome composition of backcross females between Flagstaff, Arizona (allopatric), and Mather, California (sympatric), and their respective frequency of matings with D. persimilis males. The strongest effect is observed when both sympatric X and fourth chromosomes are substituted in an allopatric background, and no significant epistatic interactions were detected between chromosomes.
Figure 2 Mean Square Chromosomal Effects on Mating Discrimination
(A) Male-parent backcross 1 shows the effects of substituting chromosomes derived from Mather, California (sympatry), into a background derived from Flagstaff, Arizona (allopatry).
(B) Male-parent backcross 2 shows the effects of substituting chromosomes derived from Mt. St. Helena, California (sympatry), into a background derived from Mesa Verde, Colorado (allopatry). *, p < 0.005; **, p < 0.001; ***, p < 0.0001.
(C) Combined chromosomal contributions to female mating discrimination. Small bars on the left represent chromosomes (X, 2, 3, and 4), while long bars on the right show the frequency of matings of backcross females with D. persimilis.
These results suggest that different alleles for reinforced mating discrimination are segregating within sympatric populations of D. pseudoobscura despite extensive gene flow within and between populations (Schaeffer and Miller 1992; Noor et al. 2000).
Fine-Mapping the Genes Causing Reinforcement
We measured female mating discrimination against D. persimilis males in 1,500 F2 individuals derived from a female-parent backcross between a line derived from Mather, California (sympatric line), and a line derived from Flagstaff, Arizona (allopatric line), and genotyped 275 to 1,500 individuals for 70 markers dispersed along the four major chromosomes in D. pseudoobscura. Our initial single-marker analyses revealed significant associations between reinforced mating discrimination and three regions defined by markers located on the right and left arms of the X chromosome (XR and XL, respectively) (XR marker X021, p < 0.0001, n = 1,129; XL marker X002, p = 0.02, n = 1,293) and the fourth chromosome (4034 marker, p < 0.0001, n = 1,434). We were not able to detect an effect of any single region on the third chromosome even though nine markers were surveyed. Effects identified on XR and Chromosome 4 reinforced mating discrimination when the sympatric allele was present (positive), while the effect from XL was negative. After our initial scan, we used composite interval mapping (CIM) to account for any inflated estimates in the absence of background correction. In addition, several markers were genotyped around the X021 and 4034 regions with the goal of refining the segments containing the quantitative trait loci (QTLs). Figure 3 shows the major results from CIM, and confirms our previous observations: one major QTL was identified on XR around X021, and a suggestive one close to the telomere of XL. In addition, one major QTL was found on the fourth chromosome. These results validate our previous findings using male-parent backcross females and provide a high-resolution definition of regions contributing to reinforced mating discrimination.
Figure 3 QTLs and Candidate Genes for Reinforced Mating Discrimination
Each panel shows CIM estimations of chromosomal region effects on mating discrimination. Arrows point to major QTL locations and are named after their candidate genes. The direction of the chromosomal region effect on mating discrimination is shown in parentheses. The y-axis, LR, is the ratio of the likelihood value under the null hypothesis of no QTL to the likelihood value under the hypothesis that there is a QTL in a given interval of adjacent markers. The likelihood ratio significance threshold reflecting a Type I error of 0.05 is 11.5 s. The indicated inversion is a fixed chromosomal inversion differentiating D. pseudoobscura and D. persimilis.
X chromosome: Candidate genes Coy-1 andCoy-3
A more careful examination of the X021 region showed that the QTL location (CIM LOD score = 5. 16), hereafter referred to as Coy-1, is estimated to lie between two additional markers, X021-A1 and X021-A4 (these markers are physically separated by 390 kb and by a recombination fraction of 4.5 centi-Morgans [cM]). According to the recently obtained genome sequence of D. pseudoobscura, there are seven genes between these two markers, one of which, bru-3, accounts for one-third of the sequence length of this region. In addition, CIM also identified another QTL, hereafter referred to as Coy-3, located between markers X021 and X021-B2, although with a weaker effect (CIM LOD score = 2. 84). There are approximately 200 kb and 30 genes between these markers and a recombination fraction of 3.5 cM. Finally, a third QTL was found near the XL telomere and, in contrast to the X021 region, showed a negative and weak additive effect (CIM LOD score = 2. 45). We tested this model for the X chromosome using multiple interval mapping and found that the strongest support is for Coy-1, followed by Coy-3. We were unable to recover any support for the QTL on XL. No epistatic interactions were detected among any QTLs.
Chromosome 4: Candidate genesCoy-2 andCoy4
Dissection of the 4034 region using CIM split the effect into two QTLs for reinforced mating discrimination; we refer to them as Coy-2 and Coy-4, respectively. These QTLs show the strongest effects (CIM LOD scores of 7.7 and 7, respectively). As with Coy-1 and Coy-3, these QTL are additive and contribute positively to the degree of mating discrimination of F2 females. Coy-2 is located next to marker 4034-A8. This marker is within a 300-kb region homologous to a D. melanogaster region containing a p-element insertion disrupting normal olfactory behavior (see Discussion for details) (Anholt et al. 2001, 2003). The D. melanogaster region contains 30 genes of which at least ten have known or predicted olfactory functions. The primary candidate gene for the disrupted olfactory behavior in the p-element mutant is CG13982. Interestingly, the D. melanogaster p-element mutation up-regulates expression of bru-3, suggesting a possible functional link between the candidate genes Coy-1 and Coy-2. The second QTL in this region, Coy-4, is defined by two markers, 4003 and 4032, on each side of 4034. CIM places the QTL between 4003 and 4034, an approximately 200-kb region containing only nine genes. Five of these nine genes are a conglomerate of UDP-glycosyltransferases, genes preferentially expressed in the Drosophila antenna and coding for biotransformation enzymes involved in detoxification and olfaction (Wang et al. 2003). However, a more careful examination of the genes shows that their sequence overlap results from the inability of BLAST homology searches to distinguish the members of this gene family, suggesting that there may be only one or few UDP-glycosyltransferase genes here. Consequently, the number of candidate genes in the region may be reduced from nine to five genes, at least one of which is involved in olfaction. As before, we tested this model using multiple interval mapping and recovered significant support for Coy-2 under stringent conditions and no evidence of significant epistasis among previously identified QTLs.
Based on these results and those for the X chromosome, we suggest that the strongest evidence for QTLs contributing to reinforced discrimination in sympatry lies with Coy-1 and Coy-2, and that Coy-3 and Coy-4 are suggestive QTLs.
Discussion
We have provided the first genetic dissection of an adaptive female preference involved in speciation by developing a QTL map for discrimination variation in Drosophila pseudoobscura. The resolution of our approach is novel to genetic studies of behavioral discrimination in that we have surveyed the genome with 70 microsatellite markers for chromosomal regions contributing to increased mating discrimination and have narrowed some of these regions to intervals containing as few as five genes. The role of these genes in reinforcing mating discrimination is supported by indirect evidence from D. melanogaster mutants: two of the major QTLs identified in our mapping experiments bear genes identified in smell impairment screenings of p-element mutants (Anholt et al. 2003). A gene in one of these intervals, CG13982 (D. melanogaster Chromosome 2L), appears to up-regulate a second gene located in the other interval, bru-3 (D. melanogaster X chromosome). Furthermore, we have shown that the chromosomal contributions to reinforced mating discrimination vary among strains of D. pseudoobscura. Finally, the chromosomal effects on mating discrimination are inherited in a dominant fashion, consistent with general theories on the evolution of adaptive characters (Haldane 1924). Below, we discuss these results in the context of several evolutionary hypotheses of reinforcement and speciation.
The Genetics of “Basal” Versus “Reinforced” Female Mating Discrimination
Most genetic studies of female preference and sexual isolation have utilized between-species genetic crosses or non-hybridizing allopatric populations. Some of these studies suggest that female preference is a polygenic character (e.g., Moehring et al. 2004; Ting et al. 2001), while other researchers have found a very simple genetic basis for female discrimination (Doi et al. 2001). A study of another behavioral trait, response to odorants, showed that many genes contribute to olfaction, and epistasis plays a fundamental role in determining the specificity of odor identification (Anholt et al. 2001, 2003). We expect the genetics of reinforced female mating discrimination to bear some similarities to the genetics of overall female species preferences and/or traits involved in response to olfactory cues.
Available genetic data on “basal” female mating discrimination in D. pseudoobscura (between-species crosses using a D. pseudoobscura line derived from areas allopatric to D. persimilis) show that all QTLs for this trait map unequivocally to two inverted chromosomal regions separating it from D. persimilis (Noor et al. 2001a), one on XL and one on Chromosome 2. This result suggests that the regions we localized as contributing to reinforced mating discrimination (on XR and Chromosome 4) are distinct from those previously identified as contributing to basal discrimination. Hence, chromosomal inversions may have been crucial in allowing these species to persist in sympatry (Noor et al. 2001a), but the rearranged regions might not have contributed directly to the subsequent reinforcement of mating discrimination. This idea is consistent with data showing that a region (DPS4003) just 400 kb away from the QTL identified on the fourth chromosome seems to have introgressed recently between D. pseudoobscura and D. persimilis (Machado et al. 2002).
This result supports either a one-allele mechanism, perhaps controlling the genetics of variation within species for female mating discrimination if there was not strong assortative mating before sympatry, or possibly a two-allele mechanism, if reinforcement took place after sympatry and strong assortment had already evolved. The definitive test will be to determine whether introgressing the different D. pseudoobscura alleles into D. persimilis affects female discrimination in the same manner.
Female Mating Discrimination Is a Composite Trait
These “layers” of female discrimination (see Figure 4) are intimately related to the genetic differences being evaluated. Genes localized within fixed chromosomal regions inverted between D. pseudoobscura and D. persimilis are responsible for the first layer, basal discrimination. In contrast, the second layer, reinforced mating discrimination, is caused by genes localized outside those inverted regions. Basal discrimination appears to stem mostly from female responses to acoustic “courtship song” signal differences between D. pseudoobscura and D. persimilis. This is suggested by both a strong correlation in mating success of backcross hybrids with song parameters (Williams et al. 2001) and in playback experiments with wingless flies (M. Lineham, M. A. F. Noor, and M. Ritchie, unpublished data). Even though we cannot discard fine-tuning of the acoustic receiver signaling system in sympatric females, the nature of the candidate genes we identified suggests that olfactory responses might play a major role in the second layer of female preference. Non-auditory cues conferring reinforced discrimination are also suggested by behavioral data collected by Mark Lineham and Michael Ritchie (personal communication) showing that the rejection exercised by D. pseudoobscura females towards D. persimilis male song is the same in lines derived from sympatry and allopatry, even though females from the two regions clearly show differences in mating discrimination (Noor 1995; this study). Finding different genetic architectures for traits involved in speciation is expected under models based on selection on many traits (Rice and Hostert 1993). These traits may be a composite response of behavioral traits, as exemplified in this study, or ecology and behavior, as evidenced by Timema walking sticks, in which traits conferring ecological adaptation and traits contributing to mating discrimination act in conjunction to increase the overall level of sexual isolation between hybridizing populations (Nosil et al. 2003).
Figure 4 Genomic Distribution of Genetic Factors Preventing Gene Flow between D. pseudoobscura and D. persimilis
Gray boxes denote fixed chromosomal inversions separating D. pseudoobscura and D. persimilis. Black boxes denote the genomic locations of QTLs for reinforced mating discrimination. Note that the third chromosome also conferrs high discrimination in sympatry, but no particular QTLs have been identified for this chromosome.
Our results suggest, albeit not conclusively, that reinforced mating discrimination is related to differences in response to olfactory cues. We have shown here that candidate regions on the fourth chromosome bear an unusual excess of olfactory genes, and some of these have been associated with specific olfactory responses in other Drosophila. Further, the fact that we mapped reinforced discrimination to two interacting gene regions involved in olfaction (bearing bru-3 and CG13982) was striking in this regard, supporting a potential role of olfactory response in reinforcement in these species.
We also observed differences among strains in the genetic architecture of reinforced mating discrimination. Such variation in genetic control may be common when populations exchanging genes differ in phenotype because of selection. Multiple alleles from different loci may have increased in frequency because of selection for discrimination, and these alleles sometimes spread into allopatry or are replaced by the allopatric alleles in sympatry. When sampling from single lines, we capture only a fraction of the genetic variation in mating discrimination, and sometimes a high-discrimination allele is even sampled from allopatry (as we observed in the QTL on XL). This observation should be typical in many QTL mapping studies that utilize strains within species with extensive gene flow among populations, as in the many studies of D. melanogaster variation.
In brief, these results show that basal and reinforced discrimination are different, species discrimination in D. pseudoobscura is a composite trait, and there is genetic variation within species in reinforced mating discrimination.
Reinforced Mating Discrimination Is Inherited As a Dominant Trait
Recessive adaptive mutations are often lost before selection can screen their effects on the phenotype. Conversely, adaptive mutations that are visible to selection in a heterozygous state will be available for selection even at very low frequencies. Therefore, we expect that most adaptive mutations reaching high frequencies in a population are dominant (Haldane 1924, but see Orr and Betancourt 2001). This process is commonly referred to as Haldane's sieve and predicts that alleles for mating discrimination that increase in frequency in response to selection should be dominant (Orr and Betancourt 2001). Our study shows that F1 female offspring of crosses between allopatric and sympatric populations of D. pseudoobscura are as reluctant to mate with D. persimilis males as are D. pseudoobscura females from sympatric populations. This result implies that high discrimination can be expressed in heterozygous individuals, suggesting a dominant basis for the phenotype. In contrast, “basal” female mating discrimination seems to be a recessive trait (F1 female hybrids from crosses between allopatric D. pseudoobscura individuals and D. persimilis do not discriminate against D. persimilis males [Noor et al. 2001a]). Taken together, these results are consistent with genetic differences between basal and reinforced female mating discrimination and with general predictions from Haldane's sieve theory.
Conclusions
This is the first study to provide a detailed description of the genetic basis of speciation by reinforcement. We conclude that, in D. pseudoobscura, (1) high discrimination in sympatry is inherited in a dominant fashion, (2) there is within-species variability for high female mating discrimination as evidenced by the different genetic architectures recovered in the male-parent backcross experiments, (3) there are multiple genes, possibly involved in olfaction, contributing to enhanced female mating discrimination, (4) some candidate genes for reinforcement identified here have been previously identified in p-element mutant screenings for smell impairment in D. melanogaster, (5) the genetic architecture of basal female mating discrimination is different from that of reinforced mating discrimination, and (6) inversions seem to play no direct role in creating or maintaining the genetic differences directly responsible for increased female mating discrimination in sympatry. However, these inversions seem to play a crucial role, as evidenced by previous studies (Noor et al. 2001a), in maintaining the identity of hybridizing species and thus providing time for selection to reinforce their sexual isolation. These results have broad evolutionary implications, as discussed above, and open exciting new avenues of research to understand the genetics of an adaptive behavioral trait involved in speciation.
Materials and Methods
Our approach is based on measuring in one species the effects of substituting chromosomal segments from a highly discriminant genome into a less discriminant genome. In particular, we (1) test for within-species variation in the genetic architecture of female mating discrimination (F1 male-parent backcrosses), (2) identify chromosomal regions contributing to reinforced mating discrimination (F1 female-parent backcross) and compare them to regions conferring basal mating discrimination, and (3) provide a set of candidate genes for increased mating discrimination.
Fly rearing and lines
D. persimilis flies were collected in 1993 from Mt. St. Helena, California. D. pseudoobscura flies were collected from Mather, California (1997), Mt. St. Helena, California (1997), Flagstaff, Arizona (1993 and 1997), and Mesa Verde National Park, Colorado (2001). Isofemale lines were established by rearing the offspring of individual females previously mated in the wild. All lines were maintained under a constant regime of temperature (20 °C) and humidity (85%) in diurnal/nocturnal cycles of 12 h and reared on a mixture of agar, dextrose, and yeast.
Reinforced mating discrimination in sympatry
Pairs of D. pseudoobscura isofemale lines from each of two populations were crossed: Mather (1997) 52 × 10 (California, sympatric with D. persimilis) and Flagstaff (1997) 16 × 17 (Arizona, allopatric to D. persimilis), respectively. Virgin F1 females from these crosses as well as D. persimilis males were routinely collected during afternoons and confined for 8 d. On the morning of the eighth day, individual females were confined with individual D. persimilis males. The rationale of this no-choice design is based on behavioral observations suggesting that females tend to copulate more often in the presence of single males than when multiple males approach them (Noor, unpublished data). Therefore, no-choice experiments should provide a more conducive setting for mating. The flies were observed for 10 min. If the male attempted fewer than three copulations, the pair was not scored, and the data were discarded. Otherwise, the pair was scored for successful copulation versus not (the male must have been on the back of the female for at least 1 min—the average copulation duration in D. pseudoobscura is 3 min). These protocols are the same used in Noor et al. (2001a). We performed Fisher exact tests to evaluate differences among D. pseudoobscura lines sympatric versus allopatric to D. persimilis. Comparisons between allopatric and sympatric populations were performed both for outbred lines and inbred lines and only between lines that were tested for the phenotype at the same time, thus controlling for environmental error. Our comparisons between the two allopatric lines and between the two sympatric lines were not temporally controlled, and therefore may have been subject to some environmental heterogeneity. We used pairs of D. pseudoobscura inbred lines that significantly differed in their degree of female mating discrimination against D. persimilis in our mapping experiments (see below).
The heritable basis of increased mating discrimination in sympatry
We measured the frequency of matings with D. persimilis males of F1 females resulting from crosses between sympatric and allopatric D. pseudoobscura lines. If F1 females discriminated as strongly as the parent derived from sympatry, then we concluded that higher (reinforced) mating discrimination was inherited as a dominant trait. Fisher exact tests where performed to evaluate this hypothesis (see Table 1).
Testing for male discrimination
D. persimilis males were tested against D. pseudoobscura females from the Mather 17 and Flagstaff 1993 strains. We measured the time to first attempted copulation, the number of attempted copulations, and the time between the first attempt to copulate and copulation itself. Analysis of variance was conducted to test for a difference between treatments.
Mapping approach
Microsatellite markers include those reported previously (Noor et al. 2000) and 100 more that were developed by scanning contig sequences produced by the D. pseudoobscura genome project (Richards et al. 2004). Microsatellites were tested for fixed allelic differences between D. pseudoobscura lines Mather 17 and Flagstaff 1993. All primer information, both for informative and non-informative markers, will be published elsewhere and is available upon request. A recombinational map with an average distance of 15 cM between markers was produced using the female-parent backcross (see below) and the multipoint-linkage approach implemented in MapMaker version 3.0 (Lander et al. 1987).
Male-parent backcross
Two male-parent backcrosses (n
1 = 900 and n
2 = 600 flies) were used to determine the chromosomal basis of reinforced mating discrimination and its natural within-species variation. Crossing over does not occur in male Drosophila, and they thus transfer whole chromosomes to their offspring (see Figure 1). Each F1 backcross female was scored for mating (as above), and its DNA was subsequently extracted. Lines used in each backcross were: for backcross 1, Mather (California) 17 and Flagstaff (Arizona) 1993, and for backcross 2, Mt. St. Helena (California) 7 and Mesa Verde (Colorado) 17. We consider strains derived from California as sympatric and strains derived from Arizona or Colorado as allopatric to D. persimilis.
We used microsatellite markers to score the origin of each chromosomal segment in backcross hybrid females. To determine the chromosomal contributions from each chromosome, we performed analyses of variance in which the dependent variable was mating discrimination and the independent variables the origin of each chromosome.
Female-parent backcross
Once we determined the chromosomal effects and their variation for mating discrimination, we scored an additional 1,500 females derived by backcrossing Mather 17 × Flagstaff 1993 F1 females to Flagstaff 1993 males (see Figure 1) for mating discrimination against D. persimilis. A total of 288 females were genotyped for all markers, and both single-marker analyses and CIM (Zeng et al. 1999) were used to identify QTLs contributing to reinforced mating discrimination. Both approaches consistently identified the same regions. An additional 1,200 females were genotyped for markers showing significant effects on mating discrimination. When implementing CIM, forward-backward stepwise regressions were used to search for target QTLs over 2-cM intervals while simultaneously fitting partial regression coefficients for background markers with a window size of 15 cM. We tested for epistatic interactions between significant QTLs using multiple interval mapping (Zeng et al. 1999). In all cases, procedures were carried out as implemented in QTL Cartographer (Basten et al. 1999). Significance threshold values were obtained by permutation analysis as described by Doerge and Churchill (Basten et al. 1996).
Supporting Information
Accession Numbers
The Flybase (flybase.bio.indiana.edu) accession numbers for the genes discussed in this paper are bru-3 (FBgn0036379) and CG13982 (FBgn0031811).
We thank Regina Beauvais, Lisa Burk, Claire Faust, Katherine Grams, Leslie Lohmiller, Jane Reiland, and Matthew Smith for technical assistance. DOB is grateful to Cristina Lopez-Gallego for continuous moral support. We are also grateful to Robb Brumfield, Matt Carling, Zac Cheviron, Joe Covi, Sheri Dixon Schully, Dale Hedges, and Patrik Nosil for helpful comments on previous versions of the paper. We are grateful to Maria Servedio, three anonymous reviewers, and one academic editor for providing important comments that significantly improved the quality of the paper. Finally, we are indebted to Mike Ritchie and Mark Lineham for sharing their unpublished behavioral data with us. This work was supported by two Sigma Xi grants-in-aid of research to DOB, two Sigma Xi grants-in-aid of research to BAC, National Science Foundation grants 9980797, 0211007, and 0314552, and Louisiana Board of Regents Governor's Biotechnology Initiative grant 005 to MAFN.
Conflicts of interest. The authors have declared that no conflicts of interest exist.
Author contributions. MAFN conceived and designed the experiments. DOB and MAFN performed the pure-species and F1 mating experiments. DOB performed all parts involving the male-parent backcrosses and the mating assays in the female-parent backcrosses. DOB built the recombinational map for D. pseudoobscura. DOB and BAC genotyped the female backcross hybrids. DOB analyzed the data. MAFN contributed reagents/materials/analysis tools. DOB wrote the paper.
Academic Editor: Nick Barton, University of Edinburgh
Citation: Ortiz-Barrientos D, Counterman BA, Noor MAF (2004) The genetics of speciation by reinforcement. PLoS Biol 2(12): e416.
Abbreviations
CIMcomposite interval mapping
cMcenti-Morgan
QTLquantitative trait locus
XLleft arm of the X chromosome
XRright arm of the X chromosome
==== Refs
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| 15550988 | PMC529318 | CC BY | 2021-01-05 08:21:17 | no | PLoS Biol. 2004 Dec 23; 2(12):e416 | utf-8 | PLoS Biol | 2,004 | 10.1371/journal.pbio.0020416 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1555098910.1371/journal.pbio.0020423Research ArticleGenetics/Genomics/Gene TherapyVirologyVirusesPrimatesDistinct Genomic Integration of MLV and SIV Vectors in Primate Hematopoietic Stem and Progenitor Cells Retroviral Integration in HSCsHematti Peiman
1
¤1Hong Bum-Kee
1
Ferguson Cole
1
Adler Rima
1
Hanawa Hideki
2
Sellers Stephanie
1
Holt Ingeborg E
3
Eckfeldt Craig E
4
Sharma Yugal
5
Schmidt Manfred
6
von Kalle Christof
7
Persons Derek A
2
Billings Eric M
5
Verfaillie Catherine M
4
Nienhuis Arthur W
2
Wolfsberg Tyra G
3
Dunbar Cynthia E [email protected]
1
Calmels Boris
1
¤21Hematology Branch, National Institutes of HealthBethesda, MarylandUnited States of America2Experimental Hematology Division, Department of Hematology/Oncology, St. Jude Children's Research HospitalMemphis, TennesseeUnited States of America3Genome Technology Branch, National Human Genome Research Institute, National Institutes of HealthBethesda, MarylandUnited States of America4Stem Cell Institute, University of MinnesotaMinneapolis, MinnesotaUnited States of America5Bioinformatics Core Facility, National Heart, Lung, and Blood Institute, National Institutes of HealthBethesda, MarylandUnited States of America6Department of Internal Medicine, University of Freiburg, Freiburg, Germany7Division of Experimental Hematology, Children's Hospital Research FoundationCincinnati, OhioUnited States of America12 2004 23 11 2004 23 11 2004 2 12 e4231 7 2004 4 10 2004 Copyright: © 2004 Hematti et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Retroviral Gene Vectors Show Clear Target Preferences
Murine leukemia virus (MLV)-derived vectors are widely used for hematopoietic stem cell (HSC) gene transfer, but lentiviral vectors such as the simian immunodeficiency virus (SIV) may allow higher efficiency transfer and better expression. Recent studies in cell lines have challenged the notion that retroviruses and retroviral vectors integrate randomly into their host genome. Medical applications using these vectors are aimed at HSCs, and thus large-scale comprehensive analysis of MLV and SIV integration in long-term repopulating HSCs is crucial to help develop improved integrating vectors. We studied integration sites in HSCs of rhesus monkeys that had been transplanted 6 mo to 6 y prior with MLV- or SIV-transduced CD34+ cells. Unique MLV (491) and SIV (501) insertions were compared to a set of in silico-generated random integration sites. While MLV integrants were located predominantly around transcription start sites, SIV integrants strongly favored transcription units and gene-dense regions of the genome. These integration patterns suggest different mechanisms for integration as well as distinct safety implications for MLV versus SIV vectors.
A primate model of gene transfer into hematopoietic stem cells demonstrated MLV integration around transcription start sites whereas SIV integrated into gene-dense regions, indicating distinct safety implications for each
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Introduction
Integration of proviral DNA into the host cell genome is an essential step in the life cycle of retroviruses. The process begins after a retrovirus enters the cell and the RNA genome is reverse transcribed into double-stranded DNA. Preintegration complexes (PICs) containing linear proviral DNA associated with several viral and cellular proteins (Bushman 1999) either enter the nucleus of nondividing cells through the nuclear pores (lentiviruses) or gain access to chromosomal DNA after dissolution of the nuclear membrane during mitosis (oncoretroviruses). When the PIC associates with the host chromosome, the virally encoded integrase directs the insertion of the proviral DNA into the cellular chromosomal DNA (Hindmarsh and Leis 1999). The provirus is then stably transmitted to all progeny of transduced cells as an integral element of the host genome. Beyond its importance to the reproduction of the virus itself, this distinctive feature of retroviruses accounts for many of the characteristics associated with retroviral infection, including latency and persistence of infection, insertional mutagenesis, and the usefulness of retroviruses as vectors for gene therapy.
Engineered replication-defective retroviruses were introduced over 20 y ago and rapidly became attractive tools for efficient and stable introduction of genes of interest, in particular into hematopoietic stem cells (HSCs). Retroviral gene therapy targeting HSCs has been aggressively pursued because of its potential to treat many congenital and acquired human diseases. Its therapeutic promise was convincingly demonstrated in children with X-linked severe combined immunodeficiency (SCID-X1) and adenosine deaminase deficiency (Aiuti et al. 2002; Hacein-Bey-Abina et al. 2002). Unfortunately, elation over this success was recently tempered when lymphoproliferative disease developed in two children who received genetically modified CD34+ cells for treatment of SCID-X1, in association with proviral activation of the LMO2 transcription factor gene (Hacein-Bey-Abina et al. 2003). These serious adverse events have galvanized investigators to further assess the potential risks associated with gene therapy protocols utilizing retroviral vectors.
For many years, researchers have been aware that retroviral insertional activation of proto-oncogenes can result in tumors. Administration of replication-competent oncoretroviruses to susceptible mouse strains led to tumor development, the result of a high number of repetitive insertion events in vivo during rapid cell proliferation, with outgrowth of a clone containing one or more proviruses activating growth control genes (Dudley 2003). While the possibility of insertional mutagenesis using replication-defective vectors has been discussed as theoretically possible (Cornetta et al. 1991), such risks have been estimated to be extremely low (Moolten and Cupples 1992) based on the assumption that proviral integration into the genome was random (Coffin et al. 1997).
With the readily accessible human genome sequence, mapping studies of retroviral integration sites in cell lines have uncovered nonrandom integration patterns, when studied using wild-type HIV, HIV-derived, or murine leukemia virus (MLV)-derived vectors (Elleder et al. 2002; Schroder et al. 2002; Laufs et al. 2003; Wu et al. 2003; Mitchell et al. 2004). However, these integration patterns have not been investigated in the most relevant primary cells for hematopoietic gene therapy, namely HSCs. HSC transduction by retroviral vectors and their subsequent vector-genome integration patterns can unequivocally be assessed only by transplanting these cells and analyzing vector-containing cells in multiple lineages in vivo long-term, since stem and progenitor cell activity are defined by functional reconstitution of hematopoiesis in vivo. Interpretation of such studies may be more complex due to the potential for integration-specific impact on engraftment or functional properties of primitive hematopoietic cells in vivo. However, large-scale analysis of retroviral integration sites in a relevant long-term large animal model is critical to fully assess the potential risks associated with proviral insertion in this population of cells, prior to implementing new gene therapy trials. These studies may also provide further insights into mechanisms of integration targeting and the impact of integration events on the behavior of hematopoietic cells.
In order to evaluate the integrating vectors currently being developed for gene therapy applications, we compared the integration patterns of MLV and simian immunodeficiency virus (SIV) vectors. MLV vectors have been utilized for over a decade in clinical trials. However, they have a number of limitations, including inefficient transduction of quiescent cells and difficulty in maintaining stable high-level expression from tissue-specific internal genetic control elements. Thus, lentiviral vectors based on HIV or SIV “backbones” have been pursued and shown to overcome these limitations, and are now moving into clinical trials.
Vector-genome junction sequences were retrieved from mature granulocytes and mononuclear cells (MNCs) from rhesus macaques transplanted 6 mo to 6 y prior with mobilized peripheral blood (PB) CD34+ cells transduced with an MLV- (Schmidt et al. 2002) or SIV-derived vector (Hanawa et al. 2004), both containing marker genes with no known impact on proliferation or survival of transduced cells (Wu et al. 1998). This model represents a unique opportunity to analyze retroviral insertion patterns in the engrafted progeny of primitive long-term repopulating cells, without interference from confounding factors such as the impact of transgene expression or an underlying hematopoietic disease.
Results
Cloning, Sequencing, and Bioinformatic Analysis of Retroviral Integration Sites
We used a modification of the sensitive linear amplification-mediated (LAM)-PCR method (Schmidt et al. 2002) to retrieve and clone the genomic regions adjacent to proviral integration sites from circulating granulocytes and MNCs sampled in rhesus macaques engrafted stably long-term, between 6 mo and 6 y after transplantation of transduced CD34+ for the MLV-transduced animals, and 6–7 mo posttransplantation for the SIV-transduced animals. In our extensive prior analysis of 46 rhesus macaques, genetic marking levels and clonal integration patterns are stable by 3–4 mo posttransplantation, and remain stable for up to 6–7 y (Kiem et al. 2004). This approach uses a frequent-cutting enzyme to generate average genomic fragments of 80 bp, thereby circumventing PCR bias against large fragments, while facilitating amplification and cloning. The average length of all analyzed genomic fragments was 159 bp (median 131 bp, range 30–728 bp).
Owing to the close phylogenetic relationship between human and rhesus macaques, we were able to directly align our sequences with the human genome assembly. We considered a sequence as a genuine retroviral integration site only if it (a) juxtaposed to the vector long terminal repeat (LTR), (b) yielded a unique best hit by BLAT software (University of California, Santa Cruz [UCSC] Genome Browser, http://genome.ucsc.edu), and (c) showed at least 90% identity to the July 2003 human genome assembly (Kent, 2002; Karolchik et al. 2003). After several analyses using different cutoffs, we decided to use a conservative alignment cutoff of 90% in order to include most of the orthologous regions between human and rhesus genomes, while discarding sequences of technically poor quality. This cutoff eliminated 5 to 10% of the retrieved sequences, and we verified that omission of these sequences with less than 90% identity from our analysis did not change the overall distribution of the integration sites (Table S1). Using these selection criteria, we have retrieved and analyzed 992 independent unequivocal retroviral integration sites (n = 491 for MLV [Dataset S1], and n = 501 for SIV [Dataset S2]).
Of the 992 integration sites analyzed, 232 (23%) were distributed among the four major classes of transposable repetitive elements, and therefore could not be mapped to a unique position in the genome. These insertions accounted for 59 of 491 (12%) and 173 of 501 (34.5%) of the MLV and SIV integration events, respectively. Human transposon-derived repeats encompass at least 45% of our genome and their distribution is highly variable, with density varying from 2% to 98% depending on the location (Lander et al. 2001).
Integration Targets Transcription Units
We correlated the 760 integration sites (432 for MLV and 328 for SIV) that unequivocally mapped to a unique position in the genome to the locations of annotated genes, using the UCSC Genome Browser Reference Sequence (RefSeq) Genes track, which displays the positions of National Center for Biotechnology Information mRNA Reference Sequences (Table 1). We observed that 212 of 432 (49%) of the MLV integrations and 241 of 328 (73%) of the SIV integrations landed between the transcription start and stop point of a RefSeq gene. As a control, we compared the coordinates of two sets of 1,000 in silico-generated random integration sites, each containing 432 or 328 coordinates (760,000 total) with the positions of known genes. Both MLV and SIV insertion patterns were significantly different from the random integration sites (Figure 1), of which only 32 % were within RefSeq genes, a percentage identical to the average estimation of the human genome content (25.5%–37.8%, median 31.6%) (Venter et al. 2001).
Figure 1 Comparison of MLV and SIV Integration Events
Shown are integrations that landed within RefSeq gene introns (arrows) in comparison to in silico-generated integration sites (bars). Black indicates MLV and gray indicates SIV. *p < 0.0001 by a Chi2 test.
Table 1 MLV and SIV Integration Sites Distribution (Reported to UCSC RefSeq Genes) Compared to In Silico-Generated Random Integration Sites
The random integration sites correspond to two sets of 1,000 sets, each containing 432 or 328 coordinates (760,000 total). Two-sided p-values were obtained by the Chi2 test
a
p < 0.0001 compared to in silico-generated random integrations
b
p < 0.0001 compared to SIV-derived integrations
10.1371/journal.pbio.0020423.t001
We next examined whether specific regions of the transcription units were more likely sites of integration than others. We analyzed the distribution of the integration events within the transcription unit by dividing the distance of each integration site from the transcription start site by the gene length. The resulting ratio, reported as the total number of integration events in RefSeq genes for each vector, provides the percentage of integrations within ten equal sections of transcription units. While SIV targets the entire transcription unit with no noticeable preference, 42 of 212 (20%) of the MLV integration sites that land within RefSeq genes, as compared 18 of 241 (7%) for SIV, are located within the first one-tenth of the transcription unit, indicating MLV's clear predilection for the 5′ portion of transcription units (p = 0.0002).
MLV Vectors Favor Integration around Transcription Start Sites, and SIV Vectors Integrate Predominantly within Transcription Units
To further explore MLV preferential integration in the vicinity of transcription start sites, we determined the distance to the nearest 5′ and 3′ ends of a RefSeq gene for each integration site. Interestingly, whereas SIV integration events do not favor locations upstream or downstream of transcription units (Table 1), 48 of 432 (11%) of the total MLV integration sites landed within a 10-kb region upstream of a RefSeq gene, as compared to 5% expected with the random integration sets (p < 0.0001). The frequency of insertions within 10 kb downstream of the 3′ end is almost identical for the MLV and the in silico-generated random sets (5.3% versus 4.8%).
We then looked at the proviral integrations within a 2-kb window on either side of transcription start sites. This survey revealed a strong tendency for MLV vectors to integrate close to transcription start sites, with 46 of 432 (11%) of the total MLV integration events occurring within 2 kb upstream or downstream, as compared to 7 of 328 (2%) for SIV (p < 0.0001). We broadened this analysis to a 60-kb window centered on transcription start sites (Figure 2). The overall distribution of the 432 MLV integration events upstream and downstream of transcription start sites is almost identical (20% versus 27%, p = 0.02), but their distribution is clearly nonrandom and favors a 10-kb window centered around transcription start sites. This pattern is markedly different from the distribution of SIV sites: Although there is no predilection for integration in the vicinity of transcription start sites, there is a strong preference for integration within transcription units, rather than upstream of them. Of the SIV-derived sites, 122 of 328 (37%) are within 30 kb downstream of the transcription start site, while only 30 of 328 (9%) are within 30 kb upstream (p < 0.0001). Taken together, these data show a distinct integration pattern between MLV- and SIV-derived vectors (p < 0.00001 using an omnibus contingency Chi2 test): While the latter appear to integrate predominantly within transcription units, MLV vectors strongly favor integration within a 10-kb window centered on transcription start sites.
Figure 2 Distribution of MLV and SIV Integration Sites within a 60-kb Window Centered on Transcription Start Sites
The vertical arrow points to 0 kb. Each gray bar corresponds to the percentage of SIV integration sites within a 5-kb interval, and black bars correspond to the percentages of MLV integration sites in a 5-kb interval. The distribution of a set of 65,000 in silico-generated random integration sites is represented by the dashed line.
SIV-Derived Vectors Favor Integration within Gene-Dense Regions of the Genome
In order to ask whether the preferential integration of SIV vectors within transcription units might be associated with physical properties of the genome such as gene density, we analyzed the overall distribution of integration sites. The highest density of SIV integration sites per Mbp are on Chromosomes 17, 19, and 22 (0.50, 0.25, and 0.27 respectively), the three most gene-dense chromosomes, with 15, 23, and 17 genes per Mbp, respectively (Venter et al. 2001). Since each chromosome is a patchwork of domains with varying gene density, we determined the number of RefSeq genes within 1 Mbp of every integration site's LTR coordinate (Figure 3A). While most (84%) of the random integration sites tended to be within regions of average gene density (0–10 genes per Mbp), MLV displayed a strong tendency to integrate within more gene-dense regions. This was particularly evident for SIV integration sites, 174 of 328 (53%) of which occurred in regions of the genome whose gene density is higher than 11 genes per Mbp, compared to 149 of 432 (34%) and 17% for the MLV and the in silico, random sets, respectively. These data point out another difference between MLV- and SIV-derived vectors, the latter exhibiting a marked tendency to target gene-rich regions of the genome (p < 0.00001 using an omnibus contingency Chi2 test).
Figure 3 Distribution and Location of Integration Sites Relative to Chromosomal Gene Density
(A) Distribution of MLV and SIV integration sites relative to gene density within a 1-Mbp window compared to in silico-generated random integration sites. Each bar corresponds to the percentage of integration sites within the corresponding gene density region.
(B) Location of MLV and SIV integration sites and gene density on human Chromosome 6. MLV and SIV integrations were aligned to Chromosome 6 (obtained from the UCSC custom annotation track feature) and shown in relation to RefSeq gene density (blue). 73% of the SIV integration events are within the 20-Mbp unique ridge of Chromosome 6, compared to 29% for MLV. Distance between thick black bars is 20 Mbp; centromere is represented by the black circle.
Recent studies have shown that about 30 highly gene dense clusters, called “ridges” (a loose acronym for “regions of increased gene expression”), are distributed among chromosomes. These ridges are characterized by typical expression levels per gene up to seven times higher than the genomic average (Caron et al. 2001). This feature is particularly evident for Chromosomes 3 and 6 (Versteeg et al. 2003). When looking at the distribution of retroviral integration sites on Chromosome 6 (Figure 3B), 22 out of the 30 SIV integration events (73%) fall within this unique ridge, a region of 20 Mbp (12% of Chromosome 6) with a density of 24 genes per Mbp, corresponding to the major histocompatibility complex region. This tendency to target gene-rich regions is less obvious for the MLV vector, which had only 7 out of 24 integration sites (29%) within this ridge (p < 0.005). Of the 22 SIV proviruses within this 20-Mbp ridge, ten were found clustered within a 2-Mbp, extremely gene-dense region (62 RefSeq genes per Mbp). Unexpectedly, only two out of these ten integration sites are inside transcription units, underscoring the strong tendency of SIV vectors to target gene-rich regions of the genome even if not within genes. Another feature of ridges is that they are noticeably enriched for short interspersed element (SINE), but depleted for long interspersed element (LINE) repeats (Versteeg et al. 2003). This correlation between SINE repeat density, GC content, and gene density has been previously reported (Bernardi et al. 1985) and may account for our observation of overrepresentation of integration events in SINE versus LINE elements, with 119 of 501 (24%) of the SIV set of integration sites being within SINE repeats.
Common Integration Sites Differ between SIV- and MLV-Derived Vectors
Given their distinct patterns of integration, we compared identified common integration sites of MLV and SIV vectors. Using the definition of a common integration site as two or more proviruses integrated within a transcription unit (Suzuki et al. 2002), we have identified 40 genes targeted more than once by MLV and/or SIV vectors (Table S2). Of the RefSeq genes targeted by MLV and SIV vectors, 16 of 199 (8%) and 19 of 222 (9%), respectively, were hit at least twice, and ten genes were identified as common integration sites because they harbor both MLV and SIV proviruses. These genes have been targeted two times (n = 32), three times (n = 6), five times (n = 1), and seven times (n = 1). Among these 40 genes, seven are known to be involved in oncogenic translocations: ARHGEF12, MDS1, MKL1, MSF, HMGA2, RAD51L1, and RUNX1. Seven independent integration events have been identified in MDS1, predominantly within the second intron, 20–180 kb upstream of the first intron of EVI1.
Discussion
A better understanding of retroviral integration patterns has evolved due to the availability of the complete murine and human genome sequences. Prior mapping studies have been performed in cell lines or in primary cells cultured short-term in vitro. However, integration site patterns may be cell type–dependent, for instance, if gene activity impacts integration site selection (Schroder et al. 2002), or if specific integrations facilitate engraftment and long-term contribution to hematopoiesis. Our aim was to provide a comprehensive comparative analysis of integration sites distribution of MLV- and SIV-derived vectors in long-term repopulating HSCs. Nonhuman primates have been shown to closely predict results in human transplantation and gene therapy clinical protocols (Donahue and Dunbar 2001) and thus represent the best currently available approach to generate information with relevance to design of future human clinical trials.
MLV-derived retroviruses are currently the most widely used vectors in clinical gene transfer protocols. Reports of proto-oncogene activation by replication-defective MLV vectors in mice and humans mandate more detailed evaluation of their potential for insertional mutagenesis. Separating the impact of overexpressing a growth-altering transgene from the insertional events themselves is particularly important to assess in primary repopulating HSCs. The main limitation of murine oncoretroviruses as gene therapy vectors is the requirement that cells pass through mitosis in order for the PIC to reach the nucleus and integrate. Since lentiviruses can transduce noncycling cells, lentivirus-based vectors have been actively developed and a clinical trial using these vectors has commenced. A detailed analysis of lentivector integration patterns is essential to assess the risk of insertional mutagenesis of these vectors compared to MLV vectors.
Although HIV-derived vectors can enter Old World monkey cells, they encounter a block prior to reverse transcription that is mediated by the dominant repressive factor TRIM5α, a component of cytoplasmic bodies (Stremlau et al. 2004), and thus are very inefficient at transducing nonhuman primate cells (An et al. 2000; Horn et al. 2002). Lentiviral vectors derived from SIV have been generated (Hanawa et al. 2004; Negre and Cosset 2002) and are useful for preclinical testing in nonhuman primates. SIV vectors may also be used to transduce human cells, and offer a number of potential advantages over HIV vectors for eventual clinical applications, such as lack of seroconversion to HIV positivity after exposure.
Only limited information exists regarding the rhesus monkey genome, but paleontological and genomic sequence data suggests that Macaca mulatta is 92.5%–95% identical to the humans at a DNA level (Page and Goodman 2001; Stewart and Disotell 1998). Moreover, the human and macaque karyotypes are virtually identical, with near absence of interchromosomal rearrangements and no detectable segments of nonhomology in euchromatic regions (Best et al. 1998; Muller and Wienberg 2001). We believe that this evolutionary information, combined with the characteristics of the sequences obtained in our study, validates our use of the human genome sequence to localize rhesus genomic insertion sites.
Analysis of SIV integration shows a striking tendency to integrate within transcription units (73% of the mapped integration events), but no propensity toward integration in any specific region of the transcription units, in contrast to MLV vectors. Although we did not observe regional hot spots for SIV integration, as previously reported in cell lines for HIV (Schroder et al. 2002), we instead noted the clustering of integrations within gene-rich regions. This penchant for integrating in so-called ridges may offer clues to a specific mechanism of integration. Loops of chromatin extending away from chromosome territories are frequently observed on the major histocompatibility complex locus of Chromosome 6, the ridge shown in Figure 3B, especially when transcription is induced (Mahy et al. 2002; Volpi et al. 2000). These data suggest that the formation of decondensed chromatin territories might be driven by transcription (Chubb and Bickmore 2003) to establish a nuclear environment accessible to transcription factors (Gilbert et al. 2004) and, therefore, to lentiviral PICs. This hypothesis is corroborated by the fact that genes targeted by SIV vectors tend to be more highly expressed in human CD34+Rholo cells, as compared to the total set of 33,000 expressed sequences analyzed on a standard expression array (Figure S1). Interestingly, functional analysis of genes identified as targets for SIV insertion using the Gene Ontology classification (Ashburner et al. 2000) and the EASE bioinformatics software (Hosack et al. 2003) shows a statistically significant overrepresentation of genes coding for transcription factors and nuclear proteins (Figure S2), suggesting either these genes are more concentrated in targeted areas of the genome or they share common genomic motifs or cellular proteins.
This striking tendency was not observed with the MLV-derived set of integration sites. While the ratio of MLV integration sites within transcription units was significantly higher than expected compared to in silico-generated random integration sites, the MLV proviruses displayed a unique and specific affinity for the region surrounding the transcription start site of annotated genes. The finding that among the 491 MLV integration sites, only 12% are within SINEs or LINEs may support the fact that MLV inserts into 5′ regulatory elements where insertions of transposable elements are probably strongly selected against. This also indicates tethering between some transcription factor(s) and MLV PIC protein(s).
These observations, consistent with previous comparative analyses in vitro (Wu et al. 2003; Mitchell et al. 2004), likely reflect the vectors' distinct mechanisms for accessing DNA and integrating, and may have implications for the relative risk of insertional mutagenesis. While replication-competent oncoretroviruses have been widely used to identify genes involved in cancer (Dudley 2003), insertional oncogenesis has, to our knowledge, never been clearly reported after lentiviral infection. Both vectors have drawbacks: MLV vector integrations near the 5′ ends of genes may be more likely to disrupt transcriptional control and result in dysregulated expression of potentially oncogenic gene products, while SIV vector insertions within transcription units might be more likely to result in frame shifts or other events abrogating production of the normal gene product. Thus, the possibility that SIV vectors are less likely than MLV vectors to induce tumorigenesis needs to be carefully evaluated in relevant animal models.
A large number of genes were identified with two or more integration events, and thus were deemed common integration sites, including ten genes that had both MLV and SIV integrations. This suggests either that these genes are particularly susceptible to integration events due to open chromatin or other factors that favor both types of viruses, or that integration events in these particular genes alter expression and favor engraftment and long-term contributions to hematopoiesis. However, the most striking finding was the occurrence of seven independent hits by MLV in the first two introns of the MDS1 gene, whereas MDS1 was not found in the SIV dataset of integration sites. MDS1 is adjacent to the EVI1 locus, which has been implicated as a retrovirally activated proto-oncogene in a number of murine leukemogenesis studies (Bartholomew et al. 1989; Bordereaux et al. 1987; Morishita et al. 1988; Li et al. 2002). This unexpected and highly nonrandom clustering raises several questions since recent mapping analyses in cell lines did not report any common integration site (Wu et al. 2003). This suggest that proviral insertion near a proto-oncogene (MDS1/EVI1) may occur at a much higher frequency than previously expected. Studies are ongoing to better understand the causes and consequences of retroviral integration within this genomic locus.
It is important to stress that the very long-term follow-up of a large cohort of nonhuman primates, including all animals in the current study, has revealed completely normal hematopoiesis and lack of any progression towards neoplasia (Kiem et al. 2004). All animals have stable polyclonal hematopoiesis from transduced cells without any progression toward oligoclonality. Despite the nonrandom nature of integration and the possible targeting of certain proto-oncogenes, the use of replication-defective MLV or SIV vectors expressing nontransforming transgenes in the setting of one or very few integrants per cell still likely carries a very low risk of oncogenesis (Baum et al. 2003). Design of safer vectors including insulating elements to decrease the risk of activation of adjacent genes, development of targeted integration systems, or use of novel vectors with different integration patterns, should allow continued progress toward safe and effective gene therapy. However, for serious disorders such as SCID, even current MLV vectors are likely justified.
Materials and Methods
Rhesus macaque autologous transplantation model
Rhesus macaques were handled in accordance with the guidelines set by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources (National Research Council 1985). Protocols were approved by the Animal Care and Use Committee of the National Heart, Lung, and Blood Institute. Details of mobilization, transduction, and transplantation were previously published (Hanawa et al. 2004; Hematti et al. 2003; Takatoku et al. 2001; Wu et al. 2000). Animals were mobilized with stem cell factor (SCF) and granulocyte colony-stimulating factor for five doses, and underwent apheresis on day 5. CD34+ cells were enriched from mobilized PB by immunoabsorption and transduced for 96 h with either amphotropic MLV vectors LNL6 and G1Na containing the neomycin resistance gene (n = 22) (Miller and Buttimore 1986), or for 48 h with amphotropic SIV vector containing the green fluorescent protein gene (n = 3) (Hanawa et al. 2004). All transduction cultures were carried out in the presence of 100 ng/ml Flt3 ligand, 100 ng/ml SCF, and either 20 ng/ml interleukin-3 and 50 ng/ml interleukin-6 (n = 12), or 100 ng/ml megakaryocyte growth and development factor (n = 10 MLV animals and all SIV animals). All animals received cells transduced on flasks coated with Retronectin (TaKara, Shiga, Japan). In addition, two MLV animals also received cells transduced on autologous marrow stromal cells (Wu et al. 2000). Cells were reinfused intravenously following 1,000 rads of total body irradiation. PB samples were collected at a minimum of 6 mo after transplantation from three animals receiving SIV-transduced cells and 22 receiving MLV-transduced cells. MNCs were isolated by density gradient centrifugation over lymphocyte separation medium (Organon Teknika, Durham, North Carolina, United States), and granulocytes were obtained as previously described (Tisdale et al. 1998).
Cloning of the integration sites by LAM-PCR
LAM-PCR and cloning of insertion site vector genomic fusion sequences was performed as described (Hanawa et al. 2004; Schmidt et al. 2002) using 5′-linker cassettes and 3′-LTR primers designed specifically for MLV- or SIV-based vectors (Hanawa et al. 2004) (Table S3). Amplicons of junctions between genomic regions and 5′-LTRs were purified from agarose gels and cloned with the TOPO TA cloning kit (Invitrogen, Carlsbad, California, United States). Cycle sequencing was performed using an ABI Prism Genetic Analyzer 3100 (Applied Biosystems, Foster City, California, United States). Sequences were analyzed using Lasergene software (Dnastar, Madison, Wisconsin, United States).
Creation of a control set of in silico-generated integration sites
For statistical comparison to the integration site sets, we computationally generated 1,000 sets of integration sites. For MLV, we made 1,000 datasets, each containing 432 randomly selected genomic coordinates; for SIV, we made 1,000 datasets of 328 points each. All human chromosome sequences were concatenated into a single long sequence. We used the random number generator function in Perl to pick a number between 1 and the total number of nucleotides in the human genome (3,098,026,039), then identified this position in the concatenated sequence and correlated this position back to its chromosomal origin. If this coordinate fell within a sequencing gap, a new number was picked. We performed an ANOVA on the in silico-generated integration sites to demonstrate that 1,000 random sets were sufficient (unpublished data).
Genomic analysis of the retroviral and in silico-generated integration sites
We used a bioinformatic pipeline (Crawford et al. 2004) to map the position of each retroviral and in silico-generated integration site relative to 20,623 National Center for Biotechnology Information mRNA RefSeqs aligned by the UCSC Genome Browser. For each integration site, we calculated the distance to the nearest 5′ and 3′ end of a RefSeq gene. We disregarded cases in which RefSeq mRNAs aligned only partially to the genome. Genomic location of all LTR coordinates are available through the UCSC Genome Browser Custom Tracks (available at http://research.nhgri.nih.gov/projects/Dunbar/May2004/). Two-sided p values were obtained using the Chi2 test.
Functional clustering and over-representation analysis of targeted genes
Genes identified as targeted by retroviral insertion were analyzed for significant functional clusters of genes using the EASE bioinformatics software (http://david.niaid.nih.gov/david/ease.htm). This software was used to rank functional clusters by statistical overrepresentation of individual genes in specific categories relative to all genes in the same category. The functional clusters used by EASE were derived from the Gene Ontology classification system (http://www.geneontology.org).
Supporting Information
Dataset S1 MLV-Derived Integration Site Sequences (FASTA Format)
(78 KB TXT).
Click here for additional data file.
Dataset S2 SIV-Derived Integration Site Sequences (FASTA Format)
(60 KB TXT).
Click here for additional data file.
Figure S1 Relative Expression of Genes with Identified MLV and SIV Integrations, Using Data from Human CD34+Rholo Cells
(38 KB PDF).
Click here for additional data file.
Figure S2 Gene Ontology Categories Statistically Overrepresented in the Genes Targeted by SIV-Derived Vector
(16 KB PDF).
Click here for additional data file.
Table S1 Comparison of Retroviral Integration Sites Distribution within Transcription Units
(12 KB PDF).
Click here for additional data file.
Table S2 RefSeq Genes Targeted More than Once by SIV, MLV, or Both Retroviral Vectors
(18 KB PDF).
Click here for additional data file.
Table S3 Primers Used for the LAM-PCR Experiments
(8 KB PDF).
Click here for additional data file.
Accession Numbers
The retroviral integration site sequences larger than 50 bp discussed in this paper have been deposited in GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) under the accession numbers AY728482 to AY728804 for SIV, and AY733679 to AY734083 for MLV.
LocusLink ID numbers (http://www.ncbi.nlm.nih.gov/LocusLink/) for the genes discussed in this paper are ARHGEF12 (23365), EVI1 (2122), HMGA2 (8091), LMO2 (4005), MDS1 (4197), MKL1 (57591), MSF (10801), RAD51L1 (5890), RUNX1 (861), andTRIM5α (85363).
We thank Bob Wesley for help with statistical analysis, Robert Donahue and the veterinary and animal support staff at the National Heart, Lung, and Blood Institute 5 Research Court primate facility for assistance with rhesus macaques, and Greg Sabino Mullane for generating the gene-density data. BC is supported by a postdoctoral fellowship from the Fondation de France, the Philippe Foundation, and the National Institutes of Health.
Conflicts of interest. The authors have declared that no conflicts of interest exist.
Author contributions. TGW, CED, and BC conceived and designed the experiments. PH, BKH, CF, RA, SS, and BC performed the experiments. TGW, CED, and BC analyzed the data. HH, IEH, CEE, YS, MS, CvK, DAP, EMB, CMV, AWN, and TGW contributed reagents/materials/analysis tools. TGW, CED, and BC wrote the paper.
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Academic Editor: Michael Emerman, Fred Hutchinson Cancer Research Center
¤1 Current address: Department of Medicine, Hematology/Bone Marrow Transplant Section, University of Wisconsin-Madison Medical School, Madison, Wisconsin, United States of America
¤2 Current address: Centre de Thérapie Cellulaire, Institut Paoli-Calmettes, Marseille, France
Citation: Hematti P, Hong BK, Ferguson C, Adler R, Hanawa H, et al. (2004) Distinct genomic integration of MLV and SIV vectors in primate hematopoietic stem and progenitor cells. PLoS Biol 2(12): e423.
Abbreviations
HSChematopoietic stem cell
LAM-PCRlinear amplification-mediated PCR
LINElong interspersed element
LTRlong terminal repeat
MLVmurine leukemia virus
MNCmononuclear cell
PBperipheral blood
PICpreintegration complex
RefSeqreference sequence
SCIDsevere combined immunodeficiency
SINEshort interspersed element
SIVsimian immunodeficiency virus
UCSCUniversity of California
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| 15550989 | PMC529319 | CC BY | 2021-01-05 08:28:08 | no | PLoS Biol. 2004 Dec 23; 2(12):e423 | utf-8 | PLoS Biol | 2,004 | 10.1371/journal.pbio.0020423 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020433SynopsisGenetics/Genomics/Gene TherapyImmunologyMolecular Biology/Structural BiologyHomo (Human)All HEPped Up about Methylation Synopsis12 2004 23 11 2004 23 11 2004 2 12 e433Copyright: © 2004 Public Library of Science.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
DNA Methylation Profiling of the Human Major Histocompatibility Complex: A Pilot Study for the Human Epigenome Project
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For a recipe to become a meal, it's often necessary to embellish or modify the basic instructions—and to keep a note of the changes that work, so that it can be just as delicious next time around. The same is true for a gene, whose basic recipe—its nucleotide sequence—can be heritably annotated to “epigenetically” influence its level of expression without altering its sequence. Among the many epigenetic influences at work in the genome, methylation of cytosine is one of the most versatile and powerful. Addition of a methyl (-CH3) group to cytosines within a gene's regulatory regions can reduce its transcription. In its extreme form, methylation is involved in silencing one of the two X chromosomes in female mammals. Aberrant methylation underlies susceptibilities to several forms of cancer, and is likely to be involved in numerous other human diseases.
The goal of the Human Epigenome Project (HEP) is to map the methylation patterns of human genes, and to determine how they vary: among individuals, among tissues within an individual, and even over time within a single tissue. In this issue, Stephan Beck and colleagues describe the execution and results of a HEP pilot project, in which they analyzed methylation within the major histocompatibility complex (MHC), the set of genes that establish an individual's self-identity within the context of immune surveillance.
The key to any such large-scale project is high throughput—a rapid, efficient set of technologies that produce the needed data with minimal human intervention. The strategy used by Rakyan et al. included bisulfite sequencing of DNA, in which unmethylated cytosines are chemically converted to uracils, while methylated cytosines are not. Software they developed detects the methylated sites and provides an overall measure of the methylation level within any given sequence. They confirmed the accuracy of their method with mass spectrometry, an alternative method also suitable for high-throughput screening.
They initially analyzed 253 sequences within 90 genes in the MHC, about two-thirds of the total, from multiple tissues in multiple individuals. They found that most genes were either completely methylated or completely unmethylated, while relatively few had an intermediate value. The significance of this distribution pattern is not yet clear, although it does confirm similar results in smaller samples from other research groups. The researchers also confirmed that so-called CpG islands, regions rich in CG dinucleotides, are relatively hypomethylated, especially when they occur at the upstream end of a gene.
Methylation levels in a region of the human genome
Rakyan et al. also found differences in methylation levels among tissues, with some suggestion that the variations influence tissue-specific alternative splicing, at least in some genes. Intriguing inter-individual differences were also found, with median methylation levels differing significantly between individuals for at least one tissue at almost half the sites analyzed. For instance, such differences were found in liver for the regulatory region for the tumor necrosis factor gene.
A major goal of the HEP is to identify methylation variable positions, sites whose methylation state is linked with some important biological state, be it tissue type, developmental stage, or disease state. The pilot project described here begins this undertaking, which will be greatly expanded as the HEP progresses. The first phase of the full-scale HEP, an analysis of 5,000 DNA sequences, is currently underway.
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020434SynopsisAnimal BehaviorBirdsAre Animals As Irrational As Humans? Synopsis12 2004 23 11 2004 23 11 2004 2 12 e434Copyright: © 2004 Public Library of Science.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
State-Dependent Decisions Cause Apparent Violations of Rationality in Animal Choice
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Animals in the wild are constantly confronted with decisions: Where to nest? Who to mate? Where's the best forage? To explore the mechanisms underlying such decisions, animal behavior studies often incorporate concepts from economic theory. Mainstream models of choice in both economics and biology predict that preferences will be rational, or consistent across contexts, as a result of being motivated by self interest or, in the case of animals, reproductive success. Yet many studies report that when making decisions people often take shortcuts, using cognitive heuristics that may lead to economically irrational decisions, with similar claims now showing up in animal behavior studies.
In a new study, Cynthia Schuck-Paim, Lorena Pompilio, and Alex Kacelnik ask whether studies applying economic rationality to animal behavior are controlling for potentially confounding effects inherent in such approaches. The authors suggest that observed “breaches of rationality” may stem from differences in the physiological state of animals “unwittingly imposed” by experimental design rather than from real irrational decisions.
Choice studies typically offer subjects a range of choices that include clearly superior and inferior alternatives. While humans can simply hear about the various alternatives and their respective properties, animals must be trained to learn about the different choices. This difference is far from trivial, Schuck-Paim et al. argue, and could well require different interpretations of results in animal and human studies. In fact, economic theory states that optimal choices depend on both the properties of the option and the chooser's state. Training animals to learn of different choices typically involves giving them food rewards, which means that an animal's energetic state—that is, hungry versus sated—will change over a day of training. A bird that's eaten an ounce of birdseed is more likely to opt for an “irrational” option—say, a choice that dispenses little food—than one that's hungry.
To examine this theoretical constraint under experimental conditions, Schuck-Paim et al. trained European starlings to choose between two rich food sources (called focal options) and one of two poorer “decoys” in different contexts. One of the focal options offered more food while the other offered a shorter delay between pecking a key and receiving the food, but their amount/delay ratios were equal. The decoys were considered less preferable because their ratio of amount to delay was lower than that of the focal options. But the decoys could potentially confound the results because repeated training to each decoy could sate a bird's appetite to different degrees: although amount/delay was equal among the decoys, their long term energetic consequences differed.
European starlings make rational decisions
The authors tested for preference between the focal options under three experimental conditions: altering the birds' food intake/energetic state with no decoys; changing the decoy and not controlling for its corresponding energetic contributions; and changing the decoy but controlling for its energetic consequence (by supplemental feeding).
Schuck-Paim and collaborators show that the birds' preferences between the focal options differed significantly between treatments, in apparent breach of economic rationality; the preference for the larger reward option over the shorter delay option was much stronger when the trial involved lower accumulated intake than when the accumulated intake was high. Introducing the decoys resulted in an “irrational” preference only when the decoys were allowed to have an effect on food intake, suggesting that the choice resulted from the birds' energetic state rather than from cognitive mechanisms of choice similar to those used to explain irrationality in human subjects.
The authors offer an evolutionary and mechanistic explanation for why animal preference might be governed by energetic state, including the possibility that animals are less motivated to focus exclusively on the richest option when they are well fed. But they are careful to disabuse the notion that “state-dependence accounts for all reported inconsistencies in animal choice” or that animals do not employ cognitive mechanisms of choice similar to those of humans. Altogether, Schuck-Paim and co-authors argue, these results warn that studies appropriating ideas from other disciplines can introduce confounding effects. And that researchers would do well to carefully examine the underlying causes of observed animal behaviors when testing ideas formulated in a nonbiological framework.
| 0 | PMC529321 | CC BY | 2021-01-05 08:28:08 | no | PLoS Biol. 2004 Dec 23; 2(12):e434 | utf-8 | PLoS Biol | 2,004 | 10.1371/journal.pbio.0020434 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020437SynopsisDevelopmentNeuroscienceMus (Mouse)A Neuron Survival Protein May Give Directions, Too Synopsis12 2004 23 11 2004 23 11 2004 2 12 e437Copyright: © 2004 Public Library of Science.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
A Chemoattractant Role for NT-3 in Proprioceptive Axon Guidance
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Anyone who's ever had a physical exam knows the monosynaptic stretch reflex arc. When the doctor taps your patellar tendon with a hammer, your quadriceps muscle briefly stretches and your knee responds with a quick kick. This involuntarily reflex is mediated by muscle spindles, specialized muscle structures containing both proprioceptive (sensory) and motor neurons. Proprioceptive neurons send information about how much the muscle is stretched to the spinal cord, and motor neurons emanating from the spinal cord tell the muscle to contract, which corrects the stretch.
This reflex circuit is established in the developing embryo, when neurons migrate through and around developing tissues and send their axons to their signaling targets. Many neurons will successfully extend their axons into a target—which might be a muscle, another neuron, or some other tissue—but not all survive the process. Whether a neuron lives or dies depends on a family of growth factor proteins called neurotrophins. If a sensory neuron doesn't get enough neurotrophin-3 (NT-3), it will die.
Proper development of the sensory/motor circuit also depends on NT-3, which is expressed in limb buds, muscle spindles, and the ventral spinal cord: muscle spindle development depends on sensory axons, and motor neuron connections depend on developing limb buds. It has not been clear, however, whether NT-3 simply ensures the survival of proprioceptive neurons or whether it also helps establish the proprioceptive reflex arc. In a new study, Reha Erzurumlu and colleagues demonstrate a clear role for NT-3 in axon guidance.
Developing nerves in the mouse spinal cord
Attempts to investigate the axon guidance theory have been difficult since sensory neurons die without NT-3. To circumvent this problem, the authors developed a “double knockout” mouse model that deletes both the NT-3 gene and the apoptosis-promoting gene Bax, which activates the cell death pathway for sensory neurons. This system removes NT-3 signaling without killing the sensory neurons, so the researchers can investigate any effects NT-3 may have on axon behavior.
Erzurumlu and colleagues showed that sensory neurons, in the absence of NT-3 signaling, project to the right places but never reach their final destination. In normal development, sensory neuron axons travel into the ventral spinal cord and form synapses with motor neuron dendrites in the ventral horn to establish the reflex arc circuit. In the double knockout mice, sensory neurons manage to extend into the spinal cord, but then get lost; they can't find the ventral horn, so they never form a synapse with the motor neuron dendrites. The failure to establish connections between the sensory axons and motor neurons in mice lacking NT-3, the authors argue, indicates that NT-3 is required for proper axon targeting.
Similarly, deprived of NT-3 signaling, sensory neuron axons fail to reach their ultimate targets in peripheral muscle. They project down toward the muscle but don't recognize the muscle and thus cannot enter or innervate it. Consequently, the muscles' sense organs, the muscle spindles, cannot differentiate. The authors argue that these findings, along with the results of tissue culture experiments, show that NT-3 acts as a short-distance cue for proprioceptive axons, which travel in the right direction but ultimately lose their way without NT-3.
Altogether these results show that proprioceptive axons require NT-3 not just for survival, but to reach their proper endpoints in the peripheral and central nervous system. NT-3 also helps proprioceptive axons initiate muscle innervation and spindle differentiation. Researchers developing therapies to treat neurodegenerative injuries have increasingly focused their attentions on growth factors like NT-3. By identifying the molecules and mechanisms that establish connections between sensory and motor neurons during development, it may be possible to engage similar processes to attenuate neurodegeneration and even repair damaged nerves.
| 0 | PMC529322 | CC BY | 2021-01-05 08:21:17 | no | PLoS Biol. 2004 Dec 23; 2(12):e437 | utf-8 | PLoS Biol | 2,004 | 10.1371/journal.pbio.0020437 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020438SynopsisCell BiologyEvolutionGenetics/Genomics/Gene TherapyMicrobiologyMolecular Biology/Structural BiologyEubacteriaIn Times of Stress, Mutate Early and Often Synopsis12 2004 23 11 2004 23 11 2004 2 12 e438Copyright: © 2004 Public Library of Science.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Adaptive Amplification and Point Mutation Are Independent Mechanisms: Evidence for Various Stress-Inducible Mutation Mechanisms
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For a human, the normal response to stress is to reduce it through some purposeful action, be it indulging in chocolate or calling in sick, at a rate which we can vary to fit the circumstances. For a strain of bacteria faced with stress, the choice is often more stark: it must mutate or die. Among evolutionary theorists, an important question has been whether the rate of mutation is fixed, or instead can adaptively increase in response to stress, thereby increasing the likelihood of a favorable mutation. Something like this latter possibility was envisioned by Darwin, but fell out of favor among some neo-Darwinists, for whom a steady rate of mutation was more in keeping with their overall model of evolutionary gradualism. This debate is taken up in a new study in this issue by P. J. Hastings and colleagues, who examined the mechanism by which Escherichia coli lacking the ability to digest lactose, called lac − mutants, regain that ability when presented with lactose as their only food source.
It has been known for some time that the reversion of lac − mutants to a lac+ state can be achieved by either of two genetic events: amplification, which creates numerous copies of the nonfunctional lac gene, and point mutations, which give rise to functional versions of the gene (many non-useful mutations also occur; thus, there is no directed mutation, in keeping with standard Darwinian evolution). According to the gradualist view, amplification precedes mutation, and the rapid appearance of lac+ cells is explained by a normal mutation rate acting on multiple copies of the gene. In contrast, according to the hypermutation view, amplification and mutation are independent events, and lac+ cells arise quickly because the mutation rate has increased.
While some results from previous studies have supported the gradualist interpretation, the experiments of Hastings et al. show that hypermutation is the most plausible explanation. A variety of procedural improvements allowed them to analyze more individual cells at an earlier stage of colony development. For instance, they analyzed colonies composed of as few as one hundred cells, rather than the ten thousand cells in prior experiments, and even nascent colonies at the two-cell stage.
Sectored colonies of lac + and lac−
E. coli
The study produced clear evidence that point mutations arise very early in the development of lac+ colonies, before amplification can account for the number of lac+ revertants observed. Amplification is not only independent from mutation, but occurs relatively late under starvation. The researchers found that amplification, but not point mutation, requires the presence of a particular DNA polymerase, further strengthening the case that amplification need not precede mutation. They also showed that amplification by itself does not induce a so-called SOS response. The SOS system includes a group of genes that cause an increase in mutation in response to stress, and one hypothesis arising from the gradualist model was that amplification turned on the SOS response.
Based on their data, the authors reject the strict gradualist model for the adaptive mutation mechanism in the Lac system. They propose that amplification and hypermutation are independent responses to stress, each of which increases the likelihood of adaptive change. They also suggest that a stress-induced increase in the rate of point mutations may have implications for a variety of mutation-related phenomena, from tumor formation to development of resistance to antibiotics and chemotherapeutic drugs.
| 0 | PMC529323 | CC BY | 2021-01-05 08:21:17 | no | PLoS Biol. 2004 Dec 23; 2(12):e438 | utf-8 | PLoS Biol | 2,004 | 10.1371/journal.pbio.0020438 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020443SynopsisGenetics/Genomics/Gene TherapyVirologyPrimatesVirusesRetroviral Gene Vectors Show Clear Target Preferences Synopsis12 2004 23 11 2004 23 11 2004 2 12 e443Copyright: © 2004 Public Library of Science.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Distinct Genomic Integration of MLV and SIV Vectors in Primate Hematopoietic Stem and Progenitor Cells
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Despite some high-profile setbacks in gene therapy over the past few years, scientists have not lost hope that targeted gene transfer will one day treat a wide range of acquired and congenital diseases. After two young gene therapy patients developed a leukemia-like disorder last year—apparently because the viral vector used to carry the corrective gene activated a cancer-causing gene—researchers redoubled their efforts to develop safer, more effective retroviral gene delivery methods. Such efforts depend on understanding how and where retroviral vectors integrate into the genome.
In a new study, Cynthia Dunbar and colleagues describe a nonhuman primate model that mimics gene therapy protocols in humans, and report the integration biases of two classes of retroviral vectors being developed for clinical trials. The retroviruses, the authors show, display clear preferences that not only suggest different genomic integration mechanisms but also have different implications for safety.
Retroviral gene therapy exploits a retrovirus's skill at entering a cell, infiltrating its genome, and hijacking its molecular machinery for its own reproductive advantage. In gene therapy, therapeutic genes largely take the place of viral genes, and so the “infected” cell churns out beneficial gene products, not viruses. Before a retrovirus can integrate into a host cell, however, it must copy its genome—which is encoded in RNA—into DNA, so the cell's copying machinery will recognize it. After generating this “pre-integration complex,” the virus must access the cell's chromosomal DNA, which lies behind a nuclear barrier. Different retroviruses accomplish this task in different ways.
Lentiviruses—which include AIDS and SIV (simian immunodeficiency virus)—can infect nondividing cells simply by slipping through nuclear pores. Oncoretroviruses—such as murine leukemia virus, or MLV, the vector type used for the vast majority of previous clinical trials, including the trial complicated by leukemia in two patients—must wait until the nuclear membrane dissolves during cell division. Once integrated into the host genome, the provirus—and its therapeutic gene—will persist through each new cell division—a trait that underlies its usefulness as a vector as well as its risk. Retroviruses that insert near proto-oncogenes can activate these genes and set the cell on the path to tumorigenesis. Until recently, researchers assumed this risk was extremely low because retroviral integration was thought to be random—an assumption recently undercut by a number of studies that mapped retroviral integration in different cell lines.
Dunbar and colleagues take these studies a step further by mapping the integration patterns of MLV and SIV vectors in hematopoietic stem cells (HSCs) of rhesus monkeys. HSCs are the cells typically used to carry these vectors for therapeutic applications involving any of the cell types, such as red blood cells, produced by the bone marrow. The monkeys had received infusions of HSCs carrying either the SIV or MLV vectors between six months and six years earlier. Two types of white blood cells (granulocytes and mononuclear cells) were harvested from the monkeys and evaluated for proviral insertion sites.
Of nearly 1,000 integration sites identified, 760 could be mapped to unique corresponding sites in the human genome (432 MLV and 328 SIV). While both MLV and SIV vectors tended to integrate within genes, MLV showed a strong preference for the starting end of genes, most likely to result in gene activation. In contrast, SIV showed a preference for genomic regions of high gene density, but not for specific sites within a gene. Surprisingly, MLV targeted one gene—known previously to be involved in spontaneous leukemias and in murine retroviral oncogenesis—seven times, a “highly nonrandom” result suggesting that such insertions may occur far more often than previously thought. About 40 genes, including seven known oncogenes, were targeted more than once by one or both vectors. Such differences, Dunbar and colleagues note, “likely reflect the vectors' distinct mechanisms for accessing DNA and integrating,” which could in turn affect their risk of causing insertional mutagenesis. Even though the vectors tend to integrate nonrandomly and can target oncogenes, however, none of the monkeys showed signs of ill effects such as leukemia.
But before any widespread applications of retroviral gene therapy can proceed, Dunbar and colleagues argue, potential risks of proviral insertion must be assessed in the specific cell types associated with different gene therapies. And with a model for long-term, genome-wide retroviral integration analysis that mimics human gene therapy protocols, the authors have made an important contribution toward that end.
| 0 | PMC529324 | CC BY | 2021-01-05 08:21:17 | no | PLoS Biol. 2004 Dec 23; 2(12):e443 | utf-8 | PLoS Biol | 2,004 | 10.1371/journal.pbio.0020443 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020444SynopsisGenetics/Genomics/Gene TherapyNeuroscienceDrosophilaThis Is Your Fly's Brain on Drugs Synopsis12 2004 23 11 2004 23 11 2004 2 12 e444Copyright: © 2004 Public Library of Science.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Lmo Mutants Reveal a Novel Role for Circadian Pacemaker Neurons in Cocaine-Induced Behaviors
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Cocaine addiction wreaks profound changes on the brain, hijacking reward circuits and depressing inhibitory loops to the point that drug seeking and taking become central drivers of behavior. Lying at the core of these behavioral changes are molecular ones; at its most basic level, addiction alters the sensitivity of neurons. While primates and rats are useful for mapping out the neural complexity of these behavioral manifestations, insights into the molecular basis of drug abuse can be garnered more easily from simpler models, such as the fruitfly, Drosophila.
The reigning model of cocaine's effects on the brain has highlighted its ability to block reuptake of dopamine by cells of a brain region called the nucleus accumbens. But numerous experiments show this is not the whole story. Ulrike Heberlein and colleagues describe their discovery of a new gene that modulates sensitivity to cocaine within the cells of the fruitfly's internal clock. They further show that the cells' role in regulating cocaine sensitivity is distinct from its function as a timekeeper.
One known effect of cocaine on Drosophila is loss of “negative geotaxis,” or wall climbing, in response to startle. Using this behavior to screen 400 different mutants, the researchers identified seven with an increased response to cocaine, and for two of these, the disrupted gene was the same, Lmo. The Lmo protein, whose levels were reduced by the mutations, is known to regulate certain transcription factors during development. Despite this, no developmental defects were detected in the loss-of-function mutants that might explain the cocaine effect. The researchers also found that a third mutation in the same gene, previously associated with disruption in wing formation, increased levels of the Lmo protein, and decreased response to cocaine. Thus, Lmo appears to play a central role in regulating cocaine sensitivity.
While Lmo is found throughout the body, it is enriched in the brain, and by expressing normal Lmo in oversensitive mutants, Heberlein and colleagues discovered that its cocaine-related effects were localized to the ventral lateral neurons (LNvs). Comprising about ten cells per hemisphere, these neurons provide the fly with an internal clock, driving circadian activities even in the absence of light. Not surprisingly, Lmo mutants had weaker circadian rhythms than normal flies.
But is increased cocaine sensitivity a simple consequence of a broken clock? Apparently not. To date, the only known output of the LNv is a small peptide called PDF, and PDF mutation causes circadian disruptions. It does not, however, alter cocaine sensitivity. Furthermore, completely obliterating the LNv, or blocking its ability to fire, disrupted circadian rhythmicity but reduced cocaine sensitivity, rather than increasing it. These results indicate than the LNv normally enhances sensitivity to cocaine, a function enhanced further by Lmo mutants, and does so independently of circadian regulation.
Based on their results, Heberlein and colleagues propose a possible model for Lmo's role in modulating cocaine sensitivity. Drawing on recent evidence that a subset of LNv cells possess dopamine receptors, they suggest that Lmo expression normally regulates the density of these receptors on LNv cells. Loss of Lmo would raise the number of receptors, thereby increasing the sensitivity to cocaine. A key prediction from their findings is that the LNv has another output, as yet undetected, in parallel with PDF that mediates responsiveness to cocaine.
Because Lmo-related proteins are found in key areas of mammalian brains, these results may have important implications for understanding innate differences in sensitivity to cocaine in humans, and potentially provide targets for development of drugs to treat or prevent addiction.
| 0 | PMC529325 | CC BY | 2021-01-05 08:21:17 | no | PLoS Biol. 2004 Dec 23; 2(12):e444 | utf-8 | PLoS Biol | 2,004 | 10.1371/journal.pbio.0020444 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1557810110.1371/journal.pmed.0010016Health in ActionNeurology/NeurosurgeryPathologyPathologyNeurologyA Free Community Approach to Classifying Disease Health in ActionGraeber Manuel B *Lowe James Radotra Bishan Manuel Graeber is a professor of neuropathology and chair of the Department of Neuropathology, Faculty of Medicine, Division of Neuroscience, Imperial College London and Hammersmith Hospitals Trust, Charing Cross Campus, London, United Kingdom. James Lowe is a professor of neuropathology and head of the Division of Pathology, School of Molecular Medical Sciences, University of Nottingham Medical School, Nottingham, United Kingdom. Bishan Radotra is an additional professor in the Department of Histopathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
Competing Interests: MBG and JL were involved in launching the International Classification of Diseases of the Nervous System and are both on the editorial board of PLoS Medicine. BR declares that he has no competing interests.
* To whom correspondence should be addressed. E-mail: [email protected] 2004 30 11 2004 1 2 e16Copyright: © 2004 Graeber et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Defining and classifying disease is at the heart of medical practice, but the process is slow and laborious. A new "open source" approach could be faster and more democratic
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Defining and classifying disease is at the heart of medical practice. But the standard approach to classification is slow and laborious. A new approach promises to revolutionise the way in which we classify disease. It involves the free and public sharing of information via the Internet—the so-called open-source, or, perhaps more appropriately termed, “free community,” approach (R. M. Stallman, personal communication).
The International Society for Neuropathology is the first worldwide professional medical organisation to adopt such an approach with its International Classification of Diseases of the Nervous System (ICDNS; see http://www.ICDNS.org). The main characteristics of the ICDNS are free collaboration via the Internet, online access to all collaborative tools via the World Wide Web, global participation, and democratic decision making [1].
Why We Need a New Approach
Before a disease can be recognised, its nature and the conditions surrounding it must be determined in order to establish criteria for its definition. The more precise a disease definition, the greater the benefit is for the patient, especially where specific treatments are available. Once individual diseases are defined, they can be classified, resulting in the creation of conceptual links that are fundamentally important for medical practice and the advancement of medical knowledge. One example is the conceptual linking of Pick disease, Alzheimer disease, progressive supranuclear palsy, and corticobasal degeneration as members of the group of tauopathies. However, the way in which medical classifications of disease traditionally evolve is problematic.
Usually, small groups of experts meet and decide on a classification that fits best with their personal experience. Classifications then change when new scientific developments are applied to link or separate different conditions. An example is the identification of several pathologically distinct types of frontotemporal dementia based on the application of immunohistochemical staining for tau protein and ubiquitin. The wider medical community subsequently validates these new schemes, provided there is agreement on the basic aspects of any new taxonomic concept. Often, however, consensus only emerges after many years and even decades of controversy and dispute. The World Health Organization's classification of brain tumours [2] is an example of a classification that has taken decades to mature. Thus, the established process is not very effective and is undoubtedly time consuming. It is also occasionally politicised, as “egos” may be unable to resist the temptation of leaving their personal mark, while ignoring the cultural benefit of consensus agreement that results in knowledge that is usable by everyone.
New disease entities are presently emerging at a much higher rate because of advances in biomedicine that were triggered by the Human Genome Project. More and more diseases are being redefined according to molecular criteria. The Lewy body diseases, which share a pathological aggregation of the protein alpha-synuclein (“alpha-synucleinopathies”), are an example of a disease subset now defined by a common molecular pathology. The large field of pathology and the neurosciences are two areas where the translation of morphological phenotypes into molecularly defined entities is already well underway. With the pace of change accelerated by advances in molecular science, we need a much more effective way to develop the debate about medical classifications.
Open Source and the ICDNS
One effective way to further develop this debate is to adopt the approach used by the free-software and open-source movements [3], which have spawned free software, free operating systems, and free scientific and medical journals. Open source has profoundly important implications for science, technology, and medicine.
The development of global computer networks and the World Wide Web, in particular, have fostered the evolution of free and global sharing of intellectual property. The Open Source Initiative (http://www.opensource.org) is a nonprofit venture dedicated to managing and promoting the open-source idea (Box 1). A related but more radical concept is propagated by the Free (as in freedom) Software Foundation (http://www.fsf.org). The creation of the GNU/Linux operating system (http://www.gnu.org and http://www.linux.org) has resulted from the work of both.
Box 1. The Open Source Initiative
“The basic idea behind open source is very simple: When programmers can read, redistribute, and modify the source code for a piece of software, the software evolves. People improve it, people adapt it, people fix bugs. And this can happen at a speed that, if one is used to the slow pace of conventional software development, seems astonishing… Open source software is an idea whose time has finally come. For twenty years it has been building momentum in the technical cultures that built the Internet and the World Wide Web. Now it is breaking out into the commercial world, and that's changing all the rules.” (It should be noted that the Open Source Software Initiative of 1998 was preceded by Richard Stallman's Free Software Movement of 1983 [5].) Source: Open Source Web site (http://www.opensource.org).
The ICDNS was inspired by the free software and open-source approach to software development. The Council of the International Society for Neuropathology approved the ICDNS as a community activity at its last meeting in Turin, Italy, in September of 2003 [4]. The implications of ICDNS go far beyond defining neurological diseases. The benefits include the standardisation of neuropathological training programmes across continents and a new means of direct, professional communication between colleagues from countries all over the globe.
How ICDNS Works
Diagnostic criteria for all recognised neuropathological diseases are being published on the Web at http://www.ICDNS.org, where the global community of neuropathologists can judge them (Figure 1). No named individual or national group is leading the initiative. Existing classifications are translated into a generic format, avoiding personal as well as institutional names to ensure consistent terminology between related disease processes.
Figure 1 Pilocytic Astrocytoma
Future brain tumour classifications will be decided in a democratic way. (Photo: Dr. F. Roncaroli, Department of Neuropathology, Imperial College London)
Although still in its early days, definitions for Lewy body disease, Alzheimer disease, and several tumours are now online. Individual ICDNS members (membership is free) as well as expert interest groups propose core definitions, which are then posted so that the global consultation process begins via the World Wide Web. After an online discussion period, ICDNS members holding a specialist certification in diagnostic neuropathology are invited to vote. Comments made online by contributors become part of the history of a disease definition so that nobody is left out and divergent views are not forgotten. The discussion process is thereby open and democratic, allowing wide participation—including from individuals in developing countries who are often excluded in traditional academic discussions.
Different countries around the world show variations in their use of diagnostic criteria and medical classifications, which, in turn, can lead to different treatment approaches. This can certainly create problems when trying to reach a global consensus. However, free access to the information held under the ICDNS open licence may help to minimise these problems by promoting and stimulating collaborative research and the exchange of scientific ideas across the globe.
Certain diseases are far more common in particular parts of the world. In India, for example, neurotuberculosis, cerebral malaria, fungal infections of the central nervous system, human rabies, encephalomyelitis, and cerebrovenous thrombosis are more common than in most developed countries. While the conventional pathology of these diseases is well known—and in some cases we also have expert knowledge on their morphological phenotypes—their exact pathogenesis is not understood, and cellular as well as molecular knowledge is missing. The ICDNS is expected to stimulate local researchers to engage in collaborative international projects in which they can receive feedback via the global consultation process.
Publication of ICDNS criteria occurs under a general public licence. This means that all text can be freely downloaded and republished, avoiding the need for defining basic facts over and over again. Both core definitions and comments may be used immediately for diagnostic purposes as outlined in the guidance section of the ICDNS Web site.
Future Directions
In the future, definitions of histopathological phenotypes may be linked to clinical as well as molecular biological datasets, such as those obtained from microarrays and combined with imaging parameters. It is obvious that expert consensus on histological phenotypes is required before “genome matching” and similar procedures can be applied to complex diseases in a meaningful way. Most diseases are presently still defined on the basis of their histopathology. Online forums to find diagnostic consensus will provide a very effective means of correlating descriptive data with molecular data. Subsequently, statistical clusters representing “signatures of disease” may be extracted from multidimensional data spaces that will be available online. This opens new roads to link with diagnostic and therapeutic approaches.
It seems reasonable to propose that the adoption of the ICDNS paradigm by other medical specialties would facilitate the development of a comprehensive, global body of medical knowledge. Free access to this knowledge would allow novel, collaborative approaches to be developed to address the most pressing medical problems through supranational concerted efforts. The rather lowly role traditionally ascribed to the exercise of defining and classifying diseases would give way to an appreciation for the key importance of this process as a potentially powerful driver of change.
Citation: Graeber MB, Lowe J, Radotra B (2004) A free community approach to classifying disease. PLoS Med 1(2): e16.
Abbreviation
ICDNSInternational Classification of Diseases of the Nervous System
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References
Achim C Auer R Bergeron C Cardozo A Deprez M Global democratic consensus on neuropathological disease criteria Lancet Neurol 2002 1 340 12849392
Kleihues P Louis DN Scheithauer BW Rorke LB Reifenberger G The WHO classification of tumors of the nervous system J Neuropathol Exp Neurol 2002 61 215 225 11895036
O'Reilly T The open source paradigm shift 2004 Sebastopol O'Reilly Publishers Available: http://www.oreillynet.com/pub/a/oreilly/tim/opensource/paradigmshift_0504.html . Accessed 23 September 2004
Vinters HV A new initiative in classification of neurologic diseases: Global consensus on diagnostic criteria? Brain Pathol 2004 14 1 14997932
Stallman RM Lessig L Gay J Free software, free society 2002 Boston Free Software Foundation 224
| 15578101 | PMC529419 | CC BY | 2021-01-05 10:38:00 | no | PLoS Med. 2004 Nov 30; 1(2):e16 | utf-8 | PLoS Med | 2,004 | 10.1371/journal.pmed.0010016 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1557810210.1371/journal.pmed.0010017Learning ForumInfectious DiseasesDermatologyDermatologyInfectious DiseasesA 33-Year-Old Man with a Facial Rash Learning ForumFleming John Lynn William A *The Learning Forum section editors are Susan Lightman and William Lynn. John Fleming is a medical student at Imperial College, London, United Kingdom. William Lynn is a consultant in infectious diseases and the medical director at Ealing Hospital, London, United Kingdom.
*To whom correspondence should be addressed. E-mail: [email protected]
Competing Interests: John Fleming declares that he has no competing interests. William Lynn is on the editorial board of PLoS Medicine and is a section editor of the Learning Forum.
11 2004 30 11 2004 1 2 e17Copyright: © 2004 Fleming and Lynn.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Test Your Knowledge: Ten Questions about Eczema Herpeticum
A learning module based around the case of a man with ezema who presents with a rash around his ear and eye. Test your knowledge with our online quiz
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DESCRIPTION of CASE
A 33-y-old man presented to his primary care practitioner with a rash beginning around the left ear and spreading into the periorbital region. He was known to have eczema (atopic dermatitis), and the diagnosis of secondary bacterial infection and periorbital cellulitis was made.
Because of a history of penicillin allergy, the patient was started on erythromycin. This treatment had no effect, and the rash extended. On day 4, he presented to the hospital accident and emergency department. He was systemically unwell, complaining of drenching sweats and rigors. On examination he was alert, he was pyrexial at 39.2 °C, his pulse was 100 bpm, and his blood pressure was 128/70 mm Hg. There was a widespread erythematous rash covering his face, chest, and arms, which was described as wet with a yellowish exudate. The rash extended around both eyes, which could not be opened (Figure 1).
Figure 1 Appearance of Part of the Face and Shoulder at Presentation to Hospital
There are multiple shallow ulcers with copious creamy exudates and some crusting. A few intact vesicles are also visible. The right eye was closed by exudates.
The patient had a history of atopic eczema (since childhood), rhinitis, and asthma. He was taking inhaled corticosteroids for asthma and had last received a course of systemic steroids one year previously. There were no risk factors for HIV infection.
Which Investigations Were Now Indicated?
In many patients with a rash, the morphology of the rash will lead to a provisional clinical diagnosis, and it may be sufficiently characteristic for no confirmatory tests to be needed. An example is the typical rash of shingles. There are two broad reasons for doing investigations in a patient presenting with fever and a rash. Firstly, specific tests—such as blood tests, cultures, or a skin biopsy—may be required to help establish the diagnosis. In this 33-y-old patient, the rash was thought likely to be a skin infection, and so the specific tests that were indicated included viral and bacterial cultures. A skin biopsy would have been considered if the diagnosis remained unclear and the patient was not responding to empirical antimicrobial therapy. Secondly, investigations are used to evaluate how ill the patient is and whether there is any organ involvement beyond the skin. In this case, we were most concerned about severe sepsis with a risk of organ failure.
Investigations showed the following: haemoglobin, 128 g/L (normal range, 135–170 g/L); white blood cell count, 9.9 × 109/l (normal range, 3.5–12.5 × 109/l); and differential white blood cell count, 90% neutrophils with a left shift. The patient was hyponatraemic (his sodium was 126 mmol/l [normal range, 135–145]), but renal and liver function tests were normal. C-reactive protein was 140 mg/l (normal level, <10 mg/l). Chest X ray was normal. Blood cultures and skin swabs were taken along with samples for viral culture and polymerase chain reaction (PCR).
What Was the Provisional Diagnosis?
A provisional diagnosis of bacterially infected eczema was made, and, in view of his history of penicillin allergy, the patient was started on intravenous vancomycin. He was admitted and referred for dermatology, infectious diseases, and ophthalmology reviews.
On closer inspection of the rash, it was apparent that there were multiple small vesicles 1–2 mm in diameter and shallow ulcers over the face and periorbital region (Figure 1). There was extensive exudate on the skin of the eyelids of both eyes. It was not possible to open the right eye, but in the left eye, the cornea was clear and there was no evidence of disease.
How Did This Information Affect the Likely Diagnosis?
In view of the characteristic picture and underlying eczema, the diagnosis was revised to one of severe eczema herpeticum. The patient denied any previous history of oral cold sores or genital ulceration. However, the severe degree of skin inflammation, high fever, raised C-reactive protein, and left-shifted neutrophils suggested co-existent bacterial sepsis.
What Was the Management Approach?
High-dose intravenous aciclovir (10 mg/kg every 8 h) was started. Aciclovir inhibits thymidine kinase, which is an essential enzyme for several herpes viruses. Aciclovir has excellent activity against herpes simplex virus (HSV), and, although less potent, it is also effective against varicella zoster virus [1]. It has only weak activity against cytomegalovirus and Epstein-Barr virus [1].
This man had severe skin involvement with a high likelihood of complicating bacterial sepsis, which is most commonly due to Staphylococcus aureus [2]. Erythromycin had originally been prescribed, but increasing erythromycin resistance makes this a poor choice in many parts of the world. In view of his penicillin allergy, vancomycin was continued and ciprofloxacin added to broaden the antibacterial spectrum. Vancomycin disrupts the peptidoglycan bonding of the gram-positive bacterial cell wall and has no activity against gram-negative bacteria. Ciprofloxacin is a DNA gyrase inhibitor with excellent activity against many gram-negative bacteria, but it is less effective for gram-positive organisms. Aciclovir and chlorampenicol were administered topically to both eyes, and this treatment was successful in preventing corneal involvement.
Two separate sets of blood cultures subsequently grew S. aureus, which was resistant to penicillin, erythromycin, and clindamycin but sensitive to all other agents. Skin and eye swabs also grew S. aureus. HSV DNA was identified by PCR on fluid taken from the face. Viral cultures were positive for HSV-1. Serology taken on admission was positive for HSV-1 IgG.
Progress
The patient rapidly defervesced, and the vesicles desquamated on day 6 (Figure 2). He was discharged after a total hospital stay of 16 d. He returned to the clinic 2 wk later, at which point the skin was healing well (Figure 3).
Figure 2 Appearance on Day 5
Most of the exudate has cleared, and all vesicles have desquamated. The eyes are now open, and the skin is beginning to heal.
Figure 3 Appearance on Day 28
All ulcers have resolved, and healing is almost complete.
What Complications Should Be Looked Out for?
In the acute stage of his presentation, our two main concerns for this patient were the extent of involvement of the eyes beneath the eyelids and the positive blood cultures for S. aureus. As mentioned above, the eyes were proactively managed to avoid corneal involvement. The S. aureus bacteraemia could have progressed to severe sepsis or led to metastatic spread of infection. Echocardiogram was normal with no evidence of endocarditis, and intravenous vancomycin was continued for a period of 14 d to minimize the risk of metastatic spread. Close attention to infection control procedures is essential to prevent hospital-acquired infection of the damaged skin or intravenous line sites.
DISCUSSION
Eczema Herpeticum
Kaposi's varicelliform eruption describes the widespread dermal infection caused predominantly by HSV-1 in patients with a variety of skin conditions [3]. It is most commonly seen in patients with pre-existing atopic eczema, in which case it is known as eczema herpeticum. Disease severity varies from mild, transient disease to a fulminating fatal disorder involving the visceral organs. Death is very rare in immunocompetent adults; fulminant disease is more likely in severely immunocompromised individuals. The incidence of eczema herpeticum is increasing, especially in the context of sexually transmitted disease [4]. Most cases are caused by reactivation of HSV rather than primary infection [3]. Our patient had no clinical history of infection with herpes, but HSV-1 was confirmed by both PCR and culture. The positive HSV-1 IgG titre suggests that this was reactivation of latent infection.
Prompt use of antiviral therapy is highly effective in controlling HSV replication. Aciclovir is the antiviral agent of choice and is usually given for 7 d. Patients with moderate to severe disease require admission for intravenous aciclovir. Oral aciclovir is effective for mild cases, which can be managed on an outpatient basis [5]. Bacterial superinfection is common in severe eczema herpeticum [2], and antibiotic therapy is required in severe cases. Valaciclovir (a pro-drug of aciclovir) and famciclovir are available in oral preparations and can be considered as alternative antivirals where oral therapy is an option [1]. In addition to the treatment of systemic infection, skin and eye lesions require very careful management, and specialised dermatology [6] and ophthalmology input are essential [7].
A proportion of patients may experience recurrent attacks of eczema herpeticum [4]. Recurrences are generally diagnosed and treated rapidly, as both the patients and their physicians have become familiar with the disease. With frequent recurrences, prophylactic aciclovir may be considered, although there are insufficient data to recommend its use routinely. Good control of eczema is also important to reduce the risk of recurrence [3].
Which Patients with Atopic Eczema Are at Risk of Eczema Herpeticum?
A recent retrospective analysis of 100 cases of eczema herpeticum in patients with atopic ezcema highlighted some predisposing factors. The analysis found that patients with eczema herpeticum had developed atopic eczema earlier and had higher total serum IgE levels than control patients with atopic eczema alone [3]. In addition, 75% of the patients with eczema herpeticum had not received corticosteroid treatment in the 4 wk preceding onset of eczema herpeticum. The authors of the analysis concluded that most cases of eczema herpeticum occurred in patients with untreated atopic eczema. Thus, effective control of eczema is important to prevent eczema herpeticum. Such control is particularly important in our patient to prevent further attacks of eczema herpeticum.
Making the Correct Diagnosis
The rapidly progressive, widespread, crusted papules, vesicles, and erosions in a patient with atopic eczema are characteristic, and an experienced physician who has seen eczema herpeticum before will make a rapid clinical diagnosis. However, some patients present with atypical or purely crusted lesions and no obvious vesicles, making a clinical diagnosis more difficult. Thus, a high index of clinical suspicion is required, and both viral and bacterial cultures are essential when a patient with atopic eczema presents with a spreading vesicular or crusting rash [4]. Rapid confirmation of cutaneous HSV can be achieved with immunofluorescent techniques or PCR, but these are not yet available in many centres [8].
Smallpox Vaccination
A similar and potentially fatal disease may follow smallpox vaccination [9,10]. Until recently, this was only of academic interest, given the global eradication of smallpox. Recent events have raised the spectre of smallpox returning, however, and some countries have begun stockpiling vaccine and inoculating key health-care workers. Smallpox vaccination involves the intradermal inoculation of live attenuated variola virus by scarification, and the site may shed live virus for 2–3 wk until the lesion heals. In a person with eczema, the variola virus can cause widespread eczema vaccinatum. As the variola virus is shed into the environment from the vaccine site, it can also spread to close contacts with eczema, which can lead to secondary cases of eczema vaccinatum. This risk is greatly diminished with the use of an appropriate occlusive dressing to the vaccination site. It is reassuring that, so far, no cases of eczema vaccinatum have been reported in the United States following the recent smallpox vaccination programme, but this disease may well return if there is need for more extensive smallpox vaccination.
Key Learning Points
Eczema herpeticum is an uncommon but potentially fatal disorder that can present to primary care practitioners or to the emergency department.
Diagnostic delay is common and may increase morbidity.
First-line therapy is with acyclovir; intravenous high-dose therapy is required in severe cases.
Patients should be referred to a dermatologist for specialist care and follow-up to optimise eczema management, so that recurrence is less likely.
Intensive nursing care is indicated to minimise scarring and promote rapid healing.
If the periorbital region is involved, refer urgently to an ophthalmologist.
Citation: Fleming J, Lynn WA (2004) A 33-year-old man with a facial rash. PLoS Med1(2): e17.
Abbreviations
HSVherpes simplex virus
PCRpolymerase chain reaction
==== Refs
References
De Clercq E Antiviral drugs in current clinical use J Clin Virol 2004 30 115 133 15125867
Brook I Secondary bacterial infections complicating skin lesions J Med Microbiol 2002 51 808 812 12435058
Wollenberg A Zoch C Wetzel S Plewig G Przybilla B Predisposing factors and clinical features of eczema herpeticum: A retrospective analysis of 100 cases J Am Acad Dermatol 2003 49 198 205 12894065
Wollenberg A Wetzel S Burgdorf WH Haas J Viral infections in atopic dermatitis: Pathogenic aspects and clinical management J Allergy Clin Immunol 2003 112 667 674 14564342
Niimura M Nishikawa T Treatment of eczema herpeticum with oral acyclovir Am J Med 1988 85 49 52
Mackley CL Adams DR Anderson B Miller JJ Eczema herpeticum: A dermatologic emergency Dermatol Nurs 2002 14 307 310, 313, 323 12430518
Margolis TP Ostler HB Treatment of ocular disease in eczema herpeticum Am J Ophthalmol 1990 110 274 279 2118723
Aldea C Alvarez CP Folgueira L Delgado R Otero JR Rapid detection of herpes simplex virus DNA in genital ulcers by real-time PCR using SYBR green I dye as the detection signal J Clin Microbiol 2002 40 1060 1062 11880439
Fulginiti VA Papier A Lane JM Neff JM Henderson DA Smallpox vaccination: A review, part II. Adverse events Clin Infect Dis 2003 37 251 271 12856218
Poland GA Neff JM Smallpox vaccine: Problems and prospects Immunol Allergy Clin North Am 2003 23 731 743 14753389
| 15578102 | PMC529420 | CC BY | 2021-01-05 10:38:08 | no | PLoS Med. 2004 Nov 30; 1(2):e17 | utf-8 | PLoS Med | 2,004 | 10.1371/journal.pmed.0010017 | oa_comm |
==== Front
PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1557810310.1371/journal.pmed.0010021Research in TranslationOtherDiabetes/Endocrinology/MetabolismHematologyPediatricsHematology (including Blood Transfusion)PediatricsClinical trialsEnzyme Replacement in Gaucher Disease Research in TranslationBeutler Ernest Ernest Beutler is chairman of the Department of Molecular and Experimental Medicine at the Scripps Research Institute, La Jolla, California, United States of America. E-mail: [email protected]
Competing Interests: The author declares that he has no competing interests.
11 2004 30 11 2004 1 2 e21Copyright: © 2004 Ernest Beutler.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The development of enzyme replacement therapy for Gaucher disease was a triumph of translational medicine. What were the key steps in its development? What are the controversies surrounding its use?
==== Body
Gaucher disease is the most common lysosomal storage disorder (Box 1). A deficiency of the enzyme glucocerebrosidase (Figure 1) causes accumulation of the glycolipid glucocerebroside in macrophages throughout the body. In the viscera, glucocerebroside arises mainly from the biodegradation of red and white blood cells. In the brain, glucocerebroside arises from the turnover of complex lipids during brain development and the formation of the myelin sheath of nerves. The disease may be discovered as an incidental finding in the elderly because of mild thrombocytopenia or splenomegaly, or it may present early in life with hepatosplenomegaly, thrombocytopenia, anemia, and bone lesions.
Box 1. What Is Gaucher Disease?
Gaucher disease is an inherited metabolic disorder in which harmful quantities of a fatty substance called glucocerebroside accumulate in the spleen, liver, lungs, bone marrow, and, in rare cases, the brain. There are three common forms.
Type 1 is the most common. Clinical features include easy bruising, anemia, low blood platelets, enlargement of the liver and spleen, bone disease, and, in some instances, lung impairment. There are no signs of brain involvement. Problems may begin early in life, be delayed until adulthood, or not occur at all.
In type 2, liver and spleen enlargement are apparent by three months of age, and there is extensive and progressive brain damage. These patients usually die by two years of age.
In type 3, liver and spleen enlargement is variable, and signs of brain involvement, such as seizures, become apparent gradually.
Figure 1 Glucocerebrosidase Cleaves a Linkage within Glucosylceramide, a Normal Intermediate in Glycolipid Metabolism
Until 1990, treatment consisted only of palliative measures such as splenectomy and hip replacement. The development of enzyme replacement therapy for Gaucher disease, that is, exogenous administration of the missing enzyme, is a triumph of translational medicine. At the same time, powerful commercial interests may have been influential in physicians adopting a high-dose rather than a low-dose treatment schedule. Moreover, the high cost of enzyme replacement therapy forces us to consider what society can afford in the way of palliative treatments for very rare diseases.
The History of Enzyme Replacement Therapy
The possibility that the therapeutic replacement of enzymes missing from lysosomes could be achieved was first raised by de Duve forty years ago when he wrote: “Any substance that is taken up intracellularly by an endocytic process is likely to end up within lysosomes. This obviously opens up many possibilities for interaction, including replacement therapy” [1].
Type 1 Gaucher disease, the most common type, seems a particularly suitable target for enzyme replacement therapy because of the lack of central nervous system involvement (visceral damage in Gaucher disease is reversible whereas the brain damage usually is not). By the 1970s, the underlying enzyme deficiency had been identified, and methods had been developed to purify the enzyme from human placenta in a high state of purity. Three groups of investigators then attempted to treat the disease by infusing exogenous enzyme.
In the United States, at the National Institutes of Health in Bethesda, Maryland, the unaltered enzyme was infused directly into the venous circulation [2]; at City of Hope in Duarte, California, it was entrapped in red cell membranes coated with antibody in an effort to direct it to macrophages [3]. In Harrow, United Kingdom, the enzyme was delivered entrapped in liposomes [4]. Although some mildly encouraging results were achieved, it was clear that none of these approaches was likely to be translated into a useful treatment.
The needed conceptual breakthrough was provided by the identification of a mannose receptor on macrophages and the suggestion that this might prove useful in replacement therapy for Gaucher disease [5]. This led to the development of a modified enzyme, processed to expose mannose, and to its production on an industrial scale from placentas. After the gene encoding the enzyme was cloned [6], a recombinant product became available.
The Pivotal Study
The first study of commercially produced mannose-enriched glucocerebrosidase was carried out in Bethesda, Maryland, on only 12 patients, presumably because of a limited supply of the enzyme [7]. Given this small cohort of patients, only a single dose (60 units/kg) was administered. This dose was given every two weeks to ten of the patients, while two patients received it weekly. This is manifestly an unusual dose schedule for a preparation with a circulating half-life of only about 12 min that is being targeted to a relatively small number of receptors. Many of the patients studied did not live near Bethesda, and it is likely that the dose schedule that was chosen was based on convenience rather than on sound pharmacokinetic principles. Since it was unlikely that a second study would be launched if the first failed, the investigators wisely used a very generous dose of enzyme to maximize the probability that the trial would be successful. Intravenous administration of the enzyme produced objective clinical improvement (such as reduced liver and spleen size and increased hemoglobin levels and platelet counts).
The enzyme was promptly approved and marketed. Since only a single dose had been tested, this was the dose that most physicians administered in clinical practice. But the preparation was extremely costly—about US$4.00 per unit. At the dose used in the pivotal trial, a 70-kg patient would receive enzyme costing US$16,800 every two weeks.
Dosage Considerations
Visceral organ responses.
But was the large dose given actually the dose required? There were no data, and many physicians were unwilling to give less than the dose that had been used in the pivotal trial. Moreover, since most physicians took care of only one or at most two patients with the disease, they were not in a position to perform a dose-ranging study. And industry had no interest in supporting studies to show that a lower dose yielded equivalent results.
But clinical trials carried out in our National Institutes of Health–sponsored General Clinical Research Center quickly established that a quarter of the dose given at more frequent intervals was fully effective [8]. By 2000, a considerable body of data had accumulated, making it possible to perform meta-analyses of the relationship between the total monthly dose, the interval at which the dose is administered, and the decrease in the size of the liver. The results were clear (Figure 2) [9]. Even a dose of only 15 units/kg/mo, one-ninth of the dose given in the pivotal trial, resulted in an excellent clinical response. Most patients were receiving a substantial overdose of an extremely costly preparation. The data indicate that when very large doses are administered, the two-week time interval is adequate to give an optimal response, but when more modest doses are administered, more frequent infusions greatly improve the response [9].
Figure 2 A Meta-Analysis of the Decrease in Liver Size in Patients with Gaucher Disease Documented for Various Doses of Enzyme Given As Replacement Therapy
The individual studies included in the meta-analysis are listed in [9].
Recent “consensus recommendations,” which were supported in part by the Genzyme Corporation, the manufacturers of recombinant human glucocerebrosidase (imiglucerase, brand name Cerezyme), suggest that children be given an initial dose of 30 to 60 units every two weeks [10]. But there is no high-quality evidence that such a costly treatment regimen provides results superior to those achieved with smaller doses. The only support for recommending this high dosage comes from uncontrolled studies showing that in some children bone lesions may progress at low dosages. However, we know from our own published observations that skeletal progression and even fractures also occur in some individuals receiving high-dose therapy [11]. Thus, I would caution against any recommendations to give high-dose therapy that have not been based on well-designed randomized, controlled trials. Having said this, I recognize that most, but not all, of the patients that were included in our meta-analyses were adults, whereas the company-sponsored consensus recommendations refer to children. However, in the absence of any evidence-based rationale for administering large, costly doses of enzyme, I believe that the use of smaller, more frequent doses is the most prudent treatment approach.
It is often assumed that patients with severe disease require larger doses of enzyme than those with mild disease, but a meta-analysis based on liver size or spleen size made it clear that this is not the case (Figure 3) [12]. Large organs shrink more rapidly than smaller ones, and this is true regardless of the dose that is used [13].
Figure 3 A Meta-Analysis of the Decrease in Liver and Spleen Size in Patients with Gaucher Disease As a Function of the Initial Organ Size
BW, body weight. Redrawn from [11].
Skeletal response.
The response of enlarged viscera to enzyme infusion is much more rapid than the response of bones. In one early study, the large dose used in the pivotal trial was given for up to four years to patients with bone disease, and although the response was slow, gradual improvement occurred [14]. Strangely, the authors concluded that large doses were required—“strangely,” because they did not give smaller doses to any patient. Subsequently, it was shown that less than a quarter of the dose (only 30 units/kg/mo) produced an equivalent response [15].
Whom to Treat
The severity of Gaucher disease is very variable. We have estimated that some 60% of patients homozygous for the common c.1226 C → G (N370S) mutation never come to medical attention [16]. Accordingly, many—possibly most—patients with Gaucher disease require no treatment. In adults, the disease is rarely progressive [11,17]. What you see is what you have, more or less. Bone fractures, of course, are not gradual events but sudden ones. But almost invariably they occur in patients who already have very substantial, demonstrable bone disease. In children, the situation is different, and progression is common. It is only with proper awareness of the natural history of the disease that one can make rational judgments regarding who needs treatment.
Individualized Treatment
Evaluating dose–response relationships in patients with Gaucher disease has been difficult for several reasons. The number of new patients requiring therapy is relatively small, and the Genzyme Corporation has done little to encourage the performance of dose–response studies, making it difficult to enroll patients. But beyond that, the response of patients to any dose is variable. Some authors have suggested that this may be due to individual differences in dose requirements—that some patients are relatively resistant and require a large dose, while others do well on a small dose [18]. This is an attractive concept, but is it correct? Another meta-analysis indicates that it is not. Rather, there are patients who respond poorly to any dose and others who respond well to any dose [19]. Moreover, quadrupling the dose does not increase the rate of response [11].
What Does the Future Hold?
The quality of life for patients with Gaucher disease has been greatly improved by the development of enzyme replacement therapy. Manufacturing and selling the enzyme has also been enormously profitable for industry. This profitability has served as a stimulus for the development of enzyme replacement treatments for diseases less common and generally less responsive to treatment than Gaucher disease. Given the small target population, these treatments are enormously costly on a per-patient basis. Treatments for Fabry disease and Hurler-Scheie disease (also called mucopolysaccharidosis I) are already licensed, and others are on the way [20,21,22]. This brings us face-to-face with a major ethical dilemma. We do not put a price on human life. Yet health-care resources are a zero-sum game. What is spent on one disease cannot be spent on another. Is it better to treat one child with Hurler-Scheie disease [22] or to provide good prenatal care to 100 women who might not otherwise obtain it, or for that matter, to feed 1,000 malnourished children? These are difficult decisions that will be forced on us as enzyme replacement and other high-technology therapies come of age.
Citation: Beutler E (2004) Enzyme replacement in Gaucher disease. PLoS Med 1(2): e21.
==== Refs
References
de Duve C From cytases to lysosomes Fed Proc 1964 23 1045 1049 14209796
Brady RO Pentchev PG Gal AE Hibbert SR Dekaban AS Replacement therapy for inherited enzyme deficiency: Use of purified glucocerebrosidase in Gaucher's disease N Engl J Med 1974 291 989 993 4415565
Beutler E Dale GL Guinto E Kuhl W Enzyme replacement therapy in Gaucher's disease: Preliminary clinical trial of a new enzyme preparation Proc Natl Acad Sci U S A 1977 74 4620 4623 200923
Belchetz PE Crawley JC Braidman IP Gregoriadis G Treatment of Gaucher's disease with liposome-entrapped glucocerebroside: Beta-glucosidase Lancet 1977 2 116 117 69198
Achord DT Brot FE Bell CE Sly WS Human beta-glucuronidase: In vivo clearance and in vitro uptake by a glycoprotein recognition system on reticuloendothelial cells Cell 1978 15 269 278 699046
Sorge J West C Westwood B Beutler E Molecular cloning and nucleotide sequence of the human glucocerebrosidase gene Proc Natl Acad Sci U S A 1985 82 7289 7293 3864160
Barton NW Brady RO Dambrosia JM Di Bisceglie AM Doppelt SH Replacement therapy for inherited enzyme deficiency—Macrophage-targeted glucocerebrosidase for Gaucher's disease N Engl J Med 1991 324 1464 1470 2023606
Figueroa ML Rosenbloom BE Kay AC Garver P Thurston DW A less costly regimen of alglucerase to treat Gaucher's disease N Engl J Med 1992 327 1632 1636 1435900
Beutler E Commentary: Dosage-response in the treatment of Gaucher disease by enzyme replacement therapy Blood Cells Mol Dis 2000 26 303 306 11042031
Charrow J Andersson HC Kaplan P Kolodny EH Mistry P Enzyme replacement therapy and monitoring for children with type 1 Gaucher disease: Consensus recommendations J Pediatr 2004 144 112 120 14722528
Beutler E Demina A Laubscher K Garver P Gelbart T The clinical course of treated and untreated Gaucher disease. A study of 45 patients Blood Cells Mol Dis 1995 21 86 108 8846048
Beutler E Gaucher disease Adv Genet 1995 32 17 49 7741022
Beutler E Enzyme replacement therapy for Gaucher disease Baillieres Clin Haematol 1997 10 711 723 9497859
Rosenthal DI Doppelt SH Mankin HJ Dambrosia JM Xavier RJ Enzyme replacement therapy for Gaucher disease: Skeletal responses to macrophage-targeted glucocerebrosidase Pediatrics 1995 96 629 637 7567322
Elstein D Hadas-Halpern I Itzchaki M Lahad A Abrahamov A Effect of low-dose enzyme replacement therapy on bones in Gaucher disease patients with severe skeletal involvement Blood Cells Mol Dis 1996 22 104 111 8931951
Beutler E Gaucher disease: New molecular approaches to diagnosis and treatment Science 1992 256 794 799 1589760
Balicki D Beutler E Gaucher disease Medicine 1995 74 305 323 7500895
Hollak CEM Aerts JMFG Goudsmit R Phoa SSKS Ek M Individualised low-dose alglucerase therapy for type 1 Gaucher's disease Lancet 1995 345 1474 1478 7769902
Beutler E Treatment regimens in Gaucher's disease Lancet 1995 346 581 582
Fan JQ A contradictory treatment for lysosomal storage disorders: Inhibitors enhance mutant enzyme activity Trends Pharmacol Sci 2003 24 355 360 12871668
Wilcox WR Banikazemi M Guffon N Waldek S Lee P Long-term safety and efficacy of enzyme replacement therapy for Fabry disease Am J Hum Genet 2004 75 65 74 15154115
Kakkis ED Muenzer J Tiller GE Waber L Belmont J Enzyme-replacement therapy in mucopolysaccharidosis I N Engl J Med 2001 344 182 188 11172140
| 15578103 | PMC529421 | CC BY | 2021-01-05 10:38:00 | no | PLoS Med. 2004 Nov 30; 1(2):e21 | utf-8 | PLoS Med | 2,004 | 10.1371/journal.pmed.0010021 | oa_comm |
==== Front
PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1557810310.1371/journal.pmed.0010021Research in TranslationOtherDiabetes/Endocrinology/MetabolismHematologyPediatricsHematology (including Blood Transfusion)PediatricsClinical trialsEnzyme Replacement in Gaucher Disease Research in TranslationBeutler Ernest Ernest Beutler is chairman of the Department of Molecular and Experimental Medicine at the Scripps Research Institute, La Jolla, California, United States of America. E-mail: [email protected]
Competing Interests: The author declares that he has no competing interests.
11 2004 30 11 2004 1 2 e21Copyright: © 2004 Ernest Beutler.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The development of enzyme replacement therapy for Gaucher disease was a triumph of translational medicine. What were the key steps in its development? What are the controversies surrounding its use?
==== Body
Gaucher disease is the most common lysosomal storage disorder (Box 1). A deficiency of the enzyme glucocerebrosidase (Figure 1) causes accumulation of the glycolipid glucocerebroside in macrophages throughout the body. In the viscera, glucocerebroside arises mainly from the biodegradation of red and white blood cells. In the brain, glucocerebroside arises from the turnover of complex lipids during brain development and the formation of the myelin sheath of nerves. The disease may be discovered as an incidental finding in the elderly because of mild thrombocytopenia or splenomegaly, or it may present early in life with hepatosplenomegaly, thrombocytopenia, anemia, and bone lesions.
Box 1. What Is Gaucher Disease?
Gaucher disease is an inherited metabolic disorder in which harmful quantities of a fatty substance called glucocerebroside accumulate in the spleen, liver, lungs, bone marrow, and, in rare cases, the brain. There are three common forms.
Type 1 is the most common. Clinical features include easy bruising, anemia, low blood platelets, enlargement of the liver and spleen, bone disease, and, in some instances, lung impairment. There are no signs of brain involvement. Problems may begin early in life, be delayed until adulthood, or not occur at all.
In type 2, liver and spleen enlargement are apparent by three months of age, and there is extensive and progressive brain damage. These patients usually die by two years of age.
In type 3, liver and spleen enlargement is variable, and signs of brain involvement, such as seizures, become apparent gradually.
Figure 1 Glucocerebrosidase Cleaves a Linkage within Glucosylceramide, a Normal Intermediate in Glycolipid Metabolism
Until 1990, treatment consisted only of palliative measures such as splenectomy and hip replacement. The development of enzyme replacement therapy for Gaucher disease, that is, exogenous administration of the missing enzyme, is a triumph of translational medicine. At the same time, powerful commercial interests may have been influential in physicians adopting a high-dose rather than a low-dose treatment schedule. Moreover, the high cost of enzyme replacement therapy forces us to consider what society can afford in the way of palliative treatments for very rare diseases.
The History of Enzyme Replacement Therapy
The possibility that the therapeutic replacement of enzymes missing from lysosomes could be achieved was first raised by de Duve forty years ago when he wrote: “Any substance that is taken up intracellularly by an endocytic process is likely to end up within lysosomes. This obviously opens up many possibilities for interaction, including replacement therapy” [1].
Type 1 Gaucher disease, the most common type, seems a particularly suitable target for enzyme replacement therapy because of the lack of central nervous system involvement (visceral damage in Gaucher disease is reversible whereas the brain damage usually is not). By the 1970s, the underlying enzyme deficiency had been identified, and methods had been developed to purify the enzyme from human placenta in a high state of purity. Three groups of investigators then attempted to treat the disease by infusing exogenous enzyme.
In the United States, at the National Institutes of Health in Bethesda, Maryland, the unaltered enzyme was infused directly into the venous circulation [2]; at City of Hope in Duarte, California, it was entrapped in red cell membranes coated with antibody in an effort to direct it to macrophages [3]. In Harrow, United Kingdom, the enzyme was delivered entrapped in liposomes [4]. Although some mildly encouraging results were achieved, it was clear that none of these approaches was likely to be translated into a useful treatment.
The needed conceptual breakthrough was provided by the identification of a mannose receptor on macrophages and the suggestion that this might prove useful in replacement therapy for Gaucher disease [5]. This led to the development of a modified enzyme, processed to expose mannose, and to its production on an industrial scale from placentas. After the gene encoding the enzyme was cloned [6], a recombinant product became available.
The Pivotal Study
The first study of commercially produced mannose-enriched glucocerebrosidase was carried out in Bethesda, Maryland, on only 12 patients, presumably because of a limited supply of the enzyme [7]. Given this small cohort of patients, only a single dose (60 units/kg) was administered. This dose was given every two weeks to ten of the patients, while two patients received it weekly. This is manifestly an unusual dose schedule for a preparation with a circulating half-life of only about 12 min that is being targeted to a relatively small number of receptors. Many of the patients studied did not live near Bethesda, and it is likely that the dose schedule that was chosen was based on convenience rather than on sound pharmacokinetic principles. Since it was unlikely that a second study would be launched if the first failed, the investigators wisely used a very generous dose of enzyme to maximize the probability that the trial would be successful. Intravenous administration of the enzyme produced objective clinical improvement (such as reduced liver and spleen size and increased hemoglobin levels and platelet counts).
The enzyme was promptly approved and marketed. Since only a single dose had been tested, this was the dose that most physicians administered in clinical practice. But the preparation was extremely costly—about US$4.00 per unit. At the dose used in the pivotal trial, a 70-kg patient would receive enzyme costing US$16,800 every two weeks.
Dosage Considerations
Visceral organ responses.
But was the large dose given actually the dose required? There were no data, and many physicians were unwilling to give less than the dose that had been used in the pivotal trial. Moreover, since most physicians took care of only one or at most two patients with the disease, they were not in a position to perform a dose-ranging study. And industry had no interest in supporting studies to show that a lower dose yielded equivalent results.
But clinical trials carried out in our National Institutes of Health–sponsored General Clinical Research Center quickly established that a quarter of the dose given at more frequent intervals was fully effective [8]. By 2000, a considerable body of data had accumulated, making it possible to perform meta-analyses of the relationship between the total monthly dose, the interval at which the dose is administered, and the decrease in the size of the liver. The results were clear (Figure 2) [9]. Even a dose of only 15 units/kg/mo, one-ninth of the dose given in the pivotal trial, resulted in an excellent clinical response. Most patients were receiving a substantial overdose of an extremely costly preparation. The data indicate that when very large doses are administered, the two-week time interval is adequate to give an optimal response, but when more modest doses are administered, more frequent infusions greatly improve the response [9].
Figure 2 A Meta-Analysis of the Decrease in Liver Size in Patients with Gaucher Disease Documented for Various Doses of Enzyme Given As Replacement Therapy
The individual studies included in the meta-analysis are listed in [9].
Recent “consensus recommendations,” which were supported in part by the Genzyme Corporation, the manufacturers of recombinant human glucocerebrosidase (imiglucerase, brand name Cerezyme), suggest that children be given an initial dose of 30 to 60 units every two weeks [10]. But there is no high-quality evidence that such a costly treatment regimen provides results superior to those achieved with smaller doses. The only support for recommending this high dosage comes from uncontrolled studies showing that in some children bone lesions may progress at low dosages. However, we know from our own published observations that skeletal progression and even fractures also occur in some individuals receiving high-dose therapy [11]. Thus, I would caution against any recommendations to give high-dose therapy that have not been based on well-designed randomized, controlled trials. Having said this, I recognize that most, but not all, of the patients that were included in our meta-analyses were adults, whereas the company-sponsored consensus recommendations refer to children. However, in the absence of any evidence-based rationale for administering large, costly doses of enzyme, I believe that the use of smaller, more frequent doses is the most prudent treatment approach.
It is often assumed that patients with severe disease require larger doses of enzyme than those with mild disease, but a meta-analysis based on liver size or spleen size made it clear that this is not the case (Figure 3) [12]. Large organs shrink more rapidly than smaller ones, and this is true regardless of the dose that is used [13].
Figure 3 A Meta-Analysis of the Decrease in Liver and Spleen Size in Patients with Gaucher Disease As a Function of the Initial Organ Size
BW, body weight. Redrawn from [11].
Skeletal response.
The response of enlarged viscera to enzyme infusion is much more rapid than the response of bones. In one early study, the large dose used in the pivotal trial was given for up to four years to patients with bone disease, and although the response was slow, gradual improvement occurred [14]. Strangely, the authors concluded that large doses were required—“strangely,” because they did not give smaller doses to any patient. Subsequently, it was shown that less than a quarter of the dose (only 30 units/kg/mo) produced an equivalent response [15].
Whom to Treat
The severity of Gaucher disease is very variable. We have estimated that some 60% of patients homozygous for the common c.1226 C → G (N370S) mutation never come to medical attention [16]. Accordingly, many—possibly most—patients with Gaucher disease require no treatment. In adults, the disease is rarely progressive [11,17]. What you see is what you have, more or less. Bone fractures, of course, are not gradual events but sudden ones. But almost invariably they occur in patients who already have very substantial, demonstrable bone disease. In children, the situation is different, and progression is common. It is only with proper awareness of the natural history of the disease that one can make rational judgments regarding who needs treatment.
Individualized Treatment
Evaluating dose–response relationships in patients with Gaucher disease has been difficult for several reasons. The number of new patients requiring therapy is relatively small, and the Genzyme Corporation has done little to encourage the performance of dose–response studies, making it difficult to enroll patients. But beyond that, the response of patients to any dose is variable. Some authors have suggested that this may be due to individual differences in dose requirements—that some patients are relatively resistant and require a large dose, while others do well on a small dose [18]. This is an attractive concept, but is it correct? Another meta-analysis indicates that it is not. Rather, there are patients who respond poorly to any dose and others who respond well to any dose [19]. Moreover, quadrupling the dose does not increase the rate of response [11].
What Does the Future Hold?
The quality of life for patients with Gaucher disease has been greatly improved by the development of enzyme replacement therapy. Manufacturing and selling the enzyme has also been enormously profitable for industry. This profitability has served as a stimulus for the development of enzyme replacement treatments for diseases less common and generally less responsive to treatment than Gaucher disease. Given the small target population, these treatments are enormously costly on a per-patient basis. Treatments for Fabry disease and Hurler-Scheie disease (also called mucopolysaccharidosis I) are already licensed, and others are on the way [20,21,22]. This brings us face-to-face with a major ethical dilemma. We do not put a price on human life. Yet health-care resources are a zero-sum game. What is spent on one disease cannot be spent on another. Is it better to treat one child with Hurler-Scheie disease [22] or to provide good prenatal care to 100 women who might not otherwise obtain it, or for that matter, to feed 1,000 malnourished children? These are difficult decisions that will be forced on us as enzyme replacement and other high-technology therapies come of age.
Citation: Beutler E (2004) Enzyme replacement in Gaucher disease. PLoS Med 1(2): e21.
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Beutler E Dale GL Guinto E Kuhl W Enzyme replacement therapy in Gaucher's disease: Preliminary clinical trial of a new enzyme preparation Proc Natl Acad Sci U S A 1977 74 4620 4623 200923
Belchetz PE Crawley JC Braidman IP Gregoriadis G Treatment of Gaucher's disease with liposome-entrapped glucocerebroside: Beta-glucosidase Lancet 1977 2 116 117 69198
Achord DT Brot FE Bell CE Sly WS Human beta-glucuronidase: In vivo clearance and in vitro uptake by a glycoprotein recognition system on reticuloendothelial cells Cell 1978 15 269 278 699046
Sorge J West C Westwood B Beutler E Molecular cloning and nucleotide sequence of the human glucocerebrosidase gene Proc Natl Acad Sci U S A 1985 82 7289 7293 3864160
Barton NW Brady RO Dambrosia JM Di Bisceglie AM Doppelt SH Replacement therapy for inherited enzyme deficiency—Macrophage-targeted glucocerebrosidase for Gaucher's disease N Engl J Med 1991 324 1464 1470 2023606
Figueroa ML Rosenbloom BE Kay AC Garver P Thurston DW A less costly regimen of alglucerase to treat Gaucher's disease N Engl J Med 1992 327 1632 1636 1435900
Beutler E Commentary: Dosage-response in the treatment of Gaucher disease by enzyme replacement therapy Blood Cells Mol Dis 2000 26 303 306 11042031
Charrow J Andersson HC Kaplan P Kolodny EH Mistry P Enzyme replacement therapy and monitoring for children with type 1 Gaucher disease: Consensus recommendations J Pediatr 2004 144 112 120 14722528
Beutler E Demina A Laubscher K Garver P Gelbart T The clinical course of treated and untreated Gaucher disease. A study of 45 patients Blood Cells Mol Dis 1995 21 86 108 8846048
Beutler E Gaucher disease Adv Genet 1995 32 17 49 7741022
Beutler E Enzyme replacement therapy for Gaucher disease Baillieres Clin Haematol 1997 10 711 723 9497859
Rosenthal DI Doppelt SH Mankin HJ Dambrosia JM Xavier RJ Enzyme replacement therapy for Gaucher disease: Skeletal responses to macrophage-targeted glucocerebrosidase Pediatrics 1995 96 629 637 7567322
Elstein D Hadas-Halpern I Itzchaki M Lahad A Abrahamov A Effect of low-dose enzyme replacement therapy on bones in Gaucher disease patients with severe skeletal involvement Blood Cells Mol Dis 1996 22 104 111 8931951
Beutler E Gaucher disease: New molecular approaches to diagnosis and treatment Science 1992 256 794 799 1589760
Balicki D Beutler E Gaucher disease Medicine 1995 74 305 323 7500895
Hollak CEM Aerts JMFG Goudsmit R Phoa SSKS Ek M Individualised low-dose alglucerase therapy for type 1 Gaucher's disease Lancet 1995 345 1474 1478 7769902
Beutler E Treatment regimens in Gaucher's disease Lancet 1995 346 581 582
Fan JQ A contradictory treatment for lysosomal storage disorders: Inhibitors enhance mutant enzyme activity Trends Pharmacol Sci 2003 24 355 360 12871668
Wilcox WR Banikazemi M Guffon N Waldek S Lee P Long-term safety and efficacy of enzyme replacement therapy for Fabry disease Am J Hum Genet 2004 75 65 74 15154115
Kakkis ED Muenzer J Tiller GE Waber L Belmont J Enzyme-replacement therapy in mucopolysaccharidosis I N Engl J Med 2001 344 182 188 11172140
| 15578104 | PMC529422 | CC BY | 2021-01-05 10:38:00 | no | PLoS Med. 2004 Nov 30; 1(2):e26 | latin-1 | PLoS Med | 2,004 | 10.1371/journal.pmed.0010026 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1557810510.1371/journal.pmed.0010028Research ArticleCancer BiologyImmunologyInfectious DiseasesAllergy/ImmunologyOncologyOncologyImmunology and allergyInfectious DiseasesDiversity and Recognition Efficiency of T Cell Responses to Cancer T Cell Responses to CancerStuge Tor B
1
Holmes Susan P
2
Saharan Sahdev
1
Tuettenberg Andrea
3
Roederer Mario
4
Weber Jeffrey S
5
Lee Peter P
1
*1Department of Medicine, Division of Hematology, Stanford UniversityStanford, CaliforniaUnited States of America2Department of Statistics, Stanford UniversityStanford, CaliforniaUnited States of America3Department of Dermatology, J. Gutenberg-UniversityMainzGermany4ImmunoTechnology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of HealthBethesda, MarylandUnited States of America5Norris Cancer Center, University of Southern CaliforniaLos Angeles, CaliforniaUnited States of AmericaMarrack Philippa Academic EditorNational Jewish Medical and Research Center/Howard Hughes Medical InstituteUnited States of America
Competing Interests: The authors have declared that no competing interests exist.
Author Contributions: TBS, SS, MR, and PPL designed the study. TBS, SPH, SS, MR, JSW, and PPL analyzed the data. MR provided key validated reagents for the study. AT and JSW enrolled patients. TBS, SPH, SS, AT, MR, JSW, and PPL contributed to writing the paper.
*To whom correspondence should be addressed. E-mail: [email protected] 2004 30 11 2004 1 2 e2816 8 2004 24 8 2004 Copyright: © 2004 Stuge et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
The Quest for a Vaccine That Yields Tumor-Killing T cells
Background
Melanoma patients vaccinated with tumor-associated antigens frequently develop measurable peptide-specific CD8+ T cell responses; however, such responses often do not confer clinical benefit. Understanding why vaccine-elicited responses are beneficial in some patients but not in others will be important to improve targeted cancer immunotherapies.
Methods and Findings
We analyzed peptide-specific CD8+ T cell responses in detail, by generating and characterizing over 200 cytotoxic T lymphocyte clones derived from T cell responses to heteroclitic peptide vaccination, and compared these responses to endogenous anti-tumor T cell responses elicited naturally (a heteroclitic peptide is a modification of a native peptide sequence involving substitution of an amino acid at an anchor residue to enhance the immunogenicity of the peptide). We found that vaccine-elicited T cells are diverse in T cell receptor variable chain beta expression and exhibit a different recognition profile for heteroclitic versus native peptide. In particular, vaccine-elicited T cells respond to native peptide with predominantly low recognition efficiency—a measure of the sensitivity of a T cell to different cognate peptide concentrations for stimulation—and, as a result, are inefficient in tumor lysis. In contrast, endogenous tumor-associated-antigen-specific T cells show a predominantly high recognition efficiency for native peptide and efficiently lyse tumor targets.
Conclusions
These results suggest that factors that shape the peptide-specific T cell repertoire after vaccination may be different from those that affect the endogenous response. Furthermore, our findings suggest that current heteroclitic peptide vaccination protocols drive expansion of peptide-specific T cells with a diverse range of recognition efficiencies, a significant proportion of which are unable to respond to melanoma cells. Therefore, it is critical that the recognition efficiency of vaccine-elicited T cells be measured, with the goal of advancing those modalities that elicit T cells with the greatest potential of tumor reactivity.
State-of-the art analysis of patients' response to melanoma vaccines yields lessons about cancer vaccines and rationale vaccine design in general
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Introduction
The immunotherapy of cancer holds promise in harnessing the host immune response to specifically target tumor cells without harming normal tissues. Strategies involve adoptive cellular therapy or active immune induction (commonly referred to as “cancer vaccination”). Cancer vaccines may consist of whole tumor cells or tumor lysates, but identification of tumor-associated antigens (TAAs) over the past decade has made possible the use of specific proteins or peptides as cancer vaccines. The anti-tumor potential of TAA-specific CD8+ T cells has been illustrated by the demonstrated capacity of adoptive T cell therapy to reduce tumor size [1]. While endogenous anti-tumor CD8+ T cell responses may already exist in some cancer patients [2], vaccination with TAA-derived peptides, and in particular heteroclitic peptide analogs, increases the frequency of TAA-specific T cell responses to detectable levels in many patients [3,4,5,6,7,8,9]. Heteroclitic peptide analogs are created by substitutions at anchor residues resulting in increased association of peptide with the major histocompatibility complex (MHC) [10]. Consequently, heteroclitic peptide analogs are predicted to be more immunogenic than their native counterparts because of more stable binding at the surface of antigen-presenting cells (APCs). Indeed, T cells capable of tumor lysis have been isolated from patients vaccinated with heteroclitic peptide [8,11,12,13]. However, the presence of TAA-specific T cells elicited by vaccination often does not correlate with clinical responses [3,14,15,16,17].
Various reasons for the paradoxical coexistence of cancer cells and TAA-specific T cells within patients have been proposed [18,19]. One possibility is that elicited TAA-specific T cells are not optimally functional in vivo [2,18]. Another possibility is that T cells inefficient in tumor recognition or lysis are induced by vaccination [20]. It is becoming recognized that antigen-specific T cells may have substantially different requirements for cognate peptide (the peptide that is recognizable to a specific T cell clone) for efficient target lysis [20,21,22,23]. “Recognition efficiency” (RE) (also known as “functional avidity”) is a measure of the sensitivity of a T cell to different peptide concentrations for stimulation [24,25,26]. We hypothesized that high antigen densities on APCs resulting from vaccination with heteroclitic peptide may paradoxically drive T cells of predominantly low RE, which are not efficiently activated by the endogenous expression levels of native peptides on tumor cells. Consequently, such T cells would be ineffective in tumor cell destruction. Support for this notion is emerging: T cells with low RE are predominantly expanded in vitro with high peptide concentration [22]. Moreover, in vitro stimulation of T cells from healthy donors with heteroclitic peptides results in expansion of cells with a wide range of RE [23]. A similar phenomenon may occur in vivo, leading to TAA-specific T cells of low RE depending on the nature of antigen stimulation [20].
While isolated T cell clones with low RE have indeed been generated from melanoma patients following heteroclitic peptide vaccination, the proportion of vaccine-elicited T cell responses these cells represent in vivo is not clear. If predominantly high-RE, tumor-cytolytic T cells are generated, then a small fraction of low-RE T cells generated would be of little consequence. However, if predominantly low-RE T cells are generated, then this low proportion of high-RE T cells may be an important factor in the observed lack of clinical effectiveness of current cancer vaccination strategies. To address this important issue, we undertook a systematic examination of the complexity of T cell responses induced by heteroclitic peptide vaccination, and compared these responses to endogenous anti-tumor T cell responses which develop in some patients. Typically, responses to vaccination are examined following in vitro expansion from patient samples, which may alter the composition of cells and consequently not reveal the proportion of cells in vivo having sufficiently high RE to lyse tumor targets. Although staining with peptide–MHC tetramers provides a direct estimate for the number of TAA-specific T cells present in vivo, and intensity of tetramer staining has been employed as a parameter for isolation of high-RE, tumor-lytic T cells [27], staining intensity does not correlate well with RE or tumor-lytic potential [28,29], and cannot be considered a reliable indicator for the functional status of TAA-specific T cells.
To analyze and compare T cell responses in melanoma patients on a single-cell level, we generated and examined a large number of cytotoxic T lymphocyte (CTL) clones derived from post-vaccination or endogenous anti-tumor T cell responses. Each clone was analyzed for T cell receptor (TCR) variable chain beta (VB) expression, RE, and ability to lyse melanoma targets. Importantly, these clones were generated directly ex vivo through tetramer-guided sorting, which minimizes the selection bias that could be introduced by prior in vitro expansion. Therefore, data from these clones could be taken to estimate the complexity of the responses in vivo.
Methods
Patients and Samples
All patients had resected stage III or IV melanoma, as determined by the 1988 modified American Joint Commission on Cancer staging system. They were required to have a magnetic resonance imaging or computed tomographic scan of the head and computed tomographic scans of the chest, abdomen, and pelvis showing no indication of disease within 4 wk of therapy to verify that they were clinically free of melanoma. Eligibility criteria included age 18 y or older, creatinine of less than 180 μmol/l, bilirubin of less than 110 μmol/l, platelet count of 100 × 109/l or more, hemoglobin of 90 g/l or more, and total white blood cell count of 3.0 × 109/l or greater. Tests for human immunodeficiency virus, hepatitis C antibody (Ab), and hepatitis B surface antigen were required to be negative, and all patients were HLA-A2 antigen positive by a microcytotoxicity assay. All patients were required to comprehend and sign an informed consent form approved by the National Cancer Institute (NCI; Bethesda, Maryland, United States) and the Los Angeles County/University of Southern California Institutional Review Board. Analysis of the patient samples was approved by Stanford University's Institutional Review Board. Peripheral blood mononuclear cell (PBMC) samples were isolated from patients after vaccination with the heteroclitic peptides MART 26–35 (27L) (ELAGIGILTV) and gp100 209–217 (210M) (IMDQVPSFV) at the University of Southern California Norris Cancer Center (Los Angeles, California, United States). Clinical-grade peptides used were provided by the Cancer Therapy Evaluation Program of the NCI under an Investigational New Drug application BB 6123 held by the NCI. Immunizations (1 mg of each peptide emulsified with incomplete Freund's adjuvant) were administered every 2 wk for 8 wk, then every 4 wk for 12 wk, and then once 8 wk later. PBMC samples were collected 4 wk after the final immunization and stored at −130 °C. Samples were thawed the day before an experiment for overnight culture in CTL medium. The following morning, viable cells were isolated by ficoll density centrifugation, washed, and resuspended to the appropriate concentration in a solution of 90% Iscove's Modified Dulbecco's Medium (IMDM) and 10% fetal bovine serum (FBS).
Flow Cytometry Analysis
For isolation and detection of peptide-specific T cells, patient PBMC samples were stained and analyzed by fluorescence-activated cell sorting (FACS) as previously described [2]. Briefly, cells were stained with anti-human CD8− fluorescein isothiocyanate (Caltag Laboratories, Burlingame, California, United States) and CD19-CyChrome (BD Biosciences, Palo Alto, California, United States) Abs, and HLA-A*0201/peptide tetramer–phycoerythrin (PE). The final staining dilution of each Ab was 1/200 and 1/80, respectively. Tetramer–PE was titrated for optimal staining, usually between 1 and 10 μg/ml. For TCR VB typing, cells were divided in seven aliquots and stained with CD8 PerCP-Cy5.5 (BD Biosciences), tetramer–PE, and a panel of two or three different anti-VB monoclonal Abs labeled with fluorescein isothiocyanate, allophycocyanin (APC), or both. Cells were incubated at room temperature for 30 min, washed, then analyzed using a two-laser, four-color FACSCalibur (Becton Dickinson, Franklin Lakes, New Jersey, United States) or sorted using a FACSVantage flow cytometer (Becton Dickinson). Lymphocytes were identified by forward and side scatter signals, then selected for CD8+ and tetramer positive. Up to one million events were acquired and analyzed using FlowJo (TreeStar, San Carlos, California, United States).
CD107 Mobilization Assays
Target cells
The HLA-A*0201-positive melanoma lines Malme-3M and A375 and the T2 cell line were purchased from
ATCC (Manassas, Virginia, United States) and maintained according to instructions provided by the
ATCC. The HLA-A*0201-positive melanoma line mel526 was obtained from the Surgery Branch of the NCI. While Malme-3M and mel526 express both MART and gp100, A375 does not express MART or gp100 and served as a negative control. Expression (or lack thereof) of these antigens by each cell line was further confirmed by immunohistochemical staining. Cells were trypsinized using Trypsin/EDTA solution (GIBCO, San Diego, California, United States) before use. T2 cells were HLA-A2.1+ and were pulsed prior to assays with peptides indicated in the text.
Effector cells
Effector cells, which include clones, cell line, and PBMC samples, were frozen and analyzed in batches. The cells were thawed the day before an experiment for overnight culture in CTL medium. The following morning, viable cells were isolated by ficoll density centrifugation, washed, and resuspended to the appropriate concentration (usually 107/ml) in CTL medium.
Experimental procedure
All assays were done at least twice, with duplicates for each condition. The effector to target (E:T) ratio used was generally 1:2, with 2 × 105 for clones or 106 for the cell line and patient PBMC samples. To each well, the following was added in order: 1 μl of 2 mM monensin (Sigma, St. Louis, Missouri, United States) in 100% EtOH, 100 μl of target cells, 100 μl of effector cells, and 1 μl of CD107-APC Abs. The cells were mixed well using a multichannel pippetor. The plate was centrifuged at 300g for 1 min to pellet cells, then placed into an incubator at 37 °C for 4 h. After the incubation, the plates were centrifuged to 500g to pellet cells, and the supernatant was removed. Cell–cell conjugates were disrupted by washing the cells with PBS supplemented with 0.02% azide and 0.5 mM EDTA, and mixed vigorously, then stained with additional Abs.
Generation of CTL Clones
CD8+ T cell clones were derived by FACSorting individual tetramer-positive cells from PBMC samples prepared for flow cytometry as described above. CD8+ tetramer-positive T cells were sorted under sterile conditions into 96-well plates, one cell per well, using a FACS Vantage (Becton Dickinson). Wells contained 100 μl of CTL IMDM, with 10% FBS, 2% human AB sera, and penicillin, streptomycin, and L-glutamine, supplemented with 100 units/ml IL-2. Sorted cells were expanded in vitro using standard protocols. Briefly, irradiated feeder cells (JY cells and fresh PBMCs) were added to wells containing the sorted T cells, and the 96-well plates were incubated at 37 °C with 7% CO2 to allow for growth. Potential clones became visible around day 14 and were then transferred to 24-well plates containing 1 ml of CTL medium with 100 units/ml IL-2. Wells were selected based on cell confluency for expansion and further analysis. Clones confirmed to be tetramer-positive were expanded in T-25 flasks containing irradiated JY cells and fresh PBMCs in 25 ml of CTL medium containing PHA. IL-2 was added to a final concentration of 50 units/ml on day 1 and then every 2 d thereafter for 2 wk.
Cytotoxic Assays
Target cells
Target cells were as described above under CD107 Mobilization Assays, and were labeled overnight with 51Chromium, washed, and resuspended to 105 cells/ml. One hundred microliters of target cells were incubated with 100 μl CTL clones at 10:1 E:T ratio for 4 h. Percent specific release of 51Chromium from target cells was calculated from 40-μl cell-free supernatants.
Determination of RE
Chromium-labeled T2 targets were pulsed with a range of peptide concentrations, generally starting at 10−7 M and decreasing by log steps to 10−13 M. T cell clones were incubated with T2 targets at 10:1 E:T ratios for 4 h, then chromium release was measured and percentage cytotoxicity calculated by standard methods. Prior to each cytotoxicity assay, clones underwent ficoll-hypaque centrifugation to remove dead feeder cells and were determined to be greater than 80% CD8+ tetramer-positive T cells by FACS. The E:T ratio was based upon live T and target cells. For each T cell clone, percent cytotoxicity was plotted against peptide concentration. The peptide concentration at which the curve crossed 40% cytotoxicity was defined as the RE of that clone [30].
Microcytotoxic assay
Cells were isolated directly from PBMCs from patient 422 by FACS as described above. Cells were collected in microfuge tubes containing 1 ml of ice-cold 90% IMDM with 10% FBS. Collected cells were washed and resuspended to 83,300 cells/ml in 90% IMDM with 10% FBS. Targets were prepared as described above and resuspended to 8,300 cells/ml in 90% IMDM with 10% FBS. A total of 2,500 sorted cells (30 μl) and 250 target cells (30 μl) were transferred to a microcentrifuge tube (VWR International, West Chester, Pennsylvania, United States), centrifuged 1 min at 200g, and incubated 4 h at 37 °C. Percent specific release of 51Chromium was calculated from 40 μl of cell-free supernatant.
TCR VB Spectratyping
RNA was extracted from clones and tetramer-positive cells using TRIzol (Invitrogen, Carlsbad, California, United States) and reverse-transcribed into cDNA using SuperScript II Reverse Transcriptase (Invitrogen). PCR was performed using 34 different 5′ primers that specifically amplify all functional TCR VB genes. Most of the 5′ primers used have been previously described [31]. These primers were used in combination with a common 3′ primer based in the beta chain constant region, BC63 (5′-
GTGTGGCCTTTTGGGTGT-3′). As an internal control, PCR for a section of the beta chain constant region was performed in parallel with VB-specific PCRs using the following primers: UpBC (5′-
CGCTGTGTTTGAGCCATC-3′) and LoBC (5′-
TGCTCAGGCAGTATCTGGA-3′). All primer concentrations were 200 nM. PCR was performed using an iCycler iQ thermic cycler equipped with a real-time detection system (Bio-Rad, Hercules, California, United States) and a QuantiTect SYBR Green PCR kit (Qiagen, Valencia, California, United States). PCR reactions were performed as follows: 94 °C for 9 min, followed by 50 cycles of 94 °C for 30 s, 58 °C for 1 min, and 72 °C for 1 min, followed by 72 °C for 10 min. Specific amplification was determined relative to constant region control PCR. For spectratyping, PCRs were performed as described above with the following VB14- and VB17-specific 5′ primers: VB14m (5′-
ACCCAAGATACCTCATCACAG-3′) and VB17 (5′-
GACAGGACCCAGGGCAAG-3′), followed by a run-off PCR with downstream VB-specific primers: VB14 (5′-
GGGCTTAAGGCAGATCTACT-3′) and VB17m (5′-
TTTCAGAAAGGAGATATAGCT-3′), and FAM6-labeled BC63 3′ primer. Run-off PCR was performed as described above except that only five cycles of PCR were run with the 55 °C annealing temperature and QuantiTect Probe PCR kit (Qiagen). Labeled PCR fragments were run on an ABI Prism 377 DNA Sequencer (Applied Biosystems, Foster City, California, United States) and analyzed using GeneScan software (Applied Biosystems).
Statistical Analysis
A standard software package (SigmaPlot 5.0, Systat Software, Richmond, California, United States) was used to provide descriptive statistical plots. Barcharts were provided with standard errors on them. Linear plots were provided with standard errors computed at each point. A linear regression (using least squares) of percent specific lysis on recognition efficiency is shown in Figure 5A and 5B.
Figure 4 High RE Recognition of Native G209n but Not G209–2M Peptide Correlates with Efficiency in Tumor Cell Lysis
CTL clones 476.105 and 132.1 were assayed for lysis of T2 cells pulsed with 10-fold dilutions of (A) native or (C) heteroclitc peptide at concentrations ranging from 100 fg/ml to 100 ng/ml. (B) Lysis of Malme-3M melanoma cells by 476.105 and 132.1 CTLs. All assays were performed in triplicate, and each clone was assayed twice. Error bars reflect variation between two separate assays.
Figure 5 Endogenous T-Cell Responses Have Higher RE Than Vaccine-Elicited Responses
CTL clones representing different tetramer-positive populations in each patient expressing different VB were assayed for lysis of T2 cells pulsed with various dilutions of G209n, G209–2M, M27, or M26 peptides in 51Chromium release cytotoxicity assays as described in Figure 4 legend. A RE score was attributed to each clone equal to the negative log10 of the peptide concentration that resulted in 40% lysis of peptide-pulsed T2 cells.
(A and B) RE scores for both (A) MART-specific and (B) gp100-specific clones from all patients were correlated with efficiency in lysing melanoma cells. Correlation coefficients were 0.66 for MART-specific clones and 0.81 for gp100-specific clones.
(C–F) Comparison of RE scores for endogenous (patients 461 and 132) and vaccine-induced (patients 517, 520, 422 and 476) responses.
(C and D) RE analysis with native peptides (C) M27 and (D) G209n. Mean RE (weighted) for each response is indicated with horizontal bars. Weighted means were based on all clones, not only those assayed, and were estimated by summing the RE of each analyzed clone multiplied by the number of total clones expressing the same VB, in each patient. Weighted means were as follows: patient 517, 5.7; patient 520, 7.0; patient 461, 7.9; patient 422, 9.7; patient 476, 9.9; and patient 132, 11.2. One-tailed T-tests demonstrated that endogenous responses had significantly higher RE than vaccine-induced responses: patient 461 versus patient 517, p = 1.8 × 10−5; patient 461 versus patient 520, p = 1.1 × 10−3; patient 132 versus patient 422, p = 6 × 10−6; and patient 132 versus patient 476, p = 4.3 × 10−4.
(E and F) RE analysis with heteroclitic peptides (E) M26 and (F) G209–2M. Weighted means were as follows: patient 517, 10.6; patient 520, 11.1; patient 461, 11.2; patient 422, 10.5; patient 476, 11.6; and patient 132, 11.3.
Results
T Cell Responses to TAAs in Patients with Melanoma
To address the complexity of T cell responses against melanoma in vivo, patients with vaccine-induced or endogenous TAA-specific responses were selected. In recent cancer vaccine trials [3,4,5], many melanoma patients who received heteroclitic peptide vaccines gp100 209–217 (210M) (IMDQVPSFV; G209–2M) and MART 26–35 (27L) (ELAGIGILTV; M26) had measurable CD8+ peptide-specific T cell responses in PBMCs detected by peptide–MHC tetramer staining. In addition, TAA-specific T cell responses could be detected in some patients without vaccination, suggesting the existence of an endogenous anti-tumor T cell response in these patients. For the current study, we selected samples from six melanoma patients from these trials—four with vaccine-elicited responses (patients 422, 476, 517, and 520) and two with endogenous T cell responses (patients 132 and 461)—for detailed analyses of TCR VB usage, RE for the target peptide, and tumor cytotoxicity. The samples from these six patient had peptide-specific T cell populations detectable with G209–2M-tetramers (patients 422, 476, and 132) or M26-tetramers (patients 517, 520, and 461) ranging from 0.1% to 2.5% of total CD8+ T cells (Figure 1A).
Figure 1 Melanoma Patient Samples Selected for Analysis of RE for Melanoma Cells
(A) Six patients with T cell responses reactive with for M26 or G209–2M tetramers were selected for analysis. PBMCs from each patient were stained with PE-conjugated peptide–MHC tetramers, G209–2M-tet PE or M26-tet PE, and co-stained with anti-CD8 fluorescein isothiocyanate and anti-CD14, -CD19, and -CD4 Cy5PE. The plots shown are gated for CD8+, CD14−, CD19−, and CD4− cells. Tetramer-positive cells are boxed and estimated for percent of total CD8+ cells: patient 422, 2.5%; patient 476, 0.31%; patient 132, 0.22%; patient 517, 0.23%; patient 520, 0.12%, and patient 461, 0.50%.
(B) Microcytotoxicity 51Chromium release assay with tetramer-positive cells isolated by FACS from the CD8+ PBMC population from patient 422. Isolated cells were assayed for lysis of T2 cells treated with relevant or irrelevant peptide, or mel526 melanoma cells. Sorted cells were combined with 250 target cells at 13:1 E:T ratios for 4 h, and supernatants were assayed for percent specific release of radiolabel.
Vaccine-Elicited T Cells Are Functional Directly Ex Vivo but of Variable Tumor Reactivity
Patient 422 had the largest detectable TAA-specific CD8+ T cell response (2.5% G209–2M-tetramer-positive) and thus sufficient numbers for examination of lytic function immediately following isolation. To test whether peptide-vaccine-induced T cell responses were functionally active directly ex vivo, T cells isolated by G209–2M-tetramer-guided cell sorting from patient 422 were tested for lysis of peptide-pulsed and melanoma target cells in microcytotoxic assays (Figure 1B). The directly isolated tetramer-positive T cells from this patient specifically lysed T2 cells pulsed with high concentrations (1 μg/ml) of G209–2M and native (G209n) peptides, but not with T2 cells pulsed with a cytomegalovirus-derived, HLA-A*0201-restricted peptide (NLVPMVATV) or melanoma targets. This suggests that while a significant portion of the vaccine-elicited T cells from patient 422 may be functional in vivo, they did not have significant tumor lysis activity.
To assess the functional status of the smaller TAA-specific CD8+ T cell responses in the other five patients—which were too small for direct cytotoxicity assays after sorting—we utilized a novel FACS assay for degranulation based on CD107 mobilization [24]. All six TAA-specific populations exhibited robust functional responses ex vivo, as measured by percentage of G2090–2M- and M26-tetramer-positive cells that mobilized CD107 and/or downregulated the CD3 complex upon incubation with T2 cells pulsed with cognate peptides (Table 1; 86%-99.6%). In response to melanoma targets mel526 and Malme-3M, which both express gp100 and MART-1 and are HLA-A*0201 positive, the two endogenous TAA-specific responses (samples from patients 132 and 461) also exhibited robust functional responses directly ex vivo (Table 1; 36.8%–87%), and these responses were specific as they had little response to A375, a HLA-A*0201-positive melanoma cell that does not express gp100 or MART-1 and served as a negative control for antigen-specific killing (Table 1; 2.7% and 3%). In contrast, the vaccine-elicited responses exhibited much lower reactivity to mel526 and Malme-3M (Table 1; 23.8%–32.5%). These data demonstrate that all six TAA-specific CD8+ T cell responses were functional ex vivo, but there were significant differences in reactivity to melanoma targets between endogenous and vaccine-elicited responses.
Table 1 Functional Status of TAA-Specific T Cell Response
Functional response was determined by percent of G209–2M- and M26-tetramer-positive cells that mobilized CD107 and/or downregulated CD3 complex in response to incubation with T2 cells pulsed with 100 ng/ml of cognate peptide (G209–2M or M26), mel526, or Malme-3M melanoma cells. A375 melanoma cells served as negative control
To substantiate the generality of these findings, we analyzed four additional patients with vaccine-elicited responses. One subject responded to G209–2M only (patient 722), one to M26 only (patient 713), and two to both G209–2M and M26 (patients 721 and 735). Similar to the first four vaccine-elicited patients, these four additional patients (six TAA-specific responses in total) exhibited variable reactivity to melanoma targets, ranging from 13% to 49.6% (Table 1).
Vaccine-Elicited T Cells Have Varied Capacity to Lyse Melanoma Targets
To confirm and further investigate the differences in tumor reactivity between endogenous and vaccine-elicited responses, we reasoned that analysis of a set of clonal CTL lines that represented the tetramer-positive population would provide an accurate estimate of the complexity of the TAA-specific T cell response in each patient. A large number of clonal CTL lines (more than 200) were generated by FACS of individual G209–2M- and M26-tetramer-positive cells directly from PBMC samples (Table 2). Up to 85% of sorted cells expanded in various sorts (data not shown). Randomly selected expanding clones and the tetramer-positive population from which they were derived were examined for TCR VB expression using TCR VB-specific monoclonal Abs and VB-specific primers in PCR. Diverse TAA-specific T cell responses were found in the four vaccinated patients, with multiple T cells expressing different TCR VB, while the two endogenous responses were less diverse. All but one clone derived from patient 132 expressed VB17, while two dominating T cell populations in patient 476 expressed VB14 and VB17 (Table 2). The clonality of the dominant populations in these patients was evaluated by PCR fragment length analysis (Table 3). Identical length fragments were demonstrated in the four selected clones from 476 BV14+ and 476 BV17+ populations. Identical length fragments were also demonstrated in all BV17+ clones from patient 132. Furthermore, analysis of sorted tetramer-positive cells from patient 476 demonstrated single fragment sizes for BV14 and BV17, which were identical to the fragment sizes generated from the selected clones, arguing for clonality of these dominant populations (Table 3).
Table 2 CTL Clones Established from Each Patient Represent a Random Selection from the Tetramer-Reactive CD8+ Parent Population
aClonal CTL lines were established from each patient
bNumber of clonal CTL lines from each patient expressing the same TCR VB chain
cPercent of G209–2M- and M26-tetramer-reactive CD8+ T cells in each patient expressing the indicated TCR VB chain
Table 3 PCR-Generated Fragment Length Analysis
PCR fragment length was determined for selected clones and for sorted G209–2M- or M26-tetramer-positive populations from which the clones were derived. Numbers indicate length in base-pairs of fragments generated by PCR with VB14 or VB17 5′ primer and BC63 constant region 3′ primer followed by a run-off reaction with VB-specific nested primers and FAM-labeled BC63 primer. Fragments were analyzed using an Applied Biosystems 377 automated sequencer and GeneScan software
Table 4 CTL Clones from Each Patient Selected for Functional Analysis
The TCR VB usage of each CTL clone was determined using a panel of 19 anti-VB monoclonal Abs by flow cytometry or by PCR with 34 VB-specific primers. All clones selected for functional analysis were assayed for lysis of melanoma cells. Some clones were also subjected to RE analysis
M, assay for lysis of melanoma cells; MR, RE analysis
Peptide specificity and CD8 expression of each clone was confirmed by staining with G209–2M- and M26-tetramers and anti-CD8 monoclonal Ab (data not shown). To obtain an accurate reflection of the total T cell population detected with tetramer in each patient, we decided to rigorously examine at least one representative clone for each subpopulation expressing a different TCR VB (Table 4). Multiple clones were analyzed to determine dominating populations. From patients 132, 517, and 461, for which fewer clones were generated, all clones were included in the analyses (Table 4).
To determine the effectiveness of tumor lysis by the different TAA-specific T cell clones that were propagated, clones were analyzed for their ability to lyse melanoma cell lines mel526 and Malme-3M. A375 cells served as a control for antigen-specific killing. In addition, each CTL clone was examined for antigen-specific lysis of T2 cells pulsed with high levels (1μg/ml) of G209–2M or M26 peptides. “Efficient lysis” in these experiments was defined as 40% or greater specific release of radiolabel from the target cells; 10% or less specific release was categorized as “low or no lysis,” and 10% to 40% was termed “intermediate lysis.” All but two of the CTL clones elicited from endogenous anti-tumor responses (from patients 132 and 461) exhibited “efficient lysis” of both the mel526 and Malme-3M melanoma cell lines (Figure 2). In contrast, only a few clones from the vaccine-elicited responses (from patients 422, 476, 520, and 517) efficiently lysed melanoma cells. The majority of clones examined from these vaccine-elicited responses either failed to lyse melanoma targets altogether or lysed them with intermediate efficiency (Figure 2). This lack of efficiency in melanoma cell lysis was not due to cellular dysfunction, since each clone efficiently lysed T2 cells pulsed with high levels of relevant, but not irrelevant, peptide (Figure 2). Overall, the majority of clones derived from endogenous anti-tumor responses (patients 132 and 461) lysed both mel526 and Malme-3M melanoma target cells more efficiently than clones from vaccine-elicited responses (patients 422, 476, 520, and 517) (Figure 3). These findings suggest that TAA-specific T cells elicited by heteroclitic peptide vaccination have different tumor-cytolytic potentials from those which develop endogenously to cancer.
Figure 2 Endogenous T Cell Responses Are More Efficient in Melanoma Lysis Than Vaccine-Elicited Responses
Cells from 87 clonal CTL lines were assayed for lysis of melanoma cells mel526, Malme-3M, and A375 in 51Chromium release cytotoxicity assays. Mel526 and Malme-3M are HLA-A2.1+ and express both gp100 and MART-1. A375 cells are HLA-A2.1+ but do not express either gp100 or MART-1 and served as a negative control. T2 cells treated with 1 μg/ml G209–2M or M26 peptides served as controls for antigen-specific lysis. The CTL clones assayed were selected to represent different tetramer-positive subsets expressing different VB. Dominating tetramer-positive populations in each patient were represented with two or more clones. Each CTL clone was assayed in triplicate wells, and the data displayed are averages of two different experiments. Clones from the same patient expressing similar VB while exhibiting different lysis potential were viewed as separate subsets. Each assay was performed at 10:1 E:T ratio as detailed in Methods. The height of each bar represents percent specific lysis, while the width represents the relative size of the tetramer-positive subpopulations (defined by VB expression) in each patient. Population size was defined as the percent of clones from each patient expressing the same VB. Error bars show standard deviation between two experiments within each clone and/or between different clones where more than one clone was analyzed.
Figure 3 Most CTL Clones Isolated from Endogenous Responses Are Efficient in Tumor Cell Lysis
CTL clones derived from each patient were classified as “efficient” (greater than 40%), “intermediate” (between 10% and 40%), or “low/no” (less than 10%) in lysis of melanoma cells based on data displayed in Figure 2. Each bar represents the portion of total clones from each patient with “efficient,” “intermediate,” or “low/no” melanoma lysis potential.
RE for Native and Heteroclitic Peptides of T Cells from Endogenous or Vaccine-Elicited Responses
We hypothesized that CTL clones that did not efficiently lyse melanoma targets may be incapable of recognizing the relatively low surface densities of native peptide present on tumor cells. CTL clones selected for analysis of tumor lysis were also assessed for RE for the native and heteroclitic peptides via a ten-log range of dilutions. This is illustrated with clones 132.1 and 476.105 (Figure 4A). There were considerable differences in killing of peptide-pulsed T2 cells by these two clones. The differences in RE for G209n native peptide displayed by the two clones highlighted in Figure 3A correlated with their ability to lyse melanoma cells: the high-RE clone 132.1 efficiently lysed melanoma targets, whereas the low-RE clone 476.105 did not (Figure 4B). In contrast to the differences in RE for G209n peptide, similar assays revealed little difference in RE of the two clones for G209–2M heteroclitic peptide (Figure 4C), suggesting that these clones recognize the native and heteroclitic peptides differently, and that RE for the native, but not heteroclitic, peptide correlates with tumor-lytic potential.
Similar RE assays were performed for the remaining clones from each patient selected for analysis. In order to compare REs of various CTL lines, each clone was assigned an RE score expressed as the negative log10 value of the peptide concentration required for 40% specific lysis at an E:T ratio of 10:1. For clones 132.1 and 476.105, these scores were 11.1 and 8.3 for assays with G209n peptide (Figure 4A), and 11.2 and 11.2 for assays with G209–2M heteroclitic peptide (Figure 4C), respectively. We compiled the data on clones from all patients, which showed a correlation between tumor-lytic potential and RE for native peptide (Figure 5A and 5B). Overall, clones generated from endogenous anti-tumor responses had higher RE for the native peptide than clones generated from post-vaccine responses (Figure 5C and 5D). We estimated the composite RE of the overall TAA-specific response (composed of a heterogeneous population of T cells) in vivo by summing the RE of each clone multiplied by its representation in the original mixture (the representation was estimated based on the proportion of TAA-specific cells expressing the same VB as the clone). These composite RE values are represented in Figure 5 as horizontal bars for each response. Clearly, the endogenous responses (patients 461 and 132) had a higher overall, and more homogeneous, RE for the native peptide than the vaccine-elicited responses (patients 422, 476, 517, and 520) (Figure 5C and 5D). Importantly, the vaccine-elicited clones also exhibited wide variations in RE even for the heteroclitic peptide, compared to the endogenous responses (Figure 5E and 5F). This suggests that the variation in RE for native peptides, and hence ability to lyse tumor cells, for vaccine-elicited responses is not merely a reflection of differential recognition of native and heteroclitic peptides by many clones. Rather, variations in RE may be a function of the manner in which these cells were elicited in vivo via vaccination.
Discussion
To achieve maximal clinical responses, the majority of T cells elicited by vaccination in cancer patients should be capable of responding to tumor targets. We have undertaken the most detailed analysis to date, on a single-cell level, of T cell responses elicited by cancer vaccination and have compared these with endogenous anti-tumor responses. To evaluate the full spectrum of T cells elicited in each patient by vaccination, we utilized tetramers made with the vaccine peptides (heteroclitic M26 and G209–2M) to isolate such cells. CTL clones were selected directly from patient PBMC samples without enrichment in culture to closely reflect the composition of the antigen-specific T cell response in vivo at the time of isolation.
Our data revealed that T cell populations induced by vaccination were significantly different from endogenous responses: while some CTLs elicited by vaccination could kill melanoma targets, most were inefficient in tumor cell lysis. In contrast, nearly all clones from endogenous responses were efficient at melanoma cell lysis. This difference was related to RE for the native peptide. Clones that did not lyse tumor cells required up to 103-fold higher concentration of peptide for similar levels of lysis of targets compared to T cell clones that were tumor-lytic. Side-by-side comparison of endogenous responses and vaccine-induced responses suggests that low RE TAA-specific T cell responses may be preferentially driven by heteroclitic peptide vaccination. Thus, high doses of peptide and/or the higher levels of expression of heteroclitic peptide on APCs may induce and actively propagate predominantly T cells with RE too low for recognition of physiological levels of the native peptide present on tumor targets. These data suggest an inverse relationship between antigen density and the RE of T cells elicited. This would be an important consideration in design of future vaccine strategies.
Differential recognition of native and heteroclitic peptides by many T cells may also account for the induction of non-tumor-lytic clones by heteroclitic peptide vaccines, which has been suggested previously [23,32]. However, our data suggest that epitope density may be the dominant driving factor for RE in vivo. In all of the vaccine-elicited T cell responses, many of the T cells generated were either of low or intermediate RE not only for the native peptide, but also for the heteroclitic peptide, and exhibited no or intermediate lysis of tumor targets. In contrast, nearly all of the clones generated from the endogenous responses were of high RE. This suggests that the high dosage of peptides administered in vaccinations and the increased binding capacity of heteroclitic peptides to MHC molecules—the very quality that provides them with increased immunogenicity—drive the induction of many T cells with low RE for both heteroclitic and native peptides.
Another implication of this study is that the number of cells measured by current methods, including ELISPOT or staining with MHC tetramers, may not correlate directly with the RE or tumor reactivity of T cell responses to vaccination. For example, of the nine clones analyzed from patient 517, none were efficient in tumor cell lysis, yet these cells were detectable by MHC tetramer staining. T cells with low RE for native TAA do not efficiently lyse tumor, and therefore are unlikely to have an impact on clinical outcome. Furthermore, it may be possible that low-RE TAA-specific T cells may interfere with elicitation of high-RE T cells, either by direct competition for antigen on APC surface [33,34] or down-modulation of peptide–MHC complexes.
Our data support the notion that not only quantity, but quality, of the T cell response elicited by vaccination may be important for clinical efficacy. There are a number of strategies to increase the magnitude of T cell responses to peptide vaccines. These include using various adjuvants, such as incomplete Freund's adjuvant and immunomodulatory agents, such as IL-12 [4], GM-CSF [5], anti-CTLA-4 Abs [35], or heat shock proteins [36]. Thus far, none of these approaches have produced improved clinical outcomes. Our data suggest that in addition to driving higher numbers of vaccine-elicited T cells, strategies to modulate the relative RE of T cell responses are also needed. While the selective activation of high- versus low-RE T cells is relatively easy to manipulate in vitro via stimulation with limiting amounts of peptides, this may be more difficult to control in vivo. It is important to bear in mind that signals needed to drive a de novo naïve T cell response may be different from those required to drive further expansion of an activated T cell population [37]. Thus, a complete vaccination strategy may involve an initial induction phase, followed by progressive shaping of the response to higher RE. Although heteroclitic peptide vaccination may drive T cells of mixed high and low RE, such a strong stimulus may be needed to induce an initial de novo T cell response. Studies in mice suggest that once activated, effector CD8+ T cells may have an increase in RE of up to 70-fold compared to naïve cells [38,39,40]. Thus, naïve TAA-specific T cells, with inadequate RE to become activated by low densities of native peptides present on tumor cells, may become efficient in tumor lysis upon vaccination with heteroclitic peptide. This notion has support from studies in tolerized mice: vaccination with a heteroclitic peptide analog recruited T cells, which were responsive to secondary stimulation with native peptide [41,42]. Therefore, optimized use of heteroclitic peptide to induce an initial peptide-specific T cell response, followed by selective expansion of the highest RE tumor-lytic T cells may be needed for an effective strategy with clear clinical application.
In summary, we have demonstrated that vaccination with heteroclitic peptide at high concentrations may drive T cell responses of variable tumor-cytolytic potential in cancer patients—and that the ability to lyse tumor cells correlates with the T cell's RE for native peptides. This represents an important—but not sole—factor in explaining the lack of correlation between immunological and clinical responses after vaccination for cancer. Importantly, the situation is different in endogenous responses, in which cells are predominantly of high RE. This suggests that the manner in which T cells are elicited in vivo are different in these two settings and may underlie their differences in biology.
Patient Summary
Why Was This Study Done?
Our immune system protects us against infectious diseases. It can also recognize and destroy early cancer cells before they form tumors. Researchers have been trying to find a way to boost the anti-cancer function of the immune system so that it can kill even established tumors. This is the idea behind developing vaccines for treating cancer—the vaccine alerts and boosts the patient's immune system and so helps to fight the cancer. The idea of enlisting the immune system against cancer has been around for a long time. There have been some spectacular successes, but it has proven difficult to find vaccines that work in more than just a few patients. And we don't yet understand why vaccines seem to work in some patients but not in others.
What Did the Researchers Do?
Peter Lee and colleagues are trying to find out why some patients respond to vaccines and others don't by looking at the immune response in vaccinated patients. In this study, using state-of-the art technology, they examined patients who received different vaccines against the skin cancer melanoma. They concentrated on the so-called killer T cells (cytotoxic T cells), which directly attack and kill tumor cells, and analyzed them in great detail.
What Did They Find?
Most cytotoxic T cells produced by patients after vaccination—including vaccination with so-called heteroclitic peptides that had been specifically designed to provoke a very strong immune response—did not kill tumor cells very well, but a few of them did. These results provide some explanation as to why cancer vaccines haven't been as successful as many had hoped, but also suggest that if it were possible to get more of the potent T cells or to expand the ones that are already produced with the current vaccines, there would be a stronger anti-tumor response.
What Next?
How to get effective cancer vaccines remains an open question. But at least technologies such as those used in this study now exist that allow researchers to analyze how the immune systems of different patients react to vaccination and hence can guide the development of better vaccines.
Additional Information.
The Cancer Research Institute: http://www.cancerresearch.org/
US Food and Drug Administration page on cancer vaccines: http://www.cancerresearch.org/
University of Michigan page on cancer vaccines: http://www.cancer.med.umich.edu/learn/cancervaccines.htm
This work was supported by National Institutes of Health R01 CA 090809 (PL) and the Damon Runyon Cancer Research Foundation (Scholar Award to PL). Part of this work was supported by research grant NSF DMS 0241246 to SPH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Citation: Stuge TB, Holmes SP, Saharan S, Tuettenberg A, Roederer M, et al. (2004) Diversity and recognition efficiency of T cell responses to cancer. PLoS Med 1(2): e28.
Abbreviations
Abantibody
APCantigen-presenting cell
CTLcytotoxic T lymphocyte
E:Teffector to target
FACSfluorescence-activated cell sorting
FBSfetal bovine serum
IMDMIscove's Modified Dulbecco's Medium
MHCmajor histocompatibility complex
NCINational Cancer Institute
PEphycoerythrin
PBMCperipheral blood mononuclear cell
RErecognition efficiency
TAAtumor-associated antigen
TCRT cell receptor
VBvariable chain beta
==== Refs
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| 15578105 | PMC529423 | CC BY | 2021-01-05 10:38:00 | no | PLoS Med. 2004 Nov 30; 1(2):e28 | utf-8 | PLoS Med | 2,004 | 10.1371/journal.pmed.0010028 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1557810610.1371/journal.pmed.0010035EssayOtherSystematic reviews and meta-analysesEvidence Based PracticeClinical researchHealth services researchDoing New Research? Don't Forget the Old EssayClarke Mike Mike Clarke is director of the United Kingdom Cochrane Centre, Oxford, United Kingdom. E-mail: [email protected]
Competing Interests: The author's employment depends upon the value and importance given to systematic reviews. He is employed by the Milton Keynes Primary Care Trust on behalf of the Department of Health in England, as director of the UK Cochrane Centre for four days per week. This is a fixed term contract, the renewal of which is dependent upon the value placed upon his work, that of the UK Cochrane Centre (and more widely, of The Cochrane Collaboration) by the Department of Health, all of which relates to the importance of systematic reviews. He is also employed by Cancer Research UK to work one day per week at the Clinical Trial Service Unit, University of Oxford, United Kingdom, primarily on systematic reviews of treatments for early breast cancer.
11 2004 30 11 2004 1 2 e35Copyright: © 2004 Mike Clarke.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Nobody should do a new research study, says Clarke, without first systematically reviewing the literature. And journal editors should insist that all research papers are accompanied by an up-to-date systematic review
Nobody should do a trial without reviewing what is known
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On May 2, 1898, George Gould used his address to the founding meeting of the Association of Medical Librarians in Philadelphia to present a vision of the future of health information. ‘I look forward,’ he said, ‘to such an organisation of the literary records of medicine that a puzzled worker in any part of the civilised world shall in an hour be able to gain a knowledge pertaining to a subject of the experience of every other man in the world’ [1]. Has his vision been realised?
Information Overload
In these early years of the 21st century, with tens of thousands, if not hundreds of thousands, of new research articles being published every year, people who need to make decisions about health care are much more overwhelmed with information than they were in 1898. Some of this information is of good quality, but some of it is not. Thus, anyone wishing to use the health literature to make well-informed decisions must both identify the relevant research from amidst this vast amount of information and then appraise it. This is an impossible task for many. Even though making access to the literature easier and cheaper will increase the ability of people to find research, it will also reveal just how much information there is out there and how daunting is the task of making sense of it.
You can get a good idea of the size of the task—and of how electronic publishing and the Internet have transformed the situation—by imagining the following four steps. How long would it take to find articles in your area of interest by paging through back copies of a relevant journal? What about by using its index? Now imagine finding articles of interest to you by going to the Internet and searching PubMed (www.ncbi.nlm.nih.gov/PubMed). What about if you searched the whole of the Internet with one or more search engines? Almost certainly, as the speed of the search increased through these four approaches, so would the number of articles retrieved—and also the time that it would take to read through them, appraise them, and decide if they were relevant to whatever decision you were trying to make.
Doctors are overloaded with information, much of it irrelevant to their practice
(Illustration: Rusty Howson, sososo design)
Should you just take a shortcut by relying on a single study in a high-profile journal? No. Sampling in this way might lead you to research whose findings are the most striking or atypical, but you would miss similar research that was ‘less fortunate’ with its results. However good the conduct of a piece of research, chance effects mean that some studies will produce an overestimate and some an underestimate of the true effects, but the easy-to-find literature is likely to be dominated by the former [2]. In addition, using a sample rather than the whole body of relevant research will have less statistical and evidential power to answer the question of interest.
The Value of Systematic Reviews
Systematic reviews provide a means to minimise these problems. In systematic reviews, the methods to be followed are stated and an attempt is made to identify, appraise, and where appropriate, statistically combine, all relevant research. In fact, a decade before George Gould's address, Lord Rayleigh, at the meeting of the British Association for the Advancement of Science in Montreal, had described such a process for scientific research in general.
‘If, as is sometimes supposed,’ he said, ‘science consisted in nothing but the laborious accumulation of facts, it would soon come to a standstill, crushed, as it were, under its own weight. The suggestion of a new idea, or the detection of a law, supersedes much that has previously been a burden on the memory, and by introducing order and coherence facilitates the retention of the remainder in an available form. Two processes are thus at work side by side, the reception of new material and the digestion and assimilation of the old. One remark, however, should be made. The work which deserves, but I am afraid does not always receive, the most credit is that in which discovery and explanation go hand in hand, in which not only are new facts presented, but their relation to old ones is pointed out’ [3].
Relating the New to the Old
If today's health researchers discussed their findings in the context of relevant, already-existing research, many of the problems of information overload would be eased. You would only need to find the most recent report of a relevant study; its discussion section would place that study within the context of an updated systematic review. This was suggested by the original CONSORT statement on the reporting of randomised trials in 1996 [4]. However, studies of five of the major general medical journals (Annals of Internal Medicine, BMJ, Lancet, JAMA, and the New England Journal of Medicine) in 1997 and 2001 found that this was not the case, at least for these journals. Only two of more than 50 reports of randomised trials in these journals in May of those two years included an updated systematic review [5,6].
Including an updated systematic review along with a report of a randomised trial (or any other piece of research) might seem too much to expect of researchers, who might not feel able or willing to do the additional work required. However, the absence of a review should raise the question: on what did the researchers base the design of their new study? To embark on a new study without first systematically reviewing what has been done before is to risk doing research for which the answer is already known. It would also mean that the researchers had denied themselves the opportunity to learn from the successes and failures of others when designing their own study. In addition, researchers have a responsibility to the participants in their research to make sure that the study is of the most appropriate design possible. To help make sure that this is the case, when designing a new study researchers should ensure that they have been adequately informed about what research has been done previously. Box 1 lists practical suggestions to researchers for making sure that their new study builds on prior knowledge.
Box 1. Practical Suggestions for Researchers
Conduct a systematic review of your research question before embarking on a new study, or identify a relevant review done by someone else.
Design your study to take account of the relevant successes and failures of the prior studies, and of the evidence within them.
Discuss the findings of your study in the context of an updated systematic review of relevant research.
Publish the systematic review within, alongside, or shortly after the report of your study.
Provide information from your study to others doing systematic reviews of similar topics.
It might even be the case that the researchers are able to draw on the work of others, who already have done a systematic review of the relevant topic. Over the last decade, The Cochrane Collaboration (Box 2) has produced more than 2,000 Cochrane systematic reviews (www.cochrane.org) [7]. There are thousands more reviews scattered throughout the literature (www.york.ac.uk/inst/crd/darefaq.htm). And with the ability to publish longer versions of articles on the Internet than are practical in print, concerns about article length should no longer be a barrier to the inclusion of a systematic review.
Box 2. The Cochrane Collaboration
The Cochrane Collaboration (www.cochrane.org) is an international, nonprofit, and independent organisation dedicated to helping people make well-informed decisions about health care by preparing, maintaining, and promoting the accessibility of systematic reviews. These reviews are published electronically in The Cochrane Library, which is available on the Internet and CD-ROM. The Cochrane Collaboration was established in 1993 and is named after the British epidemiologist, Archie Cochrane.
Updating Gould's Vision
As we progress through the 21st century, and health care information continues to become ever more plentiful, there are tremendous opportunities to make knowledge about health care more accessible. However, for this to happen without overwhelming the people who are trying to make health care decisions—for themselves or for someone else—the need for new research to be designed and reported using systematic reviews becomes ever more pressing.
Returning to George Gould's vision, but bringing it into the modern era, I hope for a system in which everyone making a decision about health care in any part of the world would be able, in 15 minutes, to obtain up-to-date, reliable evidence of the effects of interventions they might choose, based on all the relevant research. Journals, especially new ones such as PLoS Medicine, will help achieve this by only publishing a report of a new research study under the following conditions. First, the researchers must justify their study on the basis of a previous systematic review. Second, the journal should publish an updated systematic review (which incorporates the new study) within the new study, alongside it, or shortly thereafter.
I am grateful to Ian Ford for his helpful comments, which improved this essay. The views expressed in this article are those of the author and are not necessarily those of The Cochrane Collaboration.
Citation: Clarke M (2004) Doing new research? Don't forget the old. PLoS Med 1(2): e35.
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Counsell CE Clarke MJ Slattery J Sandercock PAG The miracle of DICE therapy for acute stroke: Fact or fictional product of subgroup analysis? BMJ 1994 309 1677 1681 7819982
Lord Rayleigh Address by the Rt. Hon. Lord Rayleigh Report of the fifty-fourth meeting of the British Association for the Advancement of Science; held at Montreal in August and September 1884 1885 London John Murray 3 23
Begg C Cho M Eastwood S Horton R Moher D Improving the quality of reporting of randomized controlled trials. The CONSORT statement JAMA 1996 276 637 639 8773637
Clarke M Chalmers I Discussion sections in reports of controlled trials published in five general medical journals: Islands in search of continents? JAMA 1998 280 280 282 9676682
Clarke M Alderson P Chalmers I Discussion sections in reports of controlled trials published in general medical journals JAMA 2002 287 2799 2801 12038916
Dickersin K Manheimer E The Cochrane Collaboration: Evaluation of health care and services using systematic reviews of the results of randomized controlled trials Clin Obstet Gynecol 1998 41 315 331 9646964
| 15578106 | PMC529424 | CC BY | 2021-01-05 10:38:00 | no | PLoS Med. 2004 Nov 30; 1(2):e35 | utf-8 | PLoS Med | 2,004 | 10.1371/journal.pmed.0010035 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1557810710.1371/journal.pmed.0010037The PLoS Medicine DebateEpidemiology/Public HealthHealth EconomicsHealth PolicyHIV/AIDSPrimary CareHIV Infection/AIDSMedicine in Developing CountriesPublic HealthInternational healthIs the “3 by 5” Initiative the Best Approach to Tackling the HIV Pandemic? The PLoS Medicine DebateKim Jim Yong Ammann Arthur Jim Yong Kim is the director of the Department of HIV/AIDS at the World Health Organization, Geneva, Switzerland. E-mail: [email protected]
Arthur Ammann is the president of Global Strategies for HIV Prevention (www.globalstrategies.org), San Rafael, California, United States of America. E-mail: [email protected]
Competing Interests: The authors declare that they have no competing interests
11 2004 30 11 2004 1 2 e37Copyright: © 2004 Kim and Ammann.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background to the debate: The World Health Organization (WHO) and its partners aim to treat 3 million people infected with HIV in poor and middle income countries with antiretroviral treatment by the end of 2005. The ambitious “3 by 5” initiative has had its supporters and its critics since its announcement in 2002.
Jim Yong Kim argues that getting 3 million HIV positive people on antiretroviral treatment by the end of 2005 is the right strategy. Arthur Ammann believes this approach is too narrow
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Jim Yong Kim's Viewpoint: 3 by 5 is a Point of Entry, Not an End in Itself
There are no sure prescriptions against great plagues like HIV. We must “learn by doing,” quickly assessing the inevitable missteps and false starts and using this information to improve outcomes. Our best information about the HIV pandemic suggests four clear principles.
First, treatment must be a core element. This does not mean treatment alone. Antiretroviral therapy (ART) is in no way more important than education, prevention of mother-to-child transmission, expanded access to testing and counseling, or other pieces of a comprehensive public health effort. But preventing mother-to-child transmission is a pyrrhic victory if, as the latest data suggest, AIDS will cause 15 million of those uninfected children to grow up as orphans [1]. Education is fruitless when the hope of finding work has disappeared because the loss of so many productive workers has led to the near collapse of industrial enterprise [2]. The dramatic benefits of ART for patients with advanced HIV disease (“the Lazarus effect”) engage the public imagination, helping to build political will on behalf of all interventions against the pandemic. And treatment can accelerate prevention by offering an incentive to get tested and know one's status, by reducing the stigma of an infection that leads to certain death, and by giving health workers credibility in devastated communities. Treatment is a point of entry, not an end in itself.
Second, time is of the essence. Every data point in the arcs of HIV transmission, morbidity, and mortality represents an exponential increase in human suffering and social and economic disruption. Countries that were able to respond early and comprehensively to HIV now have mortality rates comparable to those of Europe and North America and have cut transmission dramatically. Early interventions can provide large cost savings to the public sector and prevent devastating losses of human capital [3]. There may be risks associated with rapid scale-up—promotion of drug resistance or implementation of care delivery models that are not a perfect fit in all settings [4]. It is essential that we manage these risks through effective monitoring, evaluation, “real time” operational research, and knowledge management. However, the risks of action are minor compared with the certain failings of deferral.
The number of patients on ART has almost doubled in the last two years
(Photo: WHO/Michael Jensen)
Third, clear consensus targets are indispensable. An effective response to HIV demands the resources and attention of every region, state, and community. Coordinating these many different stakeholders requires clarity of purpose. All the great public projects of the modern era, such as the first manned moon expeditions of the 1960s, began with the establishment of a ringing collective priority. Like the 3 by 5 initiative, these projects were in themselves only surrogate endpoints. But they provided focus for the energies of their many participants. To reach the ultimate goal of universal access to ART we must plan in stages.
Fourth, the specific procedures developed to combat HIV must be codified and simplified. Because pandemics, by definition, afflict communities with a broad range of material resources and technical capacities, our methods must be suitable to the most disrupted and impoverished of them. This means effective training modules to increase the supply of health workers; it means inexpensive, rapid tests and diagnostics; and it means streamlined regimens and fixed-dose combinations that facilitate drug procurement and improve rates of adherence.
These principles are at the heart of the 3 by 5 effort. When it was announced in 2002, the gap between the damned and the saved had finally begun to narrow after sharp declines in the price of ART and the emergence of large-scale financing through the Global Fund, the World Bank, and the President's Emergency Plan for AIDS Relief. AIDS is unusual in the history of epidemics because proven, effective ways of interrupting the course of the disease have existed since shortly after its emergence, yet those methods were foreclosed to most of the world's population. The announcement of 3 by 5 drew from a widespread sense that this inequality presented unacceptable economic, political, moral, and epidemiological consequences.
Scaling up treatment is not only a possibility, but is already a reality. The number of patients on ART has nearly doubled in the last two years, mostly in countries where therapy had been limited to a privileged few [5]. High-burden countries are doing their part; the rest of the world must now share more fully in combating the pandemic. From sterile debates over prevention and treatment, the task has shifted to upgrading health systems in resource-poor settings to permit a comprehensive response to the epidemic.
Those who object to 3 by 5 must address this question: what would be the likely cost if it were never attempted? We can work exclusively to prevent the further spread of HIV, or aim to improve treatment access more slowly, but in the meanwhile high-burden countries will collapse at our feet. Or we can aim for 3 by 5 and move ourselves that much closer to the ultimate goal: preventing all unnecessary deaths from HIV.
Arthur Ammann's Viewpoint: The Intentions Are Good, the Approach Is Wrong
There is much that is exemplary about the basic principles outlined by WHO for treatment expansion. Improving access to life-saving ART is a moral imperative and everyone agrees that this will take lots of money [6,7]. So where is the debate?
The debate lies in the strategy. Reviewing the epidemic over the past 20 years, you would have to conclude that the current international public health approach has failed and that an urgent change is required. There are three fundamental problems with the 3 by 5 approach.
First, it is very narrow, and narrow strategies for tackling HIV often fail. For example, we have known for five years that a single dose of nevirapine can help prevent perinatal HIV transmission. The intervention is simpler and cheaper than the 3 by 5 initiative—yet today less than 5% of pregnant women infected with HIV receive any ART [8,9]. WHO should instead focus on a credible overarching public health approach that is commensurate with the severity of the epidemic. Sound global public health policy requires accountability that respects the right of individuals to be protected from fatal infections. This can only be accomplished by universal offering of HIV testing, integration of HIV prevention and treatment into all health-care arenas, contact tracing, and treatment for all who require ART [8,10]. The 3 by 5 initiative fully addresses only one of these issues—treatment—and leaves HIV-exposed individuals at risk for infection and infected contacts unaware of treatment possibilities. These are irreversible missed opportunities and represent a non-accountable approach to an out-of-control epidemic.
Second, confusion about realistic costs, sources of funding, and the relation to other critical WHO initiatives abound. Initial WHO estimates of $5 billion annually for the cost of the entire 3 by 5 initiative have been revised upward to $6 billion annually [6,7,11]. It is not clear whether the costs for this program are distinct from other WHO initiatives, are incorporated into the Global Fund, will increase dramatically and continue beyond 2006, or are ultimately incorporated into national budgets. If the true cost is $6 billion annually, this is $2,000 per individual per year, which is surely not sustainable for resource-poor countries. Further, of the 40 million individuals infected with HIV worldwide, it is likely that at least 20 million will require treatment, and there will be millions more newly diagnosed patients [11]. This would conservatively place treatment costs at over $40 billion per year. Only recently has WHO acknowledged what everyone else seemed to know from the beginning—it will take huge investments in infrastructure to sustain ART delivery [7,12,13,14].
Third, the 3 by 5 initiative is a “top down” unsustainable approach that, without a high level of government investment, fosters dependence on international aid. Countries that have taken ownership of their HIV programs have often been the most successful in tackling the epidemic [8,14,15]. Brazil has mounted an effective response to the HIV epidemic whereas South Africa has not. A key difference between these countries is that 84% of Brazil's AIDS programs are funded from domestic sources compared to 0.4% in South Africa [6]. Furthermore, there are thousands of nongovernmental organizations, clinics, and hospitals already treating patients with HIV and a “top down” approach doesn't work for these organizations. Many are up and running and require only minimal training to move ART distribution forward. The last thing they need is more internationally imposed hurdles that ensure sequestration of ART in costly and inefficient bureaucracies.
The HIV epidemic is the worst pandemic in history. Why, then, is the international public health response so disparate from public health responses to other life-threatening infectious diseases? The 3 by 5 initiative, with no requirement for contact tracing, does not ensure the right of uninfected individuals to be protected or of infected contacts to gain access to treatment. It is time to acknowledge that the severity of the epidemic requires universal offering of HIV testing and counseling, contact tracing, and integration of sound public health prevention and treatment principles into all health-care delivery systems [8,10].
Prevention efforts—including education—must be at the heart of tackling HIV
(Photo: Rick Maiman, on behalf of the David and Lucile Packard Foundation)
Thousands of existing and new teams of health-care workers should be trained in voluntary testing and counseling, and in treatment, using streamlined training courses. Mobile teams could be used to reach rural areas where as many as 50% of infected individuals may live. Cumbersome “top down” training and certification, as is currently planned, will only delay the very therapy that WHO seeks to make more available [8,14,15].
It is a paradox that HIV is one of the few diseases that is deemed to require exceptional international and national bodies overseeing access to medicines. The success of ART in developed countries was a result of making ART directly available to those who treat patients, not to governments. Widespread and timely access can only occur when health workers are able to provide ART without repressive procedures. We can distribute drugs more freely for other diseases—why not HIV?
Kim's Response to Ammann's Viewpoint
I agree with Arthur Ammann that the response of the international community to the HIV epidemic over the last 20 years has been grossly inadequate. I also agree that a more effective strategy must pay close attention to mother-to-child transmission, provide “opt out” testing and counseling integrated into key health-care settings, implement effective partner referral strategies, and train many thousands of health-care workers. These are stated WHO priorities—integral to the 3 by 5 initiative—as any attempt to investigate our position would reveal [16,17].
Dr. Ammann's other charges are also puzzling. Our latest cost projections (which he cites) in fact provide a two-year, not annual, range of $5.1 to $5.9 billion, depending on the price of drugs [7]. The accusation of a “top down” approach seems misplaced. WHO's primary contact is indeed with ministries of health, given our constitutionally mandated obligation to provide technical assistance for 192 WHO member states. But I strongly dispute that this relationship somehow prejudices the 3 by 5 agenda against nongovernmental organizations or local providers. WHO is far from the only partner in this struggle, and we actively promote broad-based collaborations within the nonprofit sector, the private sector, among traditional healers, and within civil society [18].
Finally, I must point out a contradiction in Dr. Ammann's arguments. On the one hand, he urges high-burden countries to take ownership of their HIV programs and finance these themselves. On the other, he acknowledges that if the “true cost [of ART] is $6 billion annually, this is $2,000 per individual per year, which is surely not sustainable for resource-poor countries.” The crucial question is whether $6 billion annually is “sustainable” for the world community as a whole. In facing the worst health disaster in several centuries, we simply cannot wait for the poorest countries to self-finance HIV treatment—or else we will be guilty of standing idly by as millions die and societies collapse.
Ammann's Response to Kim's Viewpoint
There are time-tested prescriptions for halting epidemics, and HIV should be no exception. But “HIV exceptionalism” persists [19]. Current public health efforts fail to insist on universal HIV testing and contact tracing, thereby limiting treatment and prevention opportunities and contributing to “feminization” of the epidemic (more and more women getting infected at an earlier age) [20]. Prevention and treatment are inextricably linked. The 3 by 5 initiative should meet the high standards—individual and organizational accountability, justice, and public good—of other public health approaches.
Prevention of perinatal HIV transmission cannot be called a victory—not even a pyrrhic one—since less than 5% of pregnant women infected with HIV receive any ART. Further, in most perinatal HIV prevention programs sexual partners are generally not identified, treatment has only recently been offered, and HIV testing has not been universally implemented. Narrowly focused, top-down programs such as the 3 by 5 initiative face similar outcomes when they adopt yet another partial approach that is not fully integrated into countrywide general health care.
The 3 by 5 initiative suggests that treatment is the major incentive for HIV testing. But getting tested has two incentives—treatment and prevention. Knowing one's HIV status is a means of saving lives, an incentive that should motivate every individual to be tested even when treatment is not available. We will destine ourselves to an ever-escalating epidemic if we delay universal testing until treatment is available.
Both Jim Yong Kim and I want to treat 3 million individuals infected with HIV and more, and we agree that time is of the essence. But why take another decade to learn what we already know from epidemics such as tuberculosis? Lateral, comprehensive approaches that equip health-care workers with testing and the required drugs work best [21,22]. Procedures to combat HIV have already been simplified by such workers in resource-poor areas—so get the tests and the drugs to them and they will do the treating.
Citation: Kim JY, Ammann A (2004) Is the “3 by 5” initiative the best approach to tackling the HIV pandemic? PLoS Med 1(2): e37.
Abbreviations
ARTantiretroviral therapy
WHOWorld Health Organization
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International Labour Organization HIV/AIDS and work: Global estimates, impact, and response 2004 Geneva International Labour Organization Available: http://www.ilo.org/public/english/protection/trav/aids/publ/global_est/ . Accessed 24 September 2004
Commission on Macroeconomics and Health Macroeconomics and health: Investing in health for economic development 2001 Geneva World Health Organization Available: http://www3.who.int/whosis/cmh/cmh_report/e/pdf/001-004.pdf . Accessed 24 September 2004
Popp D Fisher JD First, do no harm: A call for emphasizing adherence and HIV prevention interventions in active antiretroviral therapy programs in the developing world AIDS 2002 16 676 678 11873017
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Joint United Nations Programme on HIV/AIDS Global Resource Tracking Consortium Financing the expanded response to AIDS 2004 Geneva Joint United Nations Programme on HIV/AIDS 51
Gutierrez JP Johns B Adam T Bertozzi SM Edejer TT Achieving the WHO/UNAIDS antiretroviral treatment 3 by 5 goal: What will it cost? Lancet 2004 364 63 64 15234857
Global HIV Prevention Working Group HIV prevention in the era of expanded treatment access 2004 Available: http://www.gatesfoundation.org/nr/downloads/globalhealth/aids/PWG2004Report.pdf . Accessed 24 September 2004
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De Cock KM Mbori-Ngacha D Marum E Shadow on the continent: Public health and HIV/AIDS in Africa in the 21st century Lancet 2002 360 67 72 12114058
World Health Organization Joint United Nations Programme on HIV/AIDS Treating 3 million by 2005: Making it happen 2003 Geneva World Health Organization Available: http://www.who.int/3by5/publications/documents/isbn9241591129/en/ . Accessed 27 September 2004
Zarocostas J WHO puts HIV/AIDS pandemic at top of its agenda Lancet 2004 363 1615
The Body Pro WHO, UNAIDS meeting discusses 3 by 5 initiative, fixed-dose combination antiretroviral drugs 2004 Available: http://www.thebodypro.com/kaiser/2004/jul12_04/who_unaids.html . Accessed 24 September 2004
National Academies IOM report urges immediate rollout of HIV/AIDS initiatives in developing world; calls for new partnerships, ‘HIV/AIDS corps’ to address work-force crisis 2004 Available: http://www4.nationalacademies.org/news.nsf/isbn/0309092647?OpenDocument . Accessed 24 September 2004
World Health Organization Joint United Nations Programme on HIV/AIDS Emergency scale-up of antiretroviral therapy in resource-limited settings: Technical and operational recommendations to achieve 3 by 5 2004 Geneva Joint United Nations Programme on HIV/AIDS Available: http://www.who.int/3by5/publications/documents/zambia/en/ . Accessed 24 September 2004
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| 15578107 | PMC529425 | CC BY | 2021-01-05 10:38:01 | no | PLoS Med. 2004 Nov 30; 1(2):e37 | utf-8 | PLoS Med | 2,004 | 10.1371/journal.pmed.0010037 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1557810810.1371/journal.pmed.0010039Policy ForumEpidemiology/Public HealthHealth PolicyHealth care reformHealth PolicyHealth Services Administration/ManagementQuality of health careHealth care statisticsA National Health Insurance Program for the United States Policy ForumMcCanne Don R Don R. McCanne is a former president of Physicians for a National Health Program, Chicago, Illinois, United States of America. E-mail: [email protected]
Competing Interests: The author declares that he has no competing interests.
11 2004 30 11 2004 1 2 e39Copyright: © 2004 Don R. McCanne.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The US will spend $1.79 trillion on health care in 2004, yet 44 million Americans remain uninsured. What the country needs, argues McCanne, is publicly funded universal health coverage
The country must abandon its fragmented system
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The total projected spending on health care in the United States for 2004 is $1.79 trillion—15.5% of its gross domestic product [1]. That amounts to $6,167 per person, almost twice what most nations with comprehensive systems spend on care. Most policy analysts agree that this level of spending should be more than enough to provide all Americans with high quality, comprehensive health care. Yet the United States falls far short of these goals. What are the flaws in the United States health system that prevent Americans from receiving value from this huge health care investment? And what are the options for improvement?
Physicians for a National Health Program
First, I should reveal my personal bias. Physicians should be well represented in the forefront of reform. As we look back on the past half century of failed health policy decisions, we see that the dominant physicians' organization in the United States, the American Medical Association (AMA), has opposed most reform measures that would result in an equitable, affordable system for everyone. Instead, the AMA has supported an agenda that promotes physicians' freedom to maximize their personal financial reward, even though those policies may deprive tens of millions of Americans access to affordable care. The AMA agenda has contributed significantly to the current high costs of American health care and to our failure to adequately address the mediocrity that characterizes health care in the United States.
Many American physicians—including myself—believe that the funding infrastructure should be redesigned to maximize heath care resource allocation for the primary benefit of patients. Because of the failure of organized medicine to advocate on behalf of our patients, we decided that a new organization was needed. We established Physicians for a National Health Program (www.pnhp.org) [2,3].
The Uninsured and the Poorly Insured
There are 45 million Americans with no health care coverage, and not surprisingly, lack of insurance is associated with worse health outcomes [4]. About 18,000 young adults die each year because they lack health insurance [4]. The uninsured are less likely than the insured to receive the professionally recommended standard of care for their chronic diseases, such as diabetes (Figure 1) [5]. And if you have a serious health crisis while you are uninsured, you risk major debt or bankruptcy.
Figure 1 Diabetes Management among Insured and Uninsured Adults, Ages 18–64
The figure is based on data from a United States national survey of 105,764 adults in 1997 and 117,364 in 1998 [5]. The proportions have been adjusted to the demographic characteristics of the study cohort, controlling for age, sex, race/ethnicity, region, employment status, education, and income. (Reprinted, with permission, from [4], courtesy of the National Academies Press, Washington, DC, United States.)
Even the insured are inadequately covered. Employers and individuals who purchase coverage are rebelling at the high price of insurance premiums. To maintain competitive premiums, insurers are designing products that reduce the benefits they pay out by increasing the out-of-pocket portion that patients are required to pay for services received. Insured patients may have to pay cash for care until a designated amount is reached (the deductible)—which could be thousands of dollars. In addition, patients are often required to pay a dollar amount (co-payment) or a percentage of the charges (coinsurance) each time services are received.
Insurers may also exclude specified services from coverage, such as maternity benefits or mental health services. Most insurance plans now use lists of contracted physicians and hospitals, and impose severe financial penalties for using health care providers that are not contracted. All of these measures reduce the value of insurance by shifting costs from the insurers to the patients who actually need care.
Inadequate insurance coverage is making average-income Americans poorer. A recent study found that for 29% of individuals who had average or greater-than-average incomes and were continually insured, medical bills had caused significant financial problems [6]. For those who were not continuously insured, the percentages were even higher. These financial barriers are impairing access to beneficial services. The United States insurance market is now dominated by insurance plans that provide neither adequate health security nor financial security.
Does Higher Health Spending Mean Better Quality?
There is a widespread belief that the high spending in the United States means that high quality care is being delivered to the majority, who can afford both comprehensive coverage and the attendant out-of-pocket expenses. But international comparisons of industrialized nations have shown that the United States is in the bottom quartile of population health indicators such as life expectancy and infant mortality [7]. And in regional comparisons within the United States, increased levels of spending have not produced a commensurate improvement in health care outcomes. In fact, a recent study found that in a state-by-state comparison, there is an inverse relationship between spending and quality outcomes—the more expenditure, the worse the quality [8].
In 2000, the World Health Organization rated the United States first in its health expenditures per capita, but 37th in its overall health system performance, below most industrialized nations [9]. The United States is clearly not receiving adequate value for its health care investment.
Some contend that the poor performance of the United States system is due to the funding of health care in the private sector, and that all would be well if the government would just take over funding. But it is not quite that simple. The greater part of health care in the United States—59%—is already funded by the tax system. On a per capita basis, the public, taxpayer-funded health care expenditures alone total more than the health care spending of every other nation's public and private funding combined (with the exception of Switzerland, in which total spending per capita equals our public spending alone) [10].
Flaws in Funding and Allocation
How can the United States spend as much as it does and end up with such mediocre health care? Of the many reasons that exist, two are particularly important. The United States has a highly flawed system of funding health care and a flawed system of allocating its health care resources.
In the United States, a multitude of private health plans cover the lucrative sector of society—low cost, healthy workers and their healthy families. But public programs must cover the higher costs of the elderly, individuals with permanent disabilities, and some low-income individuals. Since the uninsured are frequently unable to pay for the care they receive, the costs for their care are shifted to government programs or private plans, or to the charity of providers, even if unintended. The costly administrative excesses of private health plans, especially when contrasted to government programs, have been well documented [11]. This fragmented system of funding care places an even greater administrative and financial burden on the providers of health care. Although the exact amount is disputed, most policy analysts agree that replacing this fragmented system of funding care with a single, universal, publicly administered insurance program could recover 200 billion dollars or more, which are currently being wasted on useless and sometimes detrimental administrative services [11].
And what is wrong with the way that the United States allocates its resources? Many studies have confirmed that supporting a strong primary care base provides better outcomes at a lower cost [12]. But in the United States, specialized, high-technology care is heavily marketed, and providers of that high-tech care are rewarded more generously than primary care professionals. Yet studies show that these greater expenditures result in no additional benefit—and sometimes even in worse outcomes [8,13]. Excessive resources are allocated to inappropriate expansion of high-tech facilities and to training an excessive number of specialists to provide high-tech services [8,13].
Health Care Reform
What has been the response to these deficiencies in the United States health care system? In the 1990s, the Clinton administration attempted to introduce a comprehensive system of funding universal health care. The system would have used marketplace principles in a program of managed competition, but their complicated idea pleased no one, and it was never even brought to a vote. Because of this miserable political failure, policymakers decided that any comprehensive approach should be avoided, and that reform must take place in incremental steps. To date, with the notable exception of the State Children's Health Insurance Program (http://www.cms.hhs.gov/schip), the accomplishments of these incremental health reform measures have been unimpressive.
Over the past decade, those interested in reform have been preoccupied with managed care measures and, more recently, with consumer-directed measures that increase costs to patients by requiring greater out-of-pocket spending. But these measures are designed more to control costs than to increase coverage and access. In the debate on universal coverage, three general concepts have been put forward: (1) the expansion of our current system of public and private programs, (2) the establishment of a national health service with government ownership of the system, or (3) the replacement of all current funding with a single, publicly administered, publicly funded program of social insurance that does not alter the existing ownership status of the delivery system.
The greatest political support today is for incremental expansions of our current programs, which, theoretically, would eventually result in universal coverage. There are innumerable variations of this approach. Most would increase the affordability of insurance premiums for private group and individual plans by providing financial assistance through tax policies and by modifying the benefits and coverage of the plans. Some policy analysts recommend that employers be mandated to offer coverage to their employees. Others recommend that individuals be required to purchase their own coverage. Since some individuals would be left without coverage, a public program, such as the existing Medicaid program for low-income individuals, would be used to cover everyone else. Many simulation studies have shown that these approaches could be effective in covering almost everyone, but they are the most expensive models of reform since they leave in place the administrative excesses of the fragmented system of funding care [14]. Also, to keep premiums affordable, these approaches may fall short on comprehensiveness of coverage and on the affordability of the out-of-pocket component, especially for those individuals with greater health care needs.
In contrast, simulation of both the national health service and public social insurance models of reform have shown that they would provide truly comprehensive benefits for everyone, and that they are the least expensive models [14]. By integrating funding with the health care delivery system, both models are well suited for the introduction of an integrated information technology system. Such a system would provide invaluable data to assist with decisions on resource allocation, enabling incentives to be established that would strengthen the primary care base. It would also improve capacity planning for high-tech and specialized services, thereby ensuring appropriate access without excessive queues [15].
Even with insurance, a serious health crisis can lead to major debt or bankruptcy
(Illustration: Rusty Howson, sososo design)
The political threshold for adopting a government-owned health service model in the United States is very high, since most citizens fear the specter of “socialized medicine.” In contrast, the Medicare program, an insurance program for the retired and for those with long-term disabilities, is very popular. There is an increasing public perception that we may need to accept a greater government role in health insurance if we are to adequately address the deteriorating status of our health care system. Correcting the flaws in Medicare and then using the program to cover everyone may be a concept that can gain political traction in the United States.
Conclusion
Our political process is currently dominated by those who are enticed by the siren song of the market theorists and turn a deaf ear to the health policy scientists who plead for health care justice. The debate needs to focus on defining the best role for government in ensuring that people receive the best health care value. That debate needs to be guided by a thorough understanding and diligent application of sound health policy science.
The continuing deterioration of affordability, coverage, and quality in health care makes it imperative that United States policymakers broaden their reform efforts beyond the ineffectual tinkering of incrementalism. A universal, single-payer, publicly funded and publicly administered program of social insurance would ensure access to affordable, comprehensive, high-quality health care for all. It should be the standard by which any other proposals are judged. If a better proposal can be crafted, now is the time to do it. People are dying while we delay.
Citation: McCanne DR (2004) A national health insurance program for the United States. PLoS Med 1(2): e39.
Abbreviation
(AMA)American Medical Association
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Himmelstein D Woolhandler S A national health program for the United States N Engl J Med 1989 320 102 108 2911282
Woolhandler S Himmelstein D Proposal of the Physicians' Working Group for single-payer national health insurance JAMA 2003 290 798 805 12915433
Institute of Medicine [IOM] Care without coverage: Too little, too late 2002 Washington (DC) The National Academies Press 212
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Collins SR Doty MM Davis K Schoen C Holmgren AL The affordability crisis in U. S. health care: Findings from the Commonwealth Fund Biennial Health Insurance Survey 2004 New York The Commonwealth Fund Available: http://www.cmwf.org/publications/publications_show.htm?doc_id=221454 . Accessed 23 September 2004
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| 15578108 | PMC529426 | CC BY | 2021-01-05 10:38:01 | no | PLoS Med. 2004 Nov 30; 1(2):e39 | utf-8 | PLoS Med | 2,004 | 10.1371/journal.pmed.0010039 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1557810910.1371/journal.pmed.0010041PerspectivesHIV/AIDSHIV Infection/AIDSReconsidering Early HIV Treatment and Supervised Treatment Interruptions PerspectivesKoup Richard A Richard Koup is the director of the Human Immunology Program for the Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America. E-mail: [email protected]
Competing Interests: The author declares that no competing interests exist.
11 2004 30 11 2004 1 2 e41This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose2004This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Limited Durability of Immune Control Following Treated Acute HIV Infection
Another study casts doubt on the value of early treatment and treatment interruptions. What are the implications of this study for our understanding of HIV pathogenesis, treatment, and vaccine development?
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The devastating effects of HIV infection worldwide are reason enough for AIDS researchers to grasp at thin rays of hope. But seldom has a single anecdotal case stimulated as much hope as the 1999 report of an acutely infected patient who appeared to control HIV replication after two short treatment interruptions [1]. This report generated the hypothesis that early antiretroviral treatment (during or very soon after symptomatic seroconversion) allows the incompletely damaged immune system to recover and respond appropriately to virus antigens during treatment interruptions. This, in turn, according to the hypothesis, leads to control of viral replication by a healed and appropriately stimulated immune response to the patient's HIV infection.
Consistent with this hypothesis was the prior finding that early antiretroviral therapy led to induction of HIV-specific proliferative responses similar to those that had been observed in patients with long-term, non-progressing HIV [2]. This led Rosenberg and colleagues to ask whether HIV-specific proliferative responses were a necessary and sufficient cause of long-term non-progression or just an immunologic consequence of controlled virus replication. Their report of virologic control in patients who interrupted therapy after early treatment raised hope that if HIV infection was treated early enough, the immune system could be repaired sufficiently to allow for long-term immunologic control of HIV replication [3]. Unfortunately, that's where the good news ends.
Enthusiasm Fades
A series of discoveries from clinical trials began to chip away at the enthusiasm for both early treatment of HIV infection and supervised treatment interruptions (STIs) as a way to boost the immune response.
Several small trials of STIs in chronically infected patients were carried out [4], buoyed by the reasonable desire of patients for respite from the unpleasant side effects of the drugs. These trials gave disappointing results, up to and including the emergence of antiretroviral drug resistance in patients randomized to receive STIs. HIV-specific immune responses did increase off therapy, but so did viral loads. The so-called immune boosting probably reflected an immune response to greater viral antigen load but did not represent constructive immune enhancement.
Larger trials clearly showed that STIs were of little if any benefit in chronic infection and that when therapy was stopped, viral loads invariably returned to pre-treatment levels [5]. Other studies indicated that HIV-specific CD4+ T cells were being preferentially infected, often massively, during treatment interruptions [6], and that proliferative responses were more likely to be a consequence—rather than a cause—of decreased HIV replication [7]. Despite multiple attempts, early reports of an inverse correlation between simple HIV-specific T cell responses and virologic control were not confirmed [8]. Where complex T cell functions did show such a correlation, the data indicated that viral replication was adversely affecting the character of the T cell immune response to HIV, and not the other way around [9]. Thus, no evidence of “immune boosting” during STIs and subsequent viral control in the absence of antiretroviral drugs was ever established. Finally, one of the acutely treated patients within Rosenberg's cohort became superinfected with a second strain of HIV despite excellent control of viral replication and significant recognition of the superinfecting strain by the pre-existing T cell response [10].
STIs offered patients hope of respite from taking complex regimens, but trials have been disappointing
(Photo: J Troha)
New Findings
Now comes a study in this month's PLoS Medicine that found that in 14 patients who were treated early and who had controlled viral loads for at least 90 days, the virologic control was only transient [11]. While one could look at this as a glass half full—these patients achieved a reasonable period of time off antiretroviral therapy—closer scrutiny of the data limits this view.
There was a disconnect between the low viral loads and an unexpectedly high rate of CD4+ T cell decline in several patients. While the small number of patients and the single-arm nature of the study preclude definitive comparisons, it is possible that the early treatment and STIs did not result in a delay in CD4+ T cell decline (and, therefore, initiation of antiretroviral therapy) beyond what would have occurred had the patients received no early treatment.
Implications of the Study
This study raises important questions in our understanding of HIV pathogenesis, treatment, and vaccine development.
First, why is it that early antiretroviral treatment, even if it does lead to better control of viral replication, does not protect against CD4+ T cell depletion? It is possible that by the time patients present with acute retroviral syndrome their CD4+ T cell reserves (in gut and lymphoid tissues) have been severely depleted, despite the fairly normal CD4+ profile of their peripheral blood. Thus, even low-level viral replication is then sufficient to deplete the remaining central and peripheral reserves [12].
Second, how do these findings affect treatment guidelines during acute infection? None of the current treatment guidelines in either resource-rich or resource-poor settings recommend early antiretroviral therapy. In the light of these new data [11], there does not appear to be a rationale for early antiretroviral therapy in the absence of a clinical trial to assess other interventions in concert with early therapy. The use of therapeutic vaccination is an obvious intervention that still needs to be tested, despite limited efficacy results in treated chronic infection. As such, practice guidelines should continue to caution against early treatment unless associated with a randomized clinical trial.
Finally, is this good or bad news for HIV vaccine development? Since most current vaccine strategies are based upon the hypothesis that induction of T cell immunity will lead to control of viral replication, it is difficult to be optimistic when a strong and broad immune response is unable to prevent disease progression. However, one must recall that phenotypic and functional assessments of HIV-specific T cell responses, even in antiretroviral-treated patients, show that these responses clearly differ from responses against viruses that are normally cleared or controlled by the immune system [9]. Therefore, the T cell responses in the patients treated for acute HIV infection in Kaufmann et al.'s study were induced upon a dramatically altered immune background. It remains to be determined how much this adversely affects the HIV-specific immune response, and whether an immune response generated by vaccination before any HIV replication (a prophylactic vaccine) might be better able to control virus replication. Far be it for us to stop grasping at rays of hope.
Citation: Koup RA (2004) Reconsidering early HIV treatment and supervised treatment interruptions. PLoS Med 1(2): e41.
Abbreviation
STIsupervised treatment interruption
==== Refs
References
Lisziewicz J Rosenberg E Lieberman J Jessen H Lopalco L Control of HIV despite the discontinuation of antiretroviral therapy N Engl J Med 1999 340 1683 1684 10348681
Rosenberg ES Billingsly JM Caliendo AM Boswell SL Sax PE Vigorous HIV-1-specific CD4+ T cell responses associated with control of viremia Science 1997 278 1447 1450 9367954
Rosenberg ES Altfeld M Poon SH Phillips MN Wilkes BM Immune control of HIV-1 after early treatment of acute infection Nature 2000 407 523 526 11029005
Montaner LJ Structured treatment interruptions to control HIV-1 and limit drug exposure Trends Immunol 2001 22 92 96 11286710
Oxenius A Price DA Gunthard HF Dawson SJ Fagard C Stimulation of HIV-specific cellular immunity by structured treatment interruption fails to enhance viral control in chronic HIV infection Proc Natl Acad Sci U S A 2002 99 13747 13752 12370434
Douek DC Brenchley JM Betts MR Ambrozak DR Hill BJ HIV preferentially infects HIV-specific CD4+ T-cells Nature 2002 417 95 98 11986671
McNeil AC Shupert WL Iyasere CA Hallahan CW Mican JA High-level HIV-1 viremia suppresses viral antigen-specific CD4(+) T cell proliferation Proc Natl Acad Sci U S A 2001 98 13878 13883 11717444
Betts MR Ambrozak DR Douek DC Bonhoeffer S Brenchley JM Analysis of total HIV-specific CD4+ and CD8+ T cell responses: Relationship to viral load in untreated HIV infection J Virol 2001 75 11983 11991 11711588
Harari A Petitpierre S Vallelian F Pantaleo G Skewed representation of functionally distinct populations of virus-specific CD4 T cells in HIV-1-infected subjects with progressive disease: Changes after antiretroviral therapy Blood 2004 103 966 972 12958069
Altfeld M Allen TM Yu XG Johnston MN Agrawal D HIV-1 superinfection despite broad CD8+ T-cell responses containing replication of the primary virus Nature 2002 420 434 439 12459786
Kaufmann DE Lichterfeld M Altfeld M Addo MM Johnston MN Limited durability of viral control following treated acute HIV infection PLoS Med 2004 2 e036
Douek DC Picker LJ Koup RA T cell dynamics in HIV-1 infection Annu Rev Immunol 2003 21 265 304 12524385
| 15578109 | PMC529427 | CC0 | 2021-01-05 10:38:00 | no | PLoS Med. 2004 Nov 30; 1(2):e41 | utf-8 | PLoS Med | 2,004 | 10.1371/journal.pmed.0010041 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1557811010.1371/journal.pmed.0010042EssayOtherPharmacology/Drug DiscoveryClinical PharmacologyEpidemiology/Public HealthHealth PolicyDrugs and adverse drug reactionsRegulationCommunication in Health CareHealth PolicyMedical journalsThe Intangible Magic of Celebrity Marketing EssayMoynihan Ray Ray Moynihan is an investigative reporter in Washington, District of Columbia, United States of America. His book Selling Sickness, coauthored with Alan Cassels, will be published in 2005. E-mail: [email protected]
Competing Interests: The author declares that he has no competing interests.
11 2004 30 11 2004 1 2 e42Copyright: © 2004 Ray Moynihan.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Drug industry insiders share their tips on using celebrities to market drugs and diseases
Drug industry insiders share their tips on using celebrities to expand markets
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As the Hispanic world well knows, the word in Spanish for advertising is ‘propaganda’, its meaning derived literally from the propagation of the faith, the antithesis of science's Enlightenment ideals. The old word somehow seems perfect for describing the new world of drug promotion and its growing use of the famous face. Like the catholic cardinals of the 17th century, many of the feted celebrities of the 21st are now engaged in spreading the word. Now, as then, the religion promises miraculous breakthroughs, wonder cures, and sometimes even eternal life. The difference is that this time around, the stars are earning fat fees from the marketing departments of giant pharmaceutical companies. And if the latest revelations from industry insiders are anything to go by, their hefty investments in celebrity selling are well worth it.
Celebrity Selling
The epicentre of this phenomenon is of course the United States, where companies routinely hire celebrities to attract attention to the latest drugs and the diseases that go with them. Pfizer famously paid presidential hopeful Bob Dole to promote awareness of erectile dysfunction as sildenafil (Viagra) was hitting the market. Wyeth hired supermodel Lauren Hutton to hawk hormone replacement therapy and menopause. GSK contracted football star Ricky Williams to sell social anxiety disorder, helping make paroxetine (Paxil)—briefly—the world's top-selling antidepressant. Even the dead are raising awareness, with the estate of Errol Flynn now enlisted to help promote cardiovascular disease as a household name [1]. The celebrity, living or dead, becomes integral to a drug marketing strategy that includes paid advertising and aggressive public relations campaigns that can produce media appearances on the likes of Oprah and The Today Show. According to celebrity brokers, the star's remuneration package, though always confidential, can range from $20,000 to $2 million.
Celebrities often hide their conflicts of interest
(Illustration: Margaret Shear)
‘A partnership between a celebrity and a brand has an intangible sort of magic’, writes a senior marketing executive at Amgen, in an extremely candid piece published recently in an industry trade magazine [2]. Amgen is the Californian biotech firm that hired handsome ‘West Wing’ star Rob Lowe to help market an anti-infection drug. Lowe was reportedly paid more than $1 million by Amgen, though there is speculation that part of the fee might flow to charity [3]. In her report, Amgen's Osnat Benshoshan shares some thoughtful tips with her peers among the pharmaceutical marketing fraternity: ‘use an A-list celebrity’; find a ‘news-hook’ that links the celebrity and your product; develop some simple messages; and make sure the celebrity delivers them at every appearance.
Benshoshan then reveals why on-air talk-show appearances on ‘top-tier media venues’ like The Rosie Show can be better forums for celebrities than straight advertisements, which are governed by regulations. ‘The great advantage over advertising is that the airtime is practically free, and there is no fair balance to worry about’ she writes [2]. The downside with a media interview, she laments, is that compared to a scripted ad, ‘the situation is less controllable. It can be tricky for the celebrity to ensure that all product messages are delivered….’ Her other big tip for drug-makers is to rate your prospective celebrity with a ‘Q score’, a measure of their likeability and recognisability with the public. Apparently Rob Lowe's Q score was high with women over fifty, a key target of the Amgen campaign [3].
Another recent report from within the industry draws on public opinion survey data to guide drug company marketers on the selection and ‘effective use’ of celebrity spokespersons [4]. The survey was conducted by a Seattle firm called NexCura Inc., in partnership with the trade magazine that published the study. The major findings echo the insights of the Amgen executive about credibility, and underline the importance of your star being perceived as generally trustworthy, and specifically knowledgeable about the condition on which they are hired to speak. Perhaps not surprisingly, the survey found that people diagnosed as suffering chronic conditions were far more attentive to celebrity messages on health than the general public.
The issue of credibility is important, the NexCura Inc. researchers point out, because ‘the credibility rating is used as a surrogate for “buying” behavior’—an intermediate measure of whether the star can persuade people to request the target drug from their doctor. The survey found that Bob Dole was still the most recognisable celebrity marketer with the United States public, but that the skater Dorothy Hamill—currently promoting Merck's arthritis medication rofecoxib (Vioxx)—took the lead in the credibility stakes. Significantly though, almost three-quarters of those surveyed were correctly able to identify Bob Dole with Pfizer's Viagra, despite the fact that the advertisements in which he appeared were ‘unbranded’ ads for erectile dysfunction. The researchers concluded by recommending that drug companies choose a celebrity with personal experience of the target condition; choose someone trustworthy—perhaps a newsreader or sports figure; and choose someone who will promote a single cause or brand rather than multiple ones.
Ironically, the NexCura survey also found two-thirds of medical consumers agreed with the proposition that celebrities were ‘just doing it for the money and can't be trusted’.
The Trouble with Celebrity Selling
The first problem here is that the public is often not even informed whether a celebrity is receiving money from a drug company. In the case of TV star Rob Lowe, there was no mandated requirement for him to disclose his link with Amgen when appearing on media shows watched by millions. According to one industry insider familiar with the case, who did not want to be named, ‘it depended if he remembered to say it, and whether he was asked’. The media's failure to disclose relevant conflicts of interest when covering healthcare is well established [5]. When Frasier star Kelsey Grammer and his wife were promoting irritable bowel syndrome on top-rating TV shows, viewers thought the pair were speaking on behalf of an independent foundation. In fact the couple's fee had flowed from GSK, which was at that time preparing the market for alosetron (Lotronex), a controversial new drug that carried modest benefits and severe side effects, including possible death [6].
Equally as serious is the lack of any formal requirement for stars or media outlets to spell out drug side effects along with benefits when celebrities are pushing products or conditions. Lauren Hutton can be quoted, in magazine articles read by millions of readers, as saying, ‘My No. 1 secret is estrogen’ without any need for her, or the magazine, to list the dangers of the hormone replacement therapy made by her sponsor [7]. But perhaps most troubling is the way celebrities, with their star power, can help to fundamentally shift the public debate about major health problems.
While Prince Charles's companion Camilla Parker Bowles takes no money from drug companies, she did choose to make an important public statement about the bone condition osteoporosis at an international conference funded by Lilly, a company promoting a medication for the condition [8]. Camilla's call for early intervention and greater use of expensive tests and technologies for the primary prevention of osteoporosis drew on materials sponsored by the pharmaceutical industry, and was synchronised with simplistic industry marketing messages. Camilla's high-profile intervention at a drug company sponsored forum, albeit unwittingly, helps keep the focus on biochemical causes of, and biochemical solutions to, the much wider public health problem of fractures. Moreover these simple marketing messages undermine the complexity of the cost-effectiveness arguments that are central to any rational debate about the equitable distribution of health care resources. Other high-profile figures attending the same conference eagerly accepted Lilly money, and one, former Texas Governor Ann Richards, blatantly promoted Lilly's drug during an interview on CNN's Larry King Show just days later [8].
The Future of Celebrity Selling
With pharmaceutical marketing, it is clear that nothing short of a Vatican II-style reform is required, though there are already encouraging signs of change. Scientific journals are slowly disentangling themselves from unhealthy industry influence over what they publish, and public access to clinical trial data is daily a closer reality [9]. However, a less distorted scientific record about healthcare products is meaningless without regulations on how important science is communicated to the public. Celebrities paid by drug companies to promote drugs, or ‘raise awareness’ about disease, should be subject to the same rules as direct-to-consumer advertising, which would mean prohibition in many nations and much more fulsome disclosure in the United States than is currently the case. At the very least, public disclosure of a product's risks and benefits, and the magnitude of the celebrity's fee, should be mandatory and routine. Let's see what that does to their Q rating.
Citation: Moynihan R (2004) The intangible magic of celebrity marketing. PLoS Med 1(2): e42.
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References
[Anonymous] Our Members Boomer Coalition 2004 Available: http://www.boomercoalition.org/bc3/members.asp . Accessed 26 September 2004
Benshoshan O Celebrity public relations: An alternative to DTC DTC Perspectives 2003 Available: http://www.dtcperspectives.com/content.asp?id?149 . Accessed 26 September 2004
Hamilton DP Celebrities help ‘educate’ public on new drugs Wall Street Journal 2002 April 22
NexCura and DTC Perspectives Effective use of celebrities in DTC promotions of pharmaceutical products DTC Perspectives 2003 2003 2
Moynihan R Bero L Ross-Degnan D Henry D Lee K Coverage by the news media of the benefits and risks of medications N Engl J Med 2000 342 1645 1650 10833211
Moynihan R Alosetron: A case study in regulatory capture, or a victory for patients' rights? BMJ 2002 325 592 595 12228140
[Anonymous] Celebrities reveal their secrets Parade Magazine 2000 March 19 10–12
Moynihan R Celebrity selling BMJ 2002 324 1342
Dickersin K Rennie D Registering clinical trials JAMA 2003 290 516 523 12876095
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1557811110.1371/journal.pmed.0010044Neglected DiseasesInfectious DiseasesOpthalmologyPrimary CareOphthalmologyInfectious DiseasesMicrobiologyMedicine in Developing CountriesPublic HealthBlinding Trachoma: A Disease of Poverty Neglected DiseasesKasi Pashtoon M Gilani Ahmed I Ahmad Khabir Janjua Naveed Z *All authors are at the Aga Khan University, Karachi, Pakistan. Pashtoon M. Kasi and Ahmed I. Gilani are students in the Medical College. Khabir Ahmad is a senior instructor in the Ophthalmology Section of the Department of Surgery and a research fellow in the Department of Community Health Sciences. Naveed Z. Janjua is a senior research fellow in the Department of Community Health Sciences.
*To whom correspondence should be addressed. E-mail: [email protected]
Competing Interests: The authors declare that they have no competing interests.
11 2004 30 11 2004 1 2 e44Copyright: © 2004 Kasi et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Trachoma accounts for 15% of blindness worldwide, affecting the world's poorest communities. How can the disease be controlled?
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Trachoma is almost exclusively a disease of poor families and communities living in developing countries. It accounts for 15% of blindness worldwide—around 6 million people [1]. Although the disease is avoidable, it continues to blind. With so few voices speaking out on behalf of people affected by trachoma, it remains a neglected public health issue.
Epidemiology
Trachoma, a chronic keratoconjunctivitis, is caused by episodes of infection with Chlamydia trachomatis, an obligate intracellular bacterium. Only serovars A, B, Ba, and C are implicated in trachoma.
Trachoma is the second leading cause of blindness worldwide [1]. According to the World Health Organization, currently 84 million people, mostly children, have active disease, and another 7.6 million people have trichiasis—a stage of trachoma in which the upper eyelid turns inward and one or more eyelashes rub against the eyeball [2]. An estimated 10% of the world's population lives in endemic areas and is at risk of developing trachoma. Global loss of productivity related to impaired vision and blindness from trachoma is thought to be as high as $US 5.3 billion annually [3]. More than 55 countries have been identified as endemic for trachoma, most of them in Africa and Asia (Figure 1) [4].
Figure 1 The Worldwide Distribution of Trachoma
(Map: Silvio Mariotti/WHO)
Humankind has known trachoma since antiquity. Ibn-e-Isa, an Arab physician, was the first person to describe the different stages of trachoma and noted trichiasis as one of its sequelae. So prevalent was the disease not so long ago that trachoma hospitals were established in many parts of Europe and America [5]. The disease then declined dramatically in what is now called the developed world, mainly because of socioeconomic development [4].
Transmission occurs from eye to eye via hands, clothing, and other fomites. Flies have been identified as a major vector for the infection's spread [6]. The presence of open latrines favors the vector population (Figure 2). Factors associated with trachoma include the extent to which the water supply is limited, the distance from the water source, the amount of water used for washing purposes, and overcrowding [7]. One case-control study in a Gambian village compared water use in 18 families having one or more active trachoma cases among the children with that in 16 trachoma-free families in the same village. The families with trachoma were found to use significantly less water per person per day for washing children than did the control group [8].
Figure 2 A Typical Community in Which Trachoma Is Endemic
Some of the factors linked with the continued presence of the disease in affected communities are lack of access to water, overcrowding, lack of facial hygiene, eye-seeking bazaar flies, and open latrines. (Illustration: Aslam Bashir, Aga Khan University)
The disease tends to cluster in certain communities within a village and certain families within a neighborhood. Women, especially in rural areas, are affected twice as often as men [9].
Clinical Manifestations and Grading
Trachoma initially presents in childhood as red eye—itching, redness, and pain. The essential lesion is a trachomatous follicle (lymphoid cell aggregate) occurring typically in the upper tarsal conjunctiva (Figure 3). The roughened appearance of the upper tarsal conjunctiva gives the disease its name (trachoma is the Greek word for “rough”). Trachomatous involvement of the cornea manifests itself initially as superficial keratitis. At a later stage, pannus formation (new vessel growth) may occur over the margin of the cornea, usually limited to the upper half.
Figure 3 WHO Simplified Grading System: A Guide for the Assessment of Trachoma
(Photos: from [11], with permission from WHO)
In a subgroup of individuals, fibrosis occurs because of repeated infections, resulting in scarring of the conjunctiva (scarring trachoma). In scarring trachoma, the upper eyelid is shortened and distorted (entropion) and the lashes abrade the eye (trichiasis). Blindness results from corneal opacification, which is related to the degree of entropion or trichiasis [10].
Based on the presence or absence of some of the key signs of the disease, WHO has developed a simplified grading system for the assessment of trachoma (Figure 3) [11,12]. The system can easily be used by non-specialists, after appropriate training, for the assessment of disease at the community level.
Herbert's pits (healed follicles in the superior limbus) and Arlt's line (a horizontal scar on the upper tarsal conjunctiva) are two other classical features of the disease.
Managing Trachoma: The SAFE Strategy
WHO currently recommends the “SAFE” strategy for the management of trachoma: Surgery for trichiasis, Antibiotics for active disease, Facial hygiene, Environmental improvement to reduce the transmission of the disease [13,14,15].
Surgery.
People with trachomatous trichiasis are at risk of blindness, and so treating these people is the first priority for the SAFE strategy. An evidence-based review of the SAFE strategy found that trichiasis surgery can alleviate discomfort and improve vision, though the evidence is less clear on whether such surgery prevents corneal opacification [14]. The review authors suggested that a protective effect of surgery against opacification is likely.
There are different types of surgical procedures to correct trachomatous trichiasis [16]. Their high costs and the lack of surgical expertise in endemic regions, however, restrict the use of many of these as public health interventions. On the basis of a controlled trial by Reacher and colleagues [16], WHO recommends the bilamellar tarsal rotation procedure as the preferred technique; it is easy to perform and easy to learn [17]. Surgical effectiveness is defined in terms of recurrence of trichiasis; in the controlled trial, bilamellar tarsal rotation produced a recurrence rate of around 20% at follow-up 9–21 months after surgery, while other procedures saw 60% of patients with recurrence of trichiasis in the same period [16]. In several countries, different levels of health staff, including nurses and ophthalmic assistants, have been trained to perform the bilamellar tarsal rotation procedure.
In addition to recurrence, there are other problems with the surgical approach to managing trachoma. It cannot correct all the complications, such as dry eyes. Even more important, and the main obstacle to preventing blindness from trachoma, is the low rate of uptake of surgery by communities with trachomatous trichiasis [14]. Barriers to uptake include distance to travel to surgery, perceived cost of the operation, child care duties, and lack of awareness about the treatment [14]. In Tanzania, less than a fifth of women with trichiasis opted for surgery, even when it was offered for free [18].
Offering surgery at the community level, rather than in distant medical facilities, is one strategy that could reduce travel times and costs and increase uptake. A cluster randomized controlled trial of village-based surgery versus health-center-based surgery in Gambia found a significantly higher uptake rate with the village-based service [19].
Antibiotics.
The use of antibiotics aims to treat active infection and eliminate the reservoir. WHO currently recommends two regimens for the treatment of trachoma in endemic regions. These are 1% topical tetracycline ointment (twice daily for six weeks) or a single dose of oral azithromycin (1 g in adults and 20 mg/kg in children) [20].
Although antibiotics are a cornerstone of the SAFE strategy, clinical trials of antibiotics versus control (no treatment, placebo, or vitamin tablets) have produced conflicting results and are difficult to pool because of their heterogeneity. A recent Cochrane systematic review concluded “there is some evidence that antibiotics reduce active trachoma but results are not consistent and cannot be pooled” [20]. The review also found that “oral treatment is neither more nor less effective than topical treatment” [20].
Several questions remain about the use of antibiotics, such as who should receive them and how often. Lietman and colleagues have developed a mathematical model of frequency of treatment that uses available epidemiological data from a variety of countries [21]. Based on their model, they recommend that in areas where trachoma is moderately prevalent (less than 35% of children with active infection), it should be treated annually, but in hyperendemic areas (more than 50% of children with active infection), it should be treated biannually. Such models, however, need to be validated by well-designed clinical trials.
Facial hygiene.
Good facial hygiene aims to reduce transmission, the risk of autoinfection in a community, and the risk of attracting flies [13,15]. Many cross-sectional surveys have shown that children with clean faces are less likely to have trachoma, and are less likely to have severe trachoma [14]. A recent study in Mali found dirtiness of the face to be the most important risk factor associated with trachoma [22].
A Cochrane systematic review found evidence that face washing combined with topical tetracycline can be effective in reducing severe active trachoma [23]. However, the evidence does not support a beneficial effect of face washing alone or in combination with topical tetracycline in reducing non-severe active trachoma [23].
Interventions aimed at promoting facial hygiene have not yielded expected results in all settings, as behavioral change is not always readily achievable.
Environmental improvement.
This component of the SAFE strategy also aims to reduce transmission of trachoma by eliminating or reducing its risk factors, some of which are ubiquitous while others are specific to a region. Improving access to water is a key element. Other measures, such as provision of latrines to reduce the fly population, have also been found effective in reducing transmission [6]. Such environmental improvements will also provide other health benefits to a community, such as reduction in the incidence of diarrhea.
As mentioned previously, there is an important association between water and trachoma—though the association is not a simple one. The distance to the water source constrains the amount of water used for hygiene practices. Improving access to water on its own, however, may not be enough. In the case-control study in Gambia, families with trachoma used less water per person per day for washing children than families without the disease, regardless of the amount of water available [8]. In other words, interventions aimed at increasing the availability of water should also promote its appropriate use. Getting community “buy in” for these interventions is important.
Why Is Trachoma So Neglected?
Trachoma is a disease of poor, underprivileged, and socioeconomically disadvantaged communities. It affects people who have little or no say in public decision making [24]. Investing in trachoma may sometimes mean compromising on other important issues. Many countries in which trachoma is endemic are also marred by regional conflicts, civil wars, and widespread corruption. Scarce resources are being spent on arms and debt servicing. These countries often lack the political commitment needed to fight against the disease. In addition, there is a lack of commitment by international donors.
Still, there is some room for optimism, given WHO's vision of the global elimination of trachoma by the year 2020 and the efforts of the International Trachoma Initiative and other non-governmental organizations. The implementation of the SAFE strategy to eliminate blinding trachoma has already proven effective in several countries [25]. Many countries have already started trying to eliminate trachoma themselves.
Future Directions
Although the new initiatives in trachoma control are encouraging, trachoma elimination programs clearly need to be extended to many more countries. In addition, there are three crucial steps that still need to be undertaken if blindness from trachoma is to be eliminated.
First, there needs to be more emphasis on the “F” and “E” components of the SAFE strategy. Antibiotics and surgery alone will not eliminate trachoma; work also needs to be done to eliminate the risk factors and decrease the transmission of the disease in affected communities. Such primary prevention is more likely to have a sustainable impact but requires a prolonged effort and investment [15,24]. Because elimination of trachoma requires improvement in education and hygiene practices, improved accessibility to water, and economic development of endemic regions, collaboration among departments and ministries is vital. An example of such collaboration is the recent involvement of the Water Supply and Sanitation Collaborative Council (www.wsscc.org) in trachoma control efforts. Similar partnerships need to be strengthened [25]. Socioeconomic development must be at the heart of control efforts—trachoma was eradicated from much of the developed world even before the advent of antibiotic programs for trachoma, and much of this eradication was attributable to socioeconomic development [26].
Second, research into different aspects of the disease should continue. Work on a vaccine for trachoma, although not successful thus far, should receive more attention [10]. Future research should look at risk factors for trachoma in diverse communities and at barriers to implementation of the SAFE strategy.
Third, awareness about the disease and the SAFE strategy need to be promoted globally. At the same time, local, cost-effective solutions to trachoma need to be encouraged. Provision of pit latrines to reduce fly populations is just one such measure [6].
Unless these steps are taken, trachoma will continue to be a major cause of blindness in communities in the developing world [24].
We are deeply indebted to Aslam Bashir, medical illustrator at Aga Khan University, for his work on the figures and tables, and to Joseph Cook for helping us obtain some of the relevant literature. Thanks are also due to WHO for graciously giving us the permission to reproduce some of the figures. Sylvio Mariotti at WHO provided us with the map of trachoma distribution and the latest figures on disease epidemiology. Thanks to Riaz Baloch (Bolan Medical College, Quetta, Pakistan) for sharing his clinical experience with trachoma in Pakistan and to Masoom Kassi, Ali Zaidi, Fahd Anzar, and Talha Khawar for their helpful suggestions.
Citation: Kasi PM, Gilani AI, Ahmad K, Janjua NZ (2004) Blinding trachoma: A diseaseof poverty. PLoS Med 1(2): e44.
Abbreviation
WHOWorld Health Organization
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References
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Frick KD Hanson CL Jacobson GA Global burden of trachoma and economics of the disease Am J Trop Med Hyg 2003 69 1 10
Whitcher JP Srinivasan M Upadhyay MP Corneal blindness: A global perspective Bull World Health Organ 2001 79 214 221 11285665
Mecaskey JW Knirsch CA Kumaresan JA Cook JA The possibility of eliminating blinding trachoma Lancet Infect Dis 2003 3 728 734 14592604
Emerson PM Lindsay SW Alexander N Bah M Dibba SM Role of flies and provision of latrines in trachoma control: Cluster-randomised controlled trial Lancet 2004 363 1093 1098 15064026
Prost A Negrel AD Water, trachoma and conjunctivitis Bull World Health Organ 1989 67 9 18 2650903
Bailey R Downes B Downes R Mabey D Trachoma and water use; a case control study in a Gambian village Trans R Soc Trop Med Hyg 1991 85 824 828 1801366
Regassa K Teshome T Trachoma among adults in Damot Gale District, South Ethiopia Ophthalmic Epidemiol 2004 11 9 16 14977493
West SK Trachoma: New assault on an ancient disease Prog Retin Eye Res 2004 23 381 401 15219874
Thylefors B Dawson CR Jones BR West SK Taylor HR A simple system for the assessment of trachoma and its complications Bull World Health Organ 1987 65 477 483 3500800
Munoz B West S Trachoma: The forgotten cause of blindness Epidemiol Rev 1997 19 205 217 9494783
Bailey R Lietman T The SAFE strategy for the elimination of trachoma by 2020: Will it work? Bull World Health Organ 2001 79 233 236 11285668
Kuper H Solomon AW Buchan J Zondervan M Foster A A critical review of the SAFE strategy for the prevention of blinding trachoma Lancet Infect Dis 2003 3 372 381 12781509
Emerson PM Cairncross S Bailey RL Mabey DC Review of the evidence base for the ‘F’ and ‘E’ components of the SAFE strategy for trachoma control Trop Med Int Health 2000 5 515 527 10995092
Reacher MH Munoz B Alghassany A Daar AS Elbualy M A controlled trial of surgery for trachomatous trichiasis of the upper lid Arch Ophthalmol 1992 110 667 674 1580842
Reacher M Foster A Huber J Trichiasis surgery for trachoma: The bilamellar tarsal rotation procedure Programme for the Prevention of Blindness 1993 Geneva World Health Organization Available: http://whqlibdoc.who.int/hq/1993/WHO_PBL_93.29.pdf . Accessed 28 September 2004
Oliva MS Munoz B Lynch M Mkocha H West SK Evaluation of barriers to surgical compliance in the treatment of trichiasis Int Ophthalmol 1997 21 235 241 9700012
Bowman RJ Soma OS Alexander N Milligan P Rowley J Should trichiasis surgery be offered in the village? A community randomised trial of village vs. health centre-based surgery Trop Med Int Health 2000 5 528 533 10995093
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Lietman T Porco T Dawson C Blower S Global elimination of trachoma: How frequently should we administer mass chemotherapy? Nat Med 1999 5 572 576 10229236
Schemann JF Sacko D Malvy D Momo G Traore L Risk factors for trachoma in Mali Int J Epidemiol 2002 31 194 201 11914321
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| 15578111 | PMC529429 | CC BY | 2021-01-05 10:38:00 | no | PLoS Med. 2004 Nov 30; 1(2):e44 | utf-8 | PLoS Med | 2,004 | 10.1371/journal.pmed.0010044 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1557811210.1371/journal.pmed.0010045Research ArticleAllergy/ImmunologyDiabetes/Endocrinology/MetabolismObesityDiabetesImmunology and AllergyIschemic Heart DiseaseAn Inflammatory Cascade Leading to Hyperresistinemia in Humans Inflammation and Resistin in HumansLehrke Michael
1
Reilly Muredach P
2
3
Millington Segan C
1
Iqbal Nayyar
1
Rader Daniel J
2
3
Lazar Mitchell A
1
*1Divisions of Endocrinology, Diabetesand Metabolism, University of Pennsylvania School of Medicine, Philadelphia, PennsylvaniaUnited States of America2Cardiology Department of Medicine, University of Pennsylvania School of MedicinePhiladelphia, PennsylvaniaUnited States of America3Center for Experimental Therapeutics and Penn Diabetes Center, University of Pennsylvania School of MedicinePhiladelphia, PennsylvaniaUnited States of AmericaScherer Philipp Academic EditorAlbert Einstein College of MedicineUnited States of America
Competing Interests: MPR has received research funding or honoraria from GlaxoSmithKline, Merck, Ely Lilly, and KOS Pharmaceuticals. MAL has received a research grant from GlaxoSmithKline and has a United States patent application pending for therapeutic antagonism of human resistin. MAL and the University of Pennsylvania have licensed to Linco the monoclonal antibodies used in the human resistin assay.
Author Contributions: ML, MPR, NI, DJR, and MAL designed the study. ML, MPR, and SCM performed experiments. ML, MPR, NI, and MAL analyzed the data. MPR and NI enrolled patients. ML, MPR, NI, DJR, and MAL contributed to writing the paper. MAL obtained funding for the study and provided the research environment where the studies were conducted.
*To whom correspondence should be addressed. E-mail: [email protected] 2004 30 11 2004 1 2 e4513 5 2004 28 9 2004 Copyright: © 2004 Lehrke et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Resistin Response to Inflammation
Background
Obesity, the most common cause of insulin resistance, is increasingly recognized as a low-grade inflammatory state. Adipocyte-derived resistin is a circulating protein implicated in insulin resistance in rodents, but the role of human resistin is uncertain because it is produced largely by macrophages.
Methods and Findings
The effect of endotoxin and cytokines on resistin gene and protein expression was studied in human primary blood monocytes differentiated into macrophages and in healthy human participants.
Inflammatory endotoxin induced resistin in primary human macrophages via a cascade involving the secretion of inflammatory cytokines that circulate at increased levels in individuals with obesity. Induction of resistin was attenuated by drugs with dual insulin-sensitizing and anti-inflammatory properties that converge on NF-κB. In human study participants, experimental endotoxemia, which produces an insulin-resistant state, causes a dramatic rise in circulating resistin levels. Moreover, in patients with type 2 diabetes, serum resistin levels are correlated with levels of soluble tumor necrosis factor α receptor, an inflammatory marker linked to obesity, insulin resistance, and atherosclerosis.
Conclusions
Inflammation is a hyperresistinemic state in humans, and cytokine induction of resistin may contribute to insulin resistance in endotoxemia, obesity, and other inflammatory states.
Inflammatory stimuli affect resistin expression in human macrophages and raise serum resistin levels in healthy volunteers
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Introduction
Dietary and lifestyle changes during the last century have entailed an unprecedented epidemic of obesity and associated metabolic diseases, including type 2 diabetes and atherosclerosis [1]. Many individuals suffer simultaneously from more than one of these conditions, and epidemiological studies in humans, as well as studies in animal models, suggest that obesity-related insulin resistance is a common pathogenic feature [2]. Indeed, insulin resistance is the keystone of the “metabolic syndrome,” a major cardiovascular risk factor even in the absence of demonstrable glucose intolerance or diabetes [3]. Obesity and insulin resistance are strongly associated with systemic markers of inflammation, and, indeed, inflammation may contribute to insulin resistance [4]. Similarities and overlap between obesity and inflammatory states are emerging. Inflammatory cytokines such as tumor necrosis factor α (TNF α) and interleukin (IL)-6 are produced by adipocytes as well as by monocytes and macrophages, and they circulate at increased levels in individuals with obesity [5,6]. Moreover, bone-marrow-derived macrophages home in on adipose tissue in individuals with obesity [7,8], and adipocytes and macrophages may even be interconvertible [9]. Furthermore, inflammation is increasingly recognized as a major component and predictor of atherosclerotic vascular disease, a major clinical consequence of insulin resistance [10]. Hence, the interrelationships between obesity, insulin resistance, and atherosclerosis are of great scientific and clinical interest.
We originally identified and characterized resistin as a circulating mouse adipocyte gene product that is regulated by antidiabetic drugs [11]. In rodents, resistin is derived exclusively from adipocytes [11,12], circulates at increased levels in obese animals [11], and causes dysregulated hepatic glucose production, leading to insulin resistance [13,14]. A syntenic gene exists in humans, but is expressed at higher levels in monocytes and macrophages than in adipocytes [15,16], raising questions about the relationship between resistin and human metabolic disease. Recently, several studies have suggested that metabolic abnormalities are associated with polymorphisms in the human resistin gene [17,18]. Furthermore, several studies, though not all, have reported increased serum resistin levels in patients with obesity, insulin resistance, and/or type 2 diabetes [19,20,21,22,23,24,25,26]. However, the mechanism and importance of increased resistin levels in human metabolic disease are not known.
Here we show that the endotoxin lipopolysaccharide (LPS), a potent inflammatory stimulant, dramatically increases resistin production by inducing secretion of inflammatory cytokines such as TNFα. This increase in resistin production is blocked by both aspirin and rosiglitazone, drugs that have dual anti-inflammatory and insulin-sensitizing actions and have been shown to antagonize NF-κB. Indeed, activation of NF-κB is sufficient to induce resistin expression, and loss of NF-κB function abolishes LPS induction of resistin. Resistin serum levels are increased dramatically by endotoxemia in humans, and correlate with a marker of inflammation in patients with type 2 diabetes. Thus, systemic inflammation leads to increased resistin production and circulating levels in humans. The increased level of resistin in humans with obesity is likely an indirect result of elevated levels of inflammatory cytokines characteristic of states of increased adiposity. Hence, obesity and acute inflammation are both hyperresistinemic states associated with insulin resistance.
Methods
Differentiation of Primary Human Macrophages
Peripheral blood mononuclear cells were isolated from whole blood of healthy donors following apheresis and elutriation. Greater than 90% of these monocytes expressed CD14 and HLA-DR. Cells were plated in 24-well plates at a density of 106 cells per well, allowed to adhere for 4 h, then washed with Dulbecco's Modified Eagles Medium and further cultured in 10% FBS in Dulbecco's Modified Eagles Medium supplemented with 5 ng/ml GM-CSF (Sigma, St. Louis, Missouri, United States) to promote macrophage differentiation. All experiments were performed after overnight equilibration with macrophage serum-free medium (GIBCO, San Diego, California, United States; Invitrogen, Carlsbad, California, United States) supplemented with 5 ng/ml GM-CSF. Cells were treated with LPS (Sigma), aspirin (Sigma), SN50, and/or control peptide (Biomol, Plymouth Meeting, Pennsylvania, United States), MG132, PD98059, SB20358 (Calbiochem, San Diego, California, United States), and TNFα (R&D Systems, Minneapolis, Minnesota, United States). Neutralizing antibodies to TNFα, IL-6, and anti-IL-1β, as well as control IgG, were obtained from R&D Systems. Adenovirus expressing activated IKK in pAD easy with GFP and control vector was a generous gift from Steven Shoelson.
RNA Isolation and Quantification
RNA was isolated using RNeasy Mini Kit (Qiagen, Valencia, California, United States), then subjected to DNase digestion followed by reverse transcription (Invitrogen). mRNA transcripts were quantified by the dual-labeled fluorogenic probe method for real-time PCR, using a Prism 7900 thermal cycler and sequence detector (Applied Biosystems, Foster City, California, United States). Real-time PCR was performed using Taqman Universal Polymerase Master Mix (Applied Biosystems). The primers and probes used in the real-time PCR were the following: Sense-Resistin, 5′-
AGCCATCAATGATAGGATCCA-3′; Antisense-Resistin, 5′-
TCCAGGCCAATGCTGCTTAT-3′; Resistin Probe, 5′-Fam-
AGGTCGCCGGCTCCCTAATATTTAGGG-TAMRA-3′; Sense human 36B4 sense, 5′-
TCGTGGAAGTGACATCGTCTTT-3′; Antisense 36B4, 5′-
CTGTCTTCCCTGGGCATCA-3′; and 36B4 Probe, 5′-FAM-
TGGCAATCCCTGACGCACCG-TAMRA-3′.
Primer and probe for TNFα were obtained from Applied Biosystems. The cycle number at which the transcripts of the gene of interest were detectable (CT) was normalized to the cycle number of 36B4 detection, referred to as deltaCT. The fold change in expression of the gene of interest in the compound-treated group relative to that in the vehicle-treated group was expressed as 2−deltadeltaCT, in which deltadeltaCT equals the deltaCT of the compound-treated group minus the deltaCT of the chosen control group, which was normalized to 1.
ELISA
Resistin concentrations, in cell media and human plasma, were assessed with a commercially available ELISA (Linco Research, St. Charles, Missouri, United States) and normalized to cell protein. The average correlation coefficient for standards using a four-parameter fit was 0.99. Intra-assay and inter-assay coefficients of variance were 4.7% and 9.1%, respectively. Direct comparison of standard curves generated by the Linco kit with those yielded by another commercially available resistin ELISA (Biovendor Laboratory Medicine, Brno, Czech Republic) yielded high correlation (rho = 0.99, p < 0.001), except that the Biovendor values were approximately 30% lower than those determined with the Linco assay. This appeared to be related to the standards used for calibration. Discrepant absolute values among different assays, including the Biovendor assay, were recently described by others [22]. Resistin levels in 40 plasma samples were measured using both Linco and Biovendor ELISA kits, with moderate correlation (rho = 0.66). Levels of soluble TNFα receptor 2 (sTNFR2) were measured using a commercially available immunoassay (R&D Systems). Intra-assay and inter-assay coefficients of variance were 5.1% and 9.8%, respectively.
Human Endotoxemia Study
Healthy volunteers (n = 6, three male and three female), aged 18–45 y with BMI between 20 and 30 and on no medications, were studied. The University of Pennsylvania Institutional Review Board approved the study protocol, and all participants gave written informed consent. Following screening and exclusion of individuals with any clinical or laboratory abnormalities, participants were admitted to the General Clinical Research Center at the University of Pennsylvania for a 60 h stay. Serial blood samples were collected during the 24 h prior to and 24 h following the intravenous administration of human-research-grade endotoxin (obtained from National Institutes of Health Clinical Center, reference endotoxin [CCRE] [lots 1 and 2; National Institutes of Health Clinical Center PDS #67801]) at a dose of 3 ng/kg given at 6 AM. Plasma and whole blood RNA (PAX tube isolators, Qiagen) samples were isolated from blood, and stored under appropriate conditions for subsequent assays.
Type 2 Diabetes Study
Participants with type 2 diabetes (n = 215, 167 male and 48 female), aged 35–75 y and free from clinical cardiovascular diseases, were recruited through the diabetes clinics at the University of Pennsylvania Medical Center and the Veterans Affairs Medical Center, Philadelphia, Pennsylvania, to an ongoing study of cardiovascular risk factors in type 2 diabetes. The sample was composed of 59% Caucasians and 35% African-Americans. All participants were evaluated at the University of Pennsylvania General Clinical Research Center in a fasting state at 8 AM. The University of Pennsylvania Institutional Review Board approved the study protocol, and all participants gave written informed consent. The patient population is described in more detail elsewhere [27].
Statistical Methods
Data are reported as mean and standard error of the mean (SEM) for continuous variables. Because of baseline variation in cell populations between batches of primary human monocytes isolated from multiple donors, cell culture experiments were performed in triplicate and data from representative experiments are presented. For cell culture experiments with multiple treatments, analysis of variance (ANOVA) was used to test for differences in means across treatment groups. When significant global differences were found, post hoc t-tests were used to compare specific treatment groups to the control. Data from the human endotoxemia experiment were analyzed by repeated measures ANOVA. In the type 2 diabetes study, Spearman correlations of plasma levels of resistin with plasma sTNFR2 levels are presented.
Results
Induction of Resistin Gene and Protein Expression by Endotoxin Treatment of Human Macrophages
The regulation of resistin expression was studied in primary cultures of human monocytic cells. Immediately upon plating of elutriated primary human monocytes, resistin gene expression was detectable but highly variable from experiment to experiment (data not shown). One day after plating, resistin gene expression remained detectable at low levels (Figure 1A). Subjection of the cells to a protocol leading to differentiation along the macrophage lineage led to a modest, time-dependent enhancement of resistin gene expression (Figure 1A). In agreement with a previous report [28], treatment of primary macrophages with the endotoxin LPS led to a dramatic, dose-responsive increase in resistin gene expression (Figure 1B). We also determined that this effect of LPS was paralleled by an increase in resistin protein secretion into the medium (Figure 1C). Of note, activated mouse peritoneal macrophages harvested after thioglycolate treatment did not express detectable levels of mouse resistin, even after treatment with LPS (data not shown).
Figure 1 Induction of Resistin in Human Macrophages
(A) Induction of resistin during human macrophage differentiation ex vivo. Expression of resistin on days 1, 3, and 7 following isolation and culture of human peripheral blood monocytes under macrophage differentiation conditions. Results shown are the mean (± SEM) of three separate experiments with triplicate samples. The ANOVA F statistic for change of resistin mRNA expression during differentiation was 7.06 (p < 0.01). *, p < 0.01 for post hoc t-tests.
(B) Resistin mRNA is induced by endotoxin in primary human macrophage cultures. The ANOVA F statistic for change of resistin mRNA expression in response to increasing concentration of LPS (24 h treatment) was 423.57 (p < 0.001). *, p < 0.001 for post hoc t-tests.
(C) Resistin protein secretion by human macrophages is induced by endotoxin. The ANOVA F statistic for change of resistin protein secretion in response to increasing concentration of LPS (24 h treatment) was 35.36 (p < 0.001). *, p < 0.001 for post hoc t-tests.
For LPS dose response studies, shown in (B) and (C), results (mean ± SEM) of representative experiments, with triplicate samples, are presented. Similar results were obtained in two independent experiments.
Endotoxin Induction of Resistin Is Delayed with Respect to TNFα
Induction of resistin gene expression by LPS exposure of human macrophages began between 6 and 24 h after treatment, with peak expression at 24 h (Figure 2A). This time course of resistin induction was delayed relative to induction of TNFα gene expression, which was detectable at 2 h and peaked 6 h after LPS exposure (Figure 2B). The secretion of TNFα followed a similar time course (Figure 2C). By contrast, secretion of resistin did not increase until much later, more closely following the pattern of the appearance of sTNFR2, a marker of TNFα action (Figure 2C) [29].
Figure 2 Endotoxin Induction of Resistin Occurs after Induction of TNFα
Primary cultures of human macrophages were treated with LPS (1 μg/ml) for various times.
(A) Time course of induction of resistin mRNA. The ANOVA F statistic for the change in resistin mRNA over time was 105.45 (p < 0.001).
(B) Time course of induction of TNFα mRNA. The ANOVA F statistic was 34.57 (p < 0.001).
(C) Time course of secretion of resistin, TNFα, and sTNFR2 into medium. ANOVA F statistics for the effect of LPS on resistin (66.51, p < 0.001), sTNFR2 (12.86, p < 0.001), and TNFα (20.48, p < 0.001) were highly significant. Maximal secreted protein levels were as follows: resistin, 21.9 ng/ml/mg; TNFα, 207.2 ng/ml/mg; and sTNFR2, 39.3 ng/ml/mg. Results of representative experiments with triplicate samples are expressed as mean (± SEM). Similar results were obtained in three independent experiments.
Endotoxin Induction of Resistin Is Blocked by Immunoneutralization of Multiple Cytokines
Resistin gene expression was also induced by TNFα treatment of primary human macrophages (Figure 3A) [28], and resistin secretion increased in parallel (Figure 3B). Since LPS induction of TNFα preceded the increase in resistin (see Figure 2C), we hypothesized that TNFα, or a similar cytokine produced early after LPS exposure, was responsible for the later induction of resistin. Indeed, neutralizing antibodies to TNFα markedly attenuated the increase in resistin gene expression (Figure 3C). LPS treatment also induces other cytokines, including IL-6 and IL-1β [30], and IL-6 induces resistin modestly (data not shown) [28]. Antibodies to IL-6 and IL-1β individually had minor effects on LPS stimulation of resistin (Figure 3C). However, the combination of antibodies to TNFα, IL-6, and IL-1β markedly attenuated LPS induction of resistin (Figure 3C). These data clearly show that resistin induction by endotoxin is mediated by a cascade in which the primary event is secretion of inflammatory cytokines that, in turn, induce resistin.
Figure 3 Endotoxin-Induced Cytokines Regulate Resistin Induction
(A) TNFα induces production of resistin mRNA by primary human macrophages. The ANOVA F statistic for the effect of increasing TNFα concentrations on resistin was 23.81 (p < 0.001). *, p < 0.001 for post hoc t-tests.
(B) TNFα induces resistin protein secretion by primary human macrophages. ANOVA F statistic for the effect of TNFα on resistin was 79.85 (p < 0.001). *, p < 0.005 for post hoc t-tests. Results of representative experiments with triplicate samples are expressed as the mean (± SEM). Similar results were obtained in two independent experiments.
(C) LPS (1 μg/ml) induction of resistin is abrogated by antibody neutralization of cytokines (7.5 μg/ml per antibody). ANOVA F statistic for the effect of neutralizing antibodies on resistin was 3.08 (p < 0.05). p-Values for post hoc t-tests versus IgG: *, p < 0.05; **, p < 0.001. Results are expressed as the mean (± SEM) of three separate experiments with triplicate samples.
Induction of Resistin Is Blocked by Anti-Inflammatory Insulin-Sensitizing Drugs That Target NF-κB
Mouse resistin, produced exclusively by adipocytes, is down-regulated by antidiabetic thiazolidinediones, including rosiglitazone [11]. Consistent with an earlier report [16], rosiglitazone down-regulated resistin gene expression (Figure 4A) in LPS-stimulated human macrophages. Resistin protein secretion was also significantly reduced by rosiglitazone (Figure 4B). Hence, macrophage expression of resistin and its induction by LPS is species-specific, but down-regulation of resistin by thiazolidinedione occurs both in rodents and humans. Rosiglitazone has marked anti-inflammatory effects on macrophages [31]. This led us to examine the effect of aspirin, an anti-inflammatory compound that targets IκB kinase and has insulin-sensitizing effects [32]. Remarkably, aspirin dramatically decreased endotoxin-induced resistin expression in a dose-dependent manner (Figure 4C). Both aspirin (via IκB kinase) and rosiglitazone (via PPARγ) inhibit NF-κB [31,32], which is activated by LPS. Indeed, treatment of the macrophages with the proteasome inhibitor MG132, which prevents NF-κB activation [33], abrogated endotoxin-induced activation of resistin expression (data not shown). Moreover, treatment of the macrophages with SN50, a cell-permeable peptide that specifically prevents activation of NF-κB by inhibiting its nuclear translocation [34], nearly abolished endotoxin-induced activation of resistin expression (Figure 4D). Thus, activation of NF-κB is required for LPS induction of resistin in human macrophages. Furthermore, constitutive activation of NF-κB by adenoviral expression of activated IκB kinase was sufficient to induce resistin in primary human macrophages (Figure 4E). The magnitude of this activation was less than that caused by LPS, which is known to also activate MAP-kinase (MAPK). Indeed, inhibition of either p42 MAPK by PD98059, or p38 MAPK (using SB20358) partially blocked the induction of resistin by LPS (Figure 4F). Together these results show that NF-κB activation is necessary and sufficient for resistin induction by LPS, with MAPK activation increasing the magnitude of the response.
Figure 4 Inhibition of Resistin Induction by Anti-Inflammatory Insulin Sensitizers
(A) Down-regulation of resistin mRNA by rosiglitazone. ANOVA F statistic for the effect Rosiglitazone on resistin expression was 62.52 (p < 0.001). p value for post hoc t-tests, is depicted in the Figure. *p < 0.005 versus control for post hoc t-tests.
(B) Down-regulation of resistin protein secretion by human macrophages treated with rosiglitazone. The ANOVA F statistic for the effect of rosiglitazone on resistin protein secretion was 29.44 (p < 0.001). p-Values for post hoc t-tests versus control: *, p < 0.05; **, p < 0.001. Cells were pre-treated with rosiglitazone for 24 h and with LPS (1 μg/ml) and rosiglitazone for an additional 24 h. Results of representative experiments with triplicate samples are expressed as mean (± SEM). Similar results were obtained in three independent experiments.
(C) Down-regulation of resistin gene expression by aspirin. The ANOVA F statistic for the effect of aspirin on resistin expression was 61.33 (p < 0.001). p-Values for post hoc t-tests versus no aspirin: *, p < 0.01; **, p < 0.001; ***, p < 0.0001. Cells were pre-treated with aspirin for 2 h and with LPS (1 μ g/ml) and aspirin for an additional 24 h. Results of representative experiments with triplicate samples are expressed as mean (± SEM). Similar results were obtained in two independent experiments.
(D) Down-regulation of resistin gene expression by NF-κB inhibitor SN50. *, p < 0.001 versus control peptide by t-test. Cells were pre-treated with SN50 or control peptide at 100 ug/ml for 2 h, and with LPS (1 μg/ml) and SN50 or control peptide for an additional 24 h. Results are the expressed as the mean (± SEM) of two independent experiments performed in triplicate.
(E) Induction of resistin by activation of NF-κB. *, p < 0.05 versus control virus by t-test. Cells were infected with adenovirus expressing activated IKK or control virus for 24 h. Results of representative experiments with triplicate samples are expressed as mean (± SEM). Similar results were obtained in two independent experiments.
(F) Down-regulation of resistin gene expression by inhibitors of p38 and p42 MAPK. The ANOVA F statistic for the effect of the MAPK inhibitor on resistin expression was 11.54 (p < 0.005). *, p < 0.005 versus control for post hoc t-tests. Cells were pretreated with 50 μM PD98059 or 2.5 μM SB20358 for 2 h and with LPS (1 μg/ml) and PD98059 or SB20358 for an additional 24 h. Results are expressed as the mean (± SEM) of two independent experiments performed in triplicate.
LPS Robustly Increases Circulating Resistin Levels in Healthy Humans
Next, we asked whether our findings from ex vivo studies of human macrophages would translate into in vivo observations in humans. Six healthy volunteers were injected with LPS, using a protocol similar to that shown to produce insulin resistance [35]. Baseline circulating resistin levels were approximately 4 ng/ml, and remained relatively constant for several hours prior to LPS infusion (Figure 5A). Remarkably, resistin levels rose dramatically because of endotoxemia, peaking 8–16 h after LPS administration (Figure 5A). The time course of hyperresistinemia paralleled the increase in circulating levels of sTNFR2, although the increase in resistin levels was more marked and sustained (Figure 5A). The increase in resistin protein levels correlated with increased resistin gene expression in peripheral blood mononuclear cells following systemic endotoxemia (Figure 5B).
Figure 5 Endotoxin Dramatically Induces Plasma Resistin in Humans
(A) Plasma resistin and sTNFR2 levels were measured serially in six healthy volunteers for 24 h before and after intravenous LPS (3 ng/kg) administration. The repeated measures ANOVA F statistics for the effect of LPS on plasma resistin (9.25, p < 0.001) and sTNFR2 (23.65, p < 0.001) were highly significant.
(B) Mean resistin RNA expression in whole blood cells of healthy volunteers (n = 2) before and after treatment with LPS (3 ng/kg).
Circulating Resistin Levels Correlate with the Inflammatory Marker sTNFR2 in Patients with Type 2 Diabetes
Patients with type 2 diabetes and insulin resistance, many of whom are obese, have elevated levels of several inflammatory markers, including IL-6, TNFα, and sTNFR2 [36]. LPS administration has been shown to induce acute insulin resistance in humans [37]. Given that LPS infusion increased resistin levels, we measured resistin in a cohort of 215 patients with type 2 diabetes. Circulating resistin levels were significantly correlated with levels of sTNFR (Figure 6A). Thus, there is an association between resistin levels and systemic inflammation in patients with type 2 diabetes.
Figure 6 Plasma Resistin Levels Correlate with sTNFR2 Levels in Humans with Type 2 Diabetes
(A) The correlation (Spearman coefficient rho = 0.38, p < 0.001) of plasma resistin and sTNFR2 levels in 215 humans with type 2 diabetes is presented. The line represents the linear regression fit between log-transformed plasma levels of resistin and sTNFR2.
(B) Model to explain hyperresistinemia in mice and humans with obesity despite the species differences in the source of plasma resistin. Circulating inflammatory cytokines TNFα and IL-6 are depicted because of their role in resistin induction in human macrophages and their implied role in insulin resistance. Other cytokines and inflammatory markers may also contribute to insulin resistance and/or resistin induction.
Discussion
We have demonstrated that, in human macrophages, an inflammatory cascade with secretion of cytokines, including TNFα and IL-6, is sufficient and necessary for the induction of resistin. Insulin sensitizers that have anti-inflammatory properties, including a synthetic PPARγ agonist as well as aspirin, suppress macrophage resistin expression, as does direct inhibition of NF-κB. Experimental endotoxemia in healthy volunteers, based on the well-established gram-negative bacterial inflammatory response in humans [38,39,40], induces a dramatic elevation of circulating resistin levels. Hence, resistin gene and protein expression are increased by inflammatory stimuli both ex vivo and in vivo.
In rodents, resistin is produced exclusively by adipocytes, regulates normal glucose homeostasis, and causes insulin resistance at high circulating levels [11,13]. Translation of resistin's metabolic effects from rodents to humans has been problematic because peripheral blood mononuclear cells and macrophages appear to be a primary source of resistin in humans [15,16]. This species difference in primary locus of expression is yet another example of the close and functionally overlapping relationship between adipocytes and macrophages [41]. Numerous studies have reported that circulating resistin levels are increased in human obesity [20,25,26,41] and diabetes [19,20,23,42,43]. Our data suggest that, whereas hyperresistinemia in obese rodents derives directly from adipocytes, human resistin is indirectly regulated by the inflammatory internal milieu of obesity (Figure 6B). Indeed, obesity is associated with elevated levels of cytokines whose systemic administration leads to impaired glucose homeostasis [36,44,45], such as TNFα and IL-6, which we show here to mediate the inflammatory induction of human resistin. Thus, in both species, adipose tissue is an endocrine organ containing adipocytes as well as macrophages that regulates energy metabolism and glucose homeostasis through secretion of multiple factors, including inflammatory cytokines [46].
Clearly the relationship between obesity, inflammation, and resistin expression is complex, and needs to be systematically studied in larger and varied patient populations. Intriguingly, we found a strong correlation between plasma levels of resistin and sTNFR2, the soluble cleavage product of the activated TNFα receptor, in diabetic patients. A comparable correlation between resistin and sTNFR2 (R = 0.31, p < 0.001) was found in a cohort of 879 non-diabetic individuals, in whom resistin levels independently correlated with coronary atherosclerotic disease (M. P. Reilly, M. Lehrke, M. L. Wolfe, A. Rohatgi, M. A. Lazar, and D. J. Rader, unpublished data).
LPS binds to pathogen-associated-molecular-pattern innate immune receptors, such as CD14 and Toll-like receptor 4, activating signal cascades involving NF-κB and MAPK [47] and thereby inducing the transcription and secretion of early cytokines, including TNFα and IL-1 [48]. We have shown here that these early cytokines are responsible for secondary induction or enhancement of resistin expression in macrophages. Hyperresistinemia impairs glucose homeostasis in rodents [49,50], and inflammatory states are associated with insulin resistance [36], which may serve as a physiological attempt to increase the provision of glucose to the brain under stress conditions. Indeed, induction of acute inflammation by administration of LPS causes insulin resistance in humans [37], and here we have demonstrated the concomitant induction of resistin. Interestingly, the peak in TNFα and IL-6 levels after LPS administration to humans precedes a phase of prolonged insulin resistance that begins approximately 6 h after LPS administration [37], closely approximating the time course of resistin induction. Hence resistin is a potential mediator of insulin resistance in humans with acute inflammation. Moreover, obesity is associated with activation of innate immunity [6], including the inflammatory mediators that induce resistin. In this context it is intriguing that resistin levels are increased in obesity [25,26] and that insulin-sensitizing agents such as aspirin and rosiglitazone, with disparate primary molecular targets, antagonize resistin induction. Indeed, thiazolidinedione suppression of resistin levels has recently been correlated with hepatic insulin sensitization [43]. Future work will be needed to better understand the relationship between circulating resistin levels and the insulin resistance characteristic of inflammatory states, including obesity.
Patient Summary
Why Was This Study Done?
There is a very close connection between obesity and diabetes: diabetes is more common among obese people, and people with type 2 diabetes know that weight control is an essential part of their diabetes treatment. But the link between extra body fat and diabetes remains a puzzle. Recent experiments in mice suggested that a hormone called resistin could be the missing link. One reason is that resistin levels respond to a particular class of diabetes drugs called thiazolidinediones. But studies in humans found that mice and humans are quite different when it comes to resistin. One difference is that in mice resistin is produced by fat cells, but in humans it is produced by special immune cells called macrophages that are involved inflammation. Researchers are now studying what role—if any—resistin might have in humans with obesity and diabetes and are studying the similarities in the ways in which the body reacts to obesity and inflammation.
What Did the Researchers Do?
The researchers examined what happens to resistin levels when human macrophages or human patients are exposed to substances that trigger inflammation.
What Did They Find?
The substances that trigger inflammation caused higher resistin levels, but resistin levels were lowered again by thiazolidinediones.
What Does This Mean?
Because in mice higher resistin levels (produced by fat cells) are linked to diabetes, one possibility is that obesity in humans, by being similar to inflammation, causes immune cells to make lots of resistin and hence promotes diabetes that way.
What Next?
More research is necessary to confirm these findings and to find out how important resistin is as a link between obesity and diabetes, and how resistin promotes diabetes.
Additional Information
United States National Institute of Diabetes, Digestive, and Kidney Diseases (NIDDK) information on obesity: http://www.niddk.nih.gov/health/nutrit/pubs/unders.htm
NIDDK information on diabetes: http://diabetes.niddk.nih.gov/
International Diabetes Federation: http://www.idf.org/
We thank C. Steppan for helpful discussions and for contributing to antibody development, S. Shoelson and D. Cai (Joslin Diabetes Center) for providing adenovirus expressing activated IKKα, M. Wolfe, J. Tabita-Martinez, K. Terembula, and C. Hinkle (University of Pennsylvania) for assistance with the patient-oriented studies, and J. Mistry (Linco) for help with the resistin assay. This work was funded by National Institute of Diabetes and Digestive and Kidney Diseases grants R01 DK49210 and R01 DK49780, an unrestricted Bristol-Myers Squibb Freedom to Discover Award in Metabolic Research (MAL), National Institutes of Health grants K23 RR15532 and R01 HL73278, a grant from the WW Smith Charitable Trust (MPR), a Burroughs Wellcome Fund Clinical Scientist Award in Translational Research, a Doris Duke Distinguished Clinical Investigator Award (DJR), and grant LE 1350/1–1 from the Deutsche Forschungsgemeinschaft (ML). We are indebted to the radioimmunoassay core of the Penn Diabetes and Endocrinology Research Center (National Institute of Diabetes and Digestive and Kidney Diseases grant P30 DK19525) for assistance with resistin assays, the Immunology Core of the University of Pennsylvania Center for AIDS Research for peripheral blood monocytes, and the University of Pennsylvania General Clinical Research Center (National Institutes of Health grant M01RR00040) and its nursing staff for outstanding patient care. The funders had no role in study design, data collection and analysis, decision to publish, or preparations of the manuscript.
Citation: Lehrke M, Reilly MP, Millington SC, Iqbal N, Rader DJ, et al. (2004) An inflammatory cascade leading to hyperresistinemia in humans. PLoS Med 1(2): e45.
Abbreviations
ANOVAanalysis of variance
ILinterleukin
LPSlipopolysaccharide
MAPKMAP-kinase
SEMstandard error of the mean
sTNFR2soluble tumor necrosis factor receptor 2
TNFαtumor necrosis factor α
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| 15578112 | PMC529430 | CC BY | 2021-01-05 10:38:04 | no | PLoS Med. 2004 Nov 30; 1(2):e45 | utf-8 | PLoS Med | 2,004 | 10.1371/journal.pmed.0010045 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1557811310.1371/journal.pmed.0010046EditorialOtherGeneral MedicineClinical TrialsMedical JournalsEditorial Policies (Including Conflicts of Interest)From Registration to Publication EditorialThe PLoS Medicine Editors 11 2004 30 11 2004 1 2 e46Copyright: © 2004 Public Library of Science.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The PLoS Medicine editors argue that all trial registries, reports, and databases should be freely available online
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In a compelling essay in this issue of PLoS Medicine, Mike Clarke, the director of the United Kingdom Cochrane Centre, lays down a challenge to clinical researchers and journal editors [1]. He argues that researchers should do a study only if there is a systematic review that shows that the new study is needed. If no review exists, the researchers should do one themselves before embarking on their research. And journals, he argues, should publish a study only if an updated systematic review is incorporated into the study, or published alongside it or shortly thereafter. How should editors respond to his challenge?
First, we would argue that by the time a paper is sent to a journal, it is surely too late in the process to be insisting on systematic reviews. If a clinical trial report meets our criteria for originality, importance, and quality, it makes little sense for us to reject it just because the authors failed to systematically review the literature when designing their study. The time to mandate that researchers do a review is much earlier—when they apply for funding, register their trial, or seek ethics committee approval.
There is no doubt that the best research builds on previous knowledge. But unfortunately, the current medical publishing system hides much of this knowledge behind subscription or “pay per view” charges, which discriminates against researchers who do not work for well-funded institutions. A group of researchers in Indonesia, for example, recently told a depressingly familiar story of trying to search the medical literature in preparation for a research project [2]; access barriers got in their way. So our second response to Clarke's challenge is that it will remain difficult for researchers, particularly in resource-poor settings, to do systematic reviews unless the medical literature is made a freely available public resource.
Many clinical trials, especially negative ones, remain unpublished, which prevents researchers from reviewing all the data on an important health issue. There are two main reasons why certain trials are not published: one is that the pharmaceutical industry has a long history of suppressing data that are commercially unfavorable and the second is that medical journals and the popular media favor publication of positive over negative trials (after all, negative trials do not make for a provocative newspaper headline). While we support the recent announcement on trial registration by the International Committee of Medical Journal Editors—as a condition of considering a trial for publication, member journals will require registration of the trial in a public trials registry [3]—we believe that this policy addresses only part of the problem.
The scientific literature will remain biased unless the publishing industry changes its practices and provides a place where the results from all registered trials can be published. PLoS Medicine is committed to publishing high-quality negative trials. In this issue, for example, we publish an important randomized controlled trial of a malaria vaccine in 372 Gambian men, which found that the vaccine was ineffective at reducing the natural infection rate.
The internet makes it possible for every single clinical trial to be publicly and seamlessly tracked through three tiers. The first tier is registration in a publicly available database. The second is the publication of a peer-reviewed summary of every trial, regardless of its outcome, in a traditional journal format, with annotations and critiques that help readers understand the trial's implications. The third is the deposition of detailed trial data in a structured, computable format that allows sophisticated searching and analyses across trials. This format will allow the development of better tools to help clinicians apply trial results to their practice. For trial data to be as useful as possible, all three tiers must be publicly accessible. Assessment of each trial's validity is critical, but should not stop crucial information about all trials being placed in the public domain.
Trial registries exist, such as ClinicalTrials.gov and the International Standard Randomised Controlled Trial Number registry. Moreover, many trials are registered in a semi-public database maintained by the United States Food and Drug Administration, and there are compelling arguments (which Turner articulates in an essay published online ahead of our December issue [4]) for making this a truly public resource. Publicly accessible trial databases (such as the Trial Bank Project at http://rctbank.ucsf.edu) are under development. And as a publisher committed to open access, PLoS will provide the second, essential tier—journals capable of peer reviewing and publishing an annotated report of every trial. Traditional medical journals, with their subscription-based model, are unlikely to be able to provide this service, because in order to attract subscribers they need to publish only the highest-profile trials. We believe that an open-access model—in which the research funder pays a publication fee to recover the costs of peer review and for hosting the report on a secure server—is the best mechanism for creating such venues. We are working to make that happen.
Returning to Clarke's challenge, our final response is to say that we have a bold vision of a freely accessible online world of clinical trials—from registration to annotated summaries to trial databases. That world would be even richer if every systematic review were made freely available. We challenge the Cochrane Collaboration to put the full text of all of its reviews into the public domain. We hope the Cochrane Collaboration will join us in the open-access revolution.
The senior editors for PLoS Medicine are Virginia Barbour, James Butcher, Barbara Cohen, and Gavin Yamey. E-mail: [email protected]
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 10.1371/journal.pmed.0010049SynopsisImmunologyAllergy/ImmunologyDiabetes/Endocrinology/MetabolismObesityDiabetesImmunology and AllergyResistin Response to Inflammation Synopsis11 2004 30 11 2004 1 2 e49Copyright: © 2004 Public Library of Science.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
An Inflammatory Cascade Leading to Hyperresistinemia in Humans
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Obesity, in particular visceral adiposity, is positively correlated with insulin resistance and type 2 diabetes. Although the link is well established in humans and in rodent models, the mechanisms involved in obesity-related insulin resistance are not clear. One possibility is that hormones secreted by adipocytes compromise peripheral insulin sensitivity, and a number of candidates for such adipocyte signals have been identified. One of them, resistin, was discovered a few years ago by Mitchell Lazar and colleagues, who showed that the protein is expressed by mouse adipocytes and regulated by a group of anti-diabetic drugs called thiazolidinediones. Several lines of evidence from functional studies in rodents suggested that resistin could be the missing mechanistic link between obesity and diabetes.
The human homolog of resistin has subsequently been under intense investigation, but initial studies revealed more differences than similarities between the human and rodent proteins: human resistin is mostly expressed in macrophages, not in adipocytes, and its serum levels do not correlate as clearly with obesity, insulin resistance, or diabetes. Similarly, genetic association studies between allelic variants of the resistin gene and metabolic abnormalities have so far been inconclusive. These results prompted some of the scientists in the field who had jumped on the resistin bandwagon after the initial results in rodents to jump off again. Others, including the resistin discoverers, continue their quest to uncover resistin's role in humans, and have started to think outside the framework defined by the mouse data.
Connections between obesity and inflammation
Starting with the role of macrophages in inflammation and encouraged by the fact that obesity and insulin resistance are associated with markers of systemic inflammation, Lazar and colleagues examined the resistin response to inflammatory stimulators. As they report in this issue, resistin production in macrophages and serum levels in patients are significantly increased by these stimulators. This response can be blocked by the thiazolidinedione rosiglitazone and by aspirin, two drugs that have dual anti-inflammatory and insulin-sensitizing actions and antagonize the immune regulator NF-kappaB. The researchers go on to show that activation of NF-kappaB is sufficient to induce resistin expression. And NF-kappaB is necessary for the resistin response to inflammatory stimuli.
Lazar and colleagues now view obesity as a state of chronic inflammation and speculate that in obese individuals inflammatory cytokines lead to elevated production of resistin by macrophages and elevated serum resistin levels, which in turn contribute to insulin resistance and diabetes. This is consistent with some studies that have found higher resistin levels in obese individuals and patients with insulin resistance and/or diabetes, but not all studies have found such differences.
Jeffrey Flier, an obesity researcher who was not involved in the study, calls the article “an excellent and timely paper that demonstrates the fact that inflammatory pathways induce resistin expression and levels in human monocytes ex vivo, and in intact humans. The work appears to provide a novel link between inflammation and insulin resistance, through monocyte derived resistin.” He points out, however, that “several other factors also appear to contribute directly to insulin resistance in inflammation (e.g., cytokines themselves, without invoking resistin) so the full biologic implications of the high resistin levels for insulin resistance in humans cannot be determined from this study.” Resistin, it seems, continues to resist easy interpretations.
| 0 | PMC529432 | CC BY | 2021-01-05 10:38:01 | no | PLoS Med. 2004 Nov 30; 1(2):e49 | utf-8 | PLoS Med | 2,004 | 10.1371/journal.pmed.0010049 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 10.1371/journal.pmed.0010050SynopsisCancer BiologyImmunologyInfectious DiseasesAllergy/ImmunologyOncologyOncologyImmunology and allergyInfectious DiseasesThe Quest for a Vaccine That Yields Tumor-Killing T cells Synopsis11 2004 30 11 2004 1 2 e50Copyright: © 2004 Public Library of Science.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Diversity and Recognition Efficiency of T Cell Responses to Cancer
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The immune system has a remarkable capacity for fending off infectious diseases, and it has become clear that these same defenses can recognize and destroy cancer cells. In fact, they do so on an ongoing basis, and cancer develops only when immune surveillance breaks down. Many patients with established tumors also mount an immune response against some antigens that are specific to, or enriched in, the tumor. This response, however, is rarely effective against the disease.
The idea of enlisting the immune system to fight cancer has been around for a long time, and has led to the development of various cancer vaccines designed to alert the immune system to the presence of a tumor and to induce a response that, selectively and potently, will eliminate tumor cells. Vaccines include whole tumor extracts or specific proteins and peptides that are selectively expressed or enriched in tumors, by themselves or with a variety of adjuvants.
There have been some spectacular successes, in particular with immune therapy to malignant melanoma, a tumor type that seems naturally to be more immunogenic than others. However, even in melanoma, success is usually restricted to a fraction of the patients, with no obvious explanation of why the strategy works for a particular patient and fails in most others. The emphasis has consequently shifted from clinical outcomes to monitoring a patient's immune response. What type of response is necessary and sufficient to eliminate tumor cells is still unclear, but the hope is that understanding the immune response in patients that show clinical benefit will answer that question.
Peter Lee and colleagues used state-of-the art technology to dissect the endogenous immune response to vaccination with heteroclitic melanoma peptides, i.e., melanoma-associated peptides that have been engineered to elicit a stronger immune response. They focused on cytotoxic T lymphocytes (CTLs), and compared CTL clones from four melanoma patients who had vaccine-induced T cell responses and two melanoma patients with spontaneous anti-tumor T cell responses. The researchers analyzed several hundred CTL clones (to get a sense for the complexity of the responses in individual patients) for T cell receptor variable chain beta expression, recognition efficiency, and ability to lyse target melanoma cells. Most T cells isolated from vaccinated patients were poor at tumor cell lysis compared with T cells from endogenous responses to cancer.
Melanoma—a prime target of cancer vaccines (Photo: Timothy Triche, National Cancer Institute)
The authors suggest that the high doses of peptides administered in vaccinations and the increased binding capacity of heteroclitic peptides to major histocompatibility complex molecules—the very quality that makes them more immunogenic—induce many T cells with low recognition efficiency for the native peptides they encounter on the tumor cells. Their findings also bring into question the ability to deduce the recognition efficiency and tumor reactivity of T cell responses from ELISPOT and tetramer staining assays—the two standard measures of T cell responses to vaccines—which has implications for rational vaccine design in general.
| 0 | PMC529433 | CC BY | 2021-01-05 10:38:00 | no | PLoS Med. 2004 Nov 30; 1(2):e50 | utf-8 | PLoS Med | 2,004 | 10.1371/journal.pmed.0010050 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1557811410.1371/journal.pmed.0010051Case ReportHematologyHematology (including Blood Transfusion)Drugs and adverse drug reactionsEpilepsy and seizuresPure Red Cell Aplasia after 13 Years of Sodium Valproate, and Bone Marrow Suppression after 17 Years of Carbamazepine Case ReportThe Tiong
1
Kolla Ratnavalli
2
Dawkins Fitzroy
2
Trouth Annapurni Jayam
2
1Case report fromSaint Peter's University Hospital, New BrunswickNew JerseyUnited States of America2Howard University Hospital, WashingtonDistrict of ColumiaUnited States of America
Competing Interests: The authors have declared that no competing interests exist.
Author Contributions: All authors contributed equally to preparing the case report.
* To whom correspondence should be addressed. E-mail: [email protected] 2004 30 11 2004 1 2 e5127 8 2004 4 10 2004 Copyright: © 2004 The et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.A 38-year-old woman presented with acute hematological toxicity from her anticonvulsants, even though she had been taking them for many years
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PRESENTATION of CASE
A 38-y-old woman with Down syndrome was admitted to hospital for investigation of a 6-mo history of anorexia and weight loss of 40 lbs. Six months prior to admission, her weight was 175 lbs, and her body mass index was 36. On admission, her complete blood count was normal, but over a 2-wk period she developed acute pancytopenia (Figure 1). During this acute episode, her lowest hematological parameters were as follows: hemoglobin (Hb), 80 g/l (normal range 120–160 g/l); mean cell volume (MCV), 121 fl (80–95 fl); white blood cell count (WBC), 2.9 × 106/l (4.8–10.8 × 106/l); absolute neutrophil count, 1.1 × 106/l (1.5–6.2 × 106/l); and platelet count, 76 × 106/l (150–350 × 106/l).
Figure 1 Patient's Clinical Course, Complete Blood Count, and Reticulocyte Count
The x-axis shows specific dates. In 1985, prior to starting anticonvulsants, the only abnormality was an MCV of 106 fl. On admission (a), in 2001, the only abnormality was an increased MCV of 119 fl. During the second week of admission (b), her WBC, then her platelet count, dropped to their lowest levels, while her Hb showed a gradual decline. Bone marrow biopsy showed suppression of all marrow elements. After stopping carbamazepine, there was a brisk recovery of her WBC and platelet counts. Six weeks later (c), in February 2002, her Hb had dropped to 31 g/l, and she was given a transfusion of packed cells. Eight weeks later (d), in April 2002, despite erythropoietin and steroid therapy, her Hb dropped to 53 g/l and she received another transfusion. At this time, the sodium valproate was stopped. The reticulocyte count had remained abnormally low throughout this period (a–d), and it was only after stopping the valproate that the reticulocyte count and Hb started to rise. Her MCV dropped after the first transfusion and did not rise again until there was a brisk reticulocyte response. ANC, absolute neutrophil count; retic, reticulocytes; Plat(s), platelets.
She had been taking carbamazepine for 17 y and sodium valproate for 13 y for a mixed seizure disorder. At age 22 y, before starting any anticonvulsants, her baseline hematological parameters were as follows: Hb,123 g/l; MCV, 106 fl; platelets, 296 × 106/l; and WBC, 10.7 × 106/l. After starting carbamazepine, her WBC dropped to 4.5–5.5 × 106/l, and her absolute neutrophil count dropped from 9 × 106/l to about 2.5 × 106/l. When the sodium valproate was added, her MCV increased to 112 fl, her platelet count fell to 100–150 × 106/l, and her Hb dropped to 110–120 g/l.
The patient's other medications on admission were carnitine and low-dose L-thyroxine for hypothyroidism; she had been taking both for several years. She had not been recently exposed to any new medications, environmental toxins, or over-the-counter dietary supplements. There was no family history of aplastic anemia. The patient lived at home with her mother, who cares for her and has legal guardianship.
A bone marrow biopsy (Figure 2) showed a hypocellular bone marrow with a normal myeloid:erythroid ratio and no malignant cells or megaloblastic changes. Cytogenetic study showed a Robertsonian (i.e., of the whole arms) translocation of Chromosome 14 and 21 involving the long arm of Chromosome 21. Except for mild adrenal insufficiency noted on an adrenocorticotropic hormone stimulation test, all her tests, including viral and immunological investigations to identify known causes of aplastic anemia, were negative. The patient's thyroid function tests were normal. She was started on low-dose hydrocortisone for her adrenal insufficiency and megestrol acetate to increase her appetite.
Figure 2 Bone Marrow Biopsy on Admission, Showing Predominantly Hypocellular Bone Marrow
The patient's carbamazepine was discontinued. Within 10 d, her WBC had risen to 6.7 × 106/l and her platelet count to 248 × 106/l. Serial complete blood counts showed worsening anemia over the next 15 wk, requiring two packed red blood cell transfusions at week 6 and week 14, when her Hb was 31 and 53 g/l, respectively. Throughout this period, her reticulocyte count was low, at about 0.18% (normal range 0.5%–2%), while her WBC and platelet count were normal. We made a diagnosis of pure red cell aplasia (PRCA). A second bone marrow biopsy under minimally conscious sedation was unsuccessful, and the patient declined further attempts. After the first transfusion she was started on prednisone and erythropoietin for her PRCA. Meanwhile, because of her worsening seizures, we increased her sodium valproate dose, increasing her serum valproate level from 70 mcg/ml to 110 mcg/ml. After the second transfusion, we discontinued her sodium valproate and started her on clonazepam and oxcarbazepine as alternative anticonvulsants.
The patient had a brisk reticulocyte response. Her reticulocyte count rose from 0.36% to 0.78% in the second week and to 6.61% in the fourth week after stopping the valproate. Six weeks after stopping the drug, her Hb was 125 g/l and her MCV was 106 fl, suggesting replacement of transfused red blood cells with newly formed red blood cells (which have a higher MCV than older transfused cells). Over the next 30 mo of follow-up, she had no relapse of her aplastic anemia.
We were unable to identify a specific cause for the patient's anorexia and weight loss. We found no evidence of malignancy on admission or subsequent follow-up. Within 1 wk of stopping her sodium valproate, her appetite improved and she put on 15 lbs over the next 6 wk. By the fourth month after stopping the drug, she had gained 45 lbs.
We last saw the patient in August 2004. Her seizure control had worsened, and she had developed signs of early dementia. Her current anticonvulsants are clonazepam, oxcarbazepine, and zonisamide, and she continues taking synthroid. Her last complete blood count was stable: WBC, 6.4 × 106/l; Hb, 147 g/l; MCV, 104.9 fl; and platelets, 262 × 106/l.
Discussion
Sodium valproate and carbamazepine are associated with rare but potentially lethal hematological complications. There are five case reports of PRCA shortly after initiation of sodium valproate, with the longest interval between the initiation of therapy and the onset of aplasia being 2 y [1,2,3,4,5]. Acute bone marrow suppression with leucopenia and thrombocytopenia associated with carbamazepine most often occur within 4 mo of starting treatment [6,7]. As far as we know, our case report is unique in that the patient developed acute bone marrow suppression and PRCA after 17 y of carbamazepine and 13 y of sodium valproate therapy.
Our investigations ruled out most of the known causes of acute bone marrow suppression, making the anticonvulsants the most likely cause. Malnutrition was an unlikely cause: her WBC and platelet counts had recovered before any increase in appetite or weight gain; her body mass index was normal despite her weight loss; and her bone marrow iron stores were adequate and her serum folate and vitamin B12 levels were high, suggesting adequate nutrient supply.
We considered and rejected the possibility of Down syndrome–associated aplastic anemia. There have been six case reports of this condition, and in all cases it occurred in young children, suggesting a genetic predisposition [8]. Half of these patients died, and half responded partially to androgenic steroids. Our patient was an adult, and her bone marrow had responded to the withdrawal of her anticonvulsants and not to the administration of androgenic steroid. She had failed to respond to a 6-wk course of high-dose non-androgenic steroid and erythropoietin. Furthermore, our patient did not have a relapse of her aplastic anemia in 30 mo of follow-up, suggesting a lack of genetic disposition.
The brisk return of her WBC and platelet counts upon discontinuing carbamazepine, and her brisk reticulocytosis upon discontinuing sodium valproate, were both consistent with previous reports of hematological toxicity due to these drugs [1,2,3,4,5,6,7]. What was unusual in our case was the extremely late onset of bone marrow suppression after initiation of drug therapy. The persistent suppression of erythropoietic elements for 15 wk after stopping the carbamazepine was unlikely to be due to persistent residual marrow suppression by carbamazepine, since carbamazepine-induced PRCA responds quickly (within 1–2 wk) to stopping the drug [6,9]. We believe that the continued use of sodium valproate, after stopping the carbamazepine, caused the persistent suppression of erythropoietic elements.
We found no cause for the patient's anorexia and weight loss, but her appetite returned and she gained weight after stopping the sodium valproate. There is a known association between this drug and anorexia [10].
Learning Points
Acute hematological complications of anticonvulsant therapy can still occur after many years of therapy, so continued vigilance is warranted.
Although sodium valproate is generally associated with increased appetite and weight gain, it can also be associated with anorexia, nausea, vomiting, and weight loss.
Citation: The T, Kolla R, Dawkins F, Trouth AJ (2004) Pure red cell aplasia after 13 year of sodium valproate, and bone marrow suppression after 17 years of carbamazepine. PLoS Med 1(2): E51.
Abbreviations
Hbhemoglobin
MCVmean cell volume
PRCApure red cell aplasia
WBCwhite blood cell count
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References
Farkas V Szabo M Renyi I Kohlheb O Benninger C Temporary pure red-cell aplasia during valproate monotherapy: Clinical observations and spectral electroencephalographic aspects J Child Neurol 2000 15 485 487 10921523
Anzai K Kitajima H Kubo M A case of pure red cell aplasia associated with sodium valproate therapy Rinsho Ketsueki 1994 35 286 290 8158850
Kawauchi K Miyano T Ikeda Y Tateoka N Kasai M A report of a case with pure red cell aplasia induced by sodium valproate Acta Paediatr Jpn 1989 31 615 619 2515743
Hirose Y Konda S Depakene-induced intravascular hemolysis and pure red cell aplasia Nippon Ketsueki Gakkai Zasshi 1984 47 1366 1370 6440398
MacDougall LG Pure red cell aplasia associated with sodium valproate therapy JAMA 1982 247 53 54 6796707
Tohen M Castillo J Baldessarini RJ Zarate C Kando JC Blood dyscrasias with carbamazepine and valproate: A pharmacoepidemiological study of 2,228 patients at risk Am J Psych 1995 152 413 418
7. Sobotka JL Alexander B Cook BL A review of carbamazepine's hematologic reactions and monitoring recommendations DICP 1990 24 1214 1219 2089834
Pavithran K Raji NL Aplastic anemia in Down's syndrome Am J Hematol 2003 73 213 12827661
Tagawa T Sumi K Uno R Itagaki Y Fujii F Pure red cell aplasia during carbamazepine monotherapy Brain Dev 1997 19 300 302 9187483
Abbott Laboratories Depakene product information In: Physicians' desk reference, 58th ed 2004 Montvale, New Jersey Thomson PDR 425 430
| 15578114 | PMC529434 | CC BY | 2021-01-05 10:38:00 | no | PLoS Med. 2004 Nov 30; 1(2):e51 | utf-8 | PLoS Med | 2,004 | 10.1371/journal.pmed.0010051 | oa_comm |
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BMC GastroenterolBMC Gastroenterology1471-230XBioMed Central London 1471-230X-4-251547154510.1186/1471-230X-4-25Research ArticleOxytocin and cholecystokinin secretion in women with colectomy Ohlsson Bodil [email protected] Jens F [email protected] Mary L [email protected] From the Department of Medicine, University Hospital, S-205 02 Malmö, Sweden2 Department of Clinical Biochemistry, Rigshospitalet, 2100 Copenhagen, Denmark3 Neuroendocrine Labs, Guy's King's and St Thomas Schools of Medicine, SE1 1UL London, United Kingdom2004 7 10 2004 4 25 25 22 4 2004 7 10 2004 Copyright © 2004 Ohlsson et al; licensee BioMed Central Ltd.2004Ohlsson et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Cholecystokinin (CCK) concentrations in plasma have been shown to be significantly higher in colectomised subjects compared to healthy controls. This has been ascribed to reduced inhibition of CCK release from colon. In an earlier study CCK in all but one woman who was colectomised, induced release of oxytocin, a peptide present throughout the gastrointestinal (GI) tract. The aim of this study was thus to examine if colectomised women had a different oxytocin response to CCK compared to healthy controls.
Methods
Eleven women, mean age 34.4 ± 2.3 years, who had undergone colectomy because of ulcerative colitis or constipation were studied. Eleven age-matched healthy women served as controls. All subjects were fasted overnight and given 0.2 μg/kg body weight of CCK-8 i.v. in the morning. Samples were taken ten minutes and immediately before the injection, and 10, 20, 30, 45, 60, 90 and 120 min afterwards. Plasma was collected for measurement of CCK and oxytocin concentrations.
Results
The basal oxytocin and CCK concentrations in plasma were similar in the two groups. Intravenous injection of CCK increased the release of oxytocin from 1.31 ± 0.12 and 1.64 ± 0.19 pmol/l to 2.82 ± 0.35 and 3.26 ± 0.50 pmol/l in controls and colectomised women, respectively (p < 0.001). Given the short half-life of CCK-8 in plasma, the increased concentration following injection could not be demonstrated in the controls. On the other hand, in colectomised women, an increase of CCK in plasma was observed for up to 20 minutes after the injection, concentrations increasing from 1.00 ± 0.21 to a maximum of 1.81 ± 0.26 pmol/l (p < 0.002).
Conclusion
CCK stimulates the release of oxytocin in women. There is no difference in plasma concentrations between colectomised and controls. However, colectomy seems to reduce the metabolic clearance of CCK. The hyperCCKemia in patients who had undergone colectomy is consequently not only dependent on CCK release, but may also depend on reduced clearance.
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Background
The gut hormone cholecystokinin (CCK) is synthesised in endocrine I cells in the mucosa of the upper small intestine [1] and is released into the blood after ingestion of fatty and protein-rich meals [2]. CCK has various effects on the gastrointestinal (GI) tract and acts on afferent vagal nerves [3], neurons of the myenteric plexus [4], and directly on muscle cells [5]. It is also synthesised in central neurons including hypothalamic, oxytocinergic neurons [6]. Circulating CCK is degraded in several sites, namely the kidney, liver and gut [7,8].
Oxytocin is synthesised in the supraoptic and paraventricular nuclei of the hypothalamus as part of a larger precursor polypeptide [9]. While the main effects are in the myoepithelial cells and uterine smooth muscle in the responses associated with the milk ejection reflex and parturition, the possibility has been raised that oxytocin also contributes to control of the GI motility [10,11]. Both exogenous and food-stimulated endogenous CCK stimulates the pituitary secretion of oxytocin in the rat through CCK-receptors on afferent vagal neurons [12]. In hypothalamus, both parvocellular neurons projecting to the dorsal vagal complex, and magnocellular neurons projecting to the pituitary, secrete oxytocin in response to CCK [13].
We have recently found that CCK also leads to oxytocin release in healthy women [14]. However, one of the women included was colectomised, and she was the only one who had no release of oxytocin in response to CCK [14], although colectomy leads to higher concentrations of CCK in plasma [15-17]. We have found mRNA for oxytocin and its receptor throughout the GI tract [18], as well as the fully expressed proteins (unpublished observation). We do not know if this has an autocrine and/or paracrine role in the gut, or if it also is released into the blood as a hormone. The aim of this study was therefore to examine if women who had performed a colectomy, had a different oxytocin response to CCK than otherwise healthy women with an intact GI tract.
Methods
Subjects
Eleven women from the Departments of Medicine and Surgery at Malmö University Hospital (mean age 34.4 ± 2.3 years, range 22–42 years) were studied. They had all a history of colectomy. Two were colectomised because of slow transit constipation (STC) and had undergone a subtotal colectomy with the creation of ileo-rectal anastomosis. Proctocolectomy with ileal pouch-anal anastomosis had been performed in seven of them because of ulcerative colitis, and one because of familial multiple polyposis. The last patient has an ileostomy after subtotal colectomy, saving the rectum, because of ulcerative colitis. Thus, the subjects were cured from their original conditions. The time interval between the proctocolectomy/colectomy and this study was 10–149 months, with a mean of 48.5 ± 12.6 months. Eleven age-matched healthy women with preserved GI tract served as controls. Physical examination and laboratory routine screening were all within normal limits in both groups. The body weight was 68.7 ± 5.8 kg in the patients and 73.8 ± 6.1 kg in the controls. No drugs and no oral contraceptives or other hormonal treatments were allowed in either group. The experiments were performed at no specific stage of the menstrual cycle. None of the included subjects had participated in our former study [14].
Protocols
The protocols were approved by the local Ethics committee at the University of Lund, and written informed consent was obtained from all subjects before the study was started. The possibility of pregnancy was excluded in all women.
Experimental procedure
All subjects were fasted overnight. In the morning they were given 0.2 μg/kg body weight of cholecystokinin octapeptide (CCK-8) (Clinalfa, Switzerland) as an intravenous injection. This bolus was chosen as it was the only dose giving raise to a weak, but not significant, oxytocin release in an earlier study [19]. Blood samples were taken through an intravenous catheter 10 min before and immediately before the injection, and 10, 20, 30, 45, 60, 90 and 120 min after the injection.
Hormone analysis
All blood samples consisted of 8.0 ml whole blood drawn into iced heparinised tubes. The plasma was separated and frozen at -20°C immediately after the experiment. Oxytocin was measured as described by Balment et al [20] using the Fourth International Standard for oxytocin (76/575). The lower limit of detection for this assay was 0.1 pmol/l with intra-assay and interassay variations of 5.4 and 11.8 %, respectively, at 2.5 pmol/l. The hormone was extracted from plasma using C 18 Sep Pak Columns (Waters Associates Ltd., Northwick, Middx., U.K.). The concentrations of CCK in plasma were measured using a highly specific and accurate radioimmunoassay as previously described [21]. The limit of detection for his assay is 0.1 pmol/l with intra-assay and interassay variations of less than 5 % and 15 %, respectively, at both 3.7 and 15 pmol/l concentrations.
Statistical analysis
The values are expressed as mean ± standard error of the mean (SEM). The basal value is the mean of the two fasting values. The peak value is the mean of the highest concentration in every subject after the injection. The total plasma CCK and oxytocin response was assessed by calculating the area under the plasma concentration time curve (AUC). The Kruskal-Wallis followed by Wilcoxon signed ranks test were used for assessment of the significance of the differences within and between the two groups. The Spearman rank test was used for calculating the correlation between CCK and oxytocin concentrations in plasma. Probabilities of less than 0.05 were considered significant.
Results
Plasma oxytocin concentrations
The basal oxytocin concentration in plasma was similar in the two groups. The concentration was stable before the start of the experiments. Injection of CCK-8 led to an increase of the oxytocin secretion compared to basal values in both groups (p < 0.001) (Table 1). The increase in plasma concentration of oxytocin was observed after 10 min and persisted throughout the study. The highest concentration was found after 20 min (Fig 1). There was no difference of the AUC between the two groups, neither there was any difference between each time point studied (Fig 1).
Table 1 Basal and peak plasma values of cholecystokinin (CCK) and oxytocin
CCK (pmol/l) N = 11 Oxytocin (pmol/l) N = 11
Controls
Basal 0.7 ± 0.1 1.3 ± 0.1
Peak 0.9 ± 0.1 2.8 ± 0.4***
Patients
Basal 1.0 ± 0.2 1.6 ± 0.2
Peak 1.8 ± 0.3**+ 3.3 ± 0.5***
Values are expressed as mean value ± standard error of the mean (SEM). Comparisons are made within groups; ** = p < 0.01, *** = p < 0.001, and between groups; + = p < 0.05. Wilcoxon signed rank test. The basal value is the mean of the two fasting values. The peak value is the mean of the highest concentration in every subject after the injection.
Figure 1 The plasma concentration of oxytocin before and at different time points after an injection of 0.2 μg/kg body weight of cholecystokinin-8 (CCK-8). There were 11 subjects in each group. Values are given as mean and standard error of the mean (SEM). There was no difference between the groups neither when calculating values at different time points studied nor the area under the curve (AUC). Wilcoxon signed rank test. = control, = patient.
Plasma cholecystokinin concentrations
There was a tendency towards higher basal CCK concentration in patients, although not significant (Table 1 and Fig 2). CCK-8 has a half-life in plasma of about < 1 min (8). Therefore, no increase in plasma CCK could be detected in the control group after the intravenous injection of CCK-8 (Table 1). However, in colectomised women, an increase in plasma CCK concentrations was found after the injection compared to basal values (p < 0.002). The difference of peak value between the groups was significant (p < 0.04) (Table 1). The AUC differed significantly between the two groups (p < 0.04), but no difference was observed between values at each time point studied (Fig 2).
Figure 2 The plasma concentration of cholecystokinin (CCK) before and at different time points after an injection of 0.2 μg/kg body weight of CCK-8. There were 11 subjects in each group. Values are given as mean and standard error of the mean (SEM). When calculating the area under the curve (AUC), there was a significantly increased AUC in the colectomised subjects compared to controls (p < 0.04). No difference was seen between the groups when comparing values at each time point. Wilcoxon signed rank test. = control, = patient.
There was no correlation between CCK and oxytocin concentrations (data not shown). Neither was there any difference in CCK and oxytocin concentrations between patients with different diagnosis and those who had rectum saved or resected (data not shown).
Discussion
This study shows for the first time that CCK-8 increases the secretion of oxytocin in women. We have previously shown that exogenous CCK-33 and -39, and a fatty meal with endogenous CCK release, led to enhanced concentrations of oxytocin in plasma [14]. One patient in that study was colectomised, and she was the only one in whom no increase in oxytocin release was seen after CCK stimulation. This observation prompted the present study. In this study, there was no difference in plasma concentrations of oxytocin between colectomised and healthy controls. Thus, the oxytocin secreted into the blood after CCK stimulation seems not to origin from the colon. The oxytocin recently found in the colon may participate in autocrine and/or paracrine regulation of the gut while having no endocrine effects [18]. The patient group examined in this study was not homogenous, but it was not possible to include enough young women with colectomy after ulcerative colitis. We have earlier described the presence of oxytocin and its receptor throughout the gut, without any efforts to quantify the expression [18]. Only the effect of colon on plasma concentrations of oxytocin was measured in the present study. We do not know from this study if oxytocin from some other part of the gut is released into the plasma. It is difficult to conduct an experiment to examine the origin from the oxytocin release. CCK acts on receptors on afferent vagal nerves to stimulate the oxytocin release from the pituarity [12], and these receptors are present throughout the GI tract [3-5]. Therefore it is not possible to use CCK-receptor antagonists to distinguish between central or local CCK effects.
CCK has been shown to stimulate oxytocin secretion in mammals in many studies [12-14]. However, in a previous study, intravenous injection of CCK-8, in the same dose as in our study, did not increase the concentration of oxytocin in plasma [19]. This may depend on methodological differences. Another possible explanation to the difference is the effect of gonadal hormones on the regulation of the oxytocin release from the posterior pituitary gland. In our study, only women were included, whereas Miaskiewicz et al [19] examined 13 men and one woman. Orally administered estrogen stimulates oxytocin secretion, and progesterone also affects release [22]. Lower plasma levels of these hormones in men may explain the absence of increased oxytocin secretion in men. In addition, one study has shown that testosterone inhibits the secretion of oxytocin from the pituitary gland [23].
Oxytocin is present in plasma in men, although at lower concentrations [24,25], and shows a circadian rythm [26]. Oxytocin may have similar effects on the GI tract in men and women, although the plasma concentrations differ. The effects of oxytocin have been only rudimentary examined. However, oxytocin has in one study been shown to enhance gastric emptying [10], and in a yet unpublished study, we have found that an oxytocin-receptor antagonist delayed the gastric emptying rate (unpublished observation). Further, we have demonstrated increased colonic peristaltis after oxytocin stimulation in healthy women [11]. Our finding of oxytocin release in response to a meal [14], and the presence of oxytocin receptors on the cells that regulate the gut motility (unpublished observation), suggest oxytocin to play a physiological role in the GI function.
Several studies have reported that after colectomy in different species there are higher concentrations of CCK in plasma, both basal and postprandial, compared to healthy controls [15-17]. It has been suggested that this is due to depletion of an inhibitory factor of CCK secretion which is released from the colon. Peptide YY (PYY) is secreted from distal ileum and colon, and CCK is known to stimulate PYY secretion from the hindgut [27-29]. PYY then inhibits further CCK secretion [30,31]. As PYY is secreted from the hindgut, this peptide is substantially reduced after colectomy [26]. Thus, the reduced PYY concentration may explain the hyperCCKemia. In this study, the elevated CCK concentrations in the group of colectomised women were not due to increased secretion of CCK, as CCK was injected exogenously. Instead, the hyperCCKemia in the group of colectomised patients seems to be due to reduced degradation of the peptide injected. CCK is degraded in the kidney, liver and gut [7,8]. Our hypothesis is that PYY could contribute to the degradation as well as the secretion of CCK. Receptors for PYY have been found in the kidney and on hepatocytes, and PYY influences the renal and hepatocyte metabolism [32-34]. Alternatively, the different CCK concentrations could be due to reduced degradation in the colon in addition to the kidney and liver, as CCK-8 has been shown to be degraded in the gut in pigs [8]. It remains to be determined which mechanism contributes most to the hyperCCKemia observed after colectomy; increased CCK secretion, or decreased clearance. In the present study, the basal levels of CCK did not differ significantly in colectomised, as observed in earlier studies [15-17].
CCK has a wide range of effects on the GI tract. Three physiological effects on gut motility have been identified; contraction of the gallbladder [2], relaxation of the sphincter Oddi [35] and inhibition of gastric emptying [2]. Its role on colonic motility is controversial. While Barone et al [36] were able to demonstrate contractions, Niederau et al could find no effect of CCK [37]. Further, CCK increases pancreatic enzyme secretion [2]. It is not known if the hyperCCKemia observed in colectomised patients [15-17] has any impact on GI motility or health.
Conclusions
CCK stimulates the release of oxytocin in women, probably via an effect on the neurohypophysial system. There is no difference in plasma concentrations between colectomised women and women with intact GI tract. The hyperCCKemia observed in patients who have undergone colectomy is dependent not only on an increase in CCK release, but may also depend on a reduced degradation. It was beyond the aim of our study to determine the clearance of CCK. However, this should be evaluated further.
Abbreviations
AUC = area under the curve
CCK = cholecystokinin
GI = gastrointestinal
PYY = peptide YY
SEM = standard error of the mean
Authors' contributions
BO designed the study, included patients, paid for the most, performed the statistical analysis and drafted the manuscript
JR carried out the radioimmunoassay for CCK and participated in the writing process
MF carried out the radioimmunoassay for oxytocin and participated in the writing process
All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We thank Mariette Bengtsson for technical assistance, Kirsten Dahl and Mikael Truedsson for recruiting subjects, and Jan-Åke Nilsson for expert statistical calculations. This study was supported by grants from Nio Meter Liv, Ruth and Richard Juhlin and Åke Wiberg. The Development Foundation of Region Skåne and the drug company Novo Nordic paid our salaries while working with this project.
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| 15471545 | PMC529435 | CC BY | 2021-01-04 16:29:55 | no | BMC Gastroenterol. 2004 Oct 7; 4:25 | utf-8 | BMC Gastroenterol | 2,004 | 10.1186/1471-230X-4-25 | oa_comm |
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BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-5-1541549149910.1186/1471-2105-5-154Research ArticleInformation assessment on predicting protein-protein interactions Lin Nan [email protected] Baolin [email protected] Ronald [email protected] Mark [email protected] Hongyu [email protected] Department of Mathematics, Washington University in St. Louis, St. Louis, MO 63130, USA2 Division of Biostatistics, School of Public Health, University of Minnesota, Minneapolis, MN 55455, USA3 Computational Biology Center, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA4 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA5 Department of Computer Science, Yale University, New Haven, CT 06520, USA6 Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT 06520, USA7 Department of Genetics, Yale University School of Medicine, New Haven, CT 06520, USA2004 18 10 2004 5 154 154 31 5 2004 18 10 2004 Copyright © 2004 Lin et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Identifying protein-protein interactions is fundamental for understanding the molecular machinery of the cell. Proteome-wide studies of protein-protein interactions are of significant value, but the high-throughput experimental technologies suffer from high rates of both false positive and false negative predictions. In addition to high-throughput experimental data, many diverse types of genomic data can help predict protein-protein interactions, such as mRNA expression, localization, essentiality, and functional annotation. Evaluations of the information contributions from different evidences help to establish more parsimonious models with comparable or better prediction accuracy, and to obtain biological insights of the relationships between protein-protein interactions and other genomic information.
Results
Our assessment is based on the genomic features used in a Bayesian network approach to predict protein-protein interactions genome-wide in yeast. In the special case, when one does not have any missing information about any of the features, our analysis shows that there is a larger information contribution from the functional-classification than from expression correlations or essentiality. We also show that in this case alternative models, such as logistic regression and random forest, may be more effective than Bayesian networks for predicting interactions.
Conclusions
In the restricted problem posed by the complete-information subset, we identified that the MIPS and Gene Ontology (GO) functional similarity datasets as the dominating information contributors for predicting the protein-protein interactions under the framework proposed by Jansen et al. Random forests based on the MIPS and GO information alone can give highly accurate classifications. In this particular subset of complete information, adding other genomic data does little for improving predictions. We also found that the data discretizations used in the Bayesian methods decreased classification performance.
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Background
Proteins transmit regulatory signals throughout the cell, catalyze large numbers of chemical reactions, and are crucial for the stability of numerous cellular structures. Interactions among proteins are key for cell functioning and identifying such interactions is crucial for deciphering the fundamental molecular mechanisms of the cell. As relevant genomic information is exponentially increasing both in quantity and complexity, in silico predictions of protein-protein interactions have been possible but also challenging. A number of techniques have been developed that exploit combinations of protein features in training data and can predict protein-protein interactions when applied to novel proteins. Our study is motivated by a study by Jansen et al. [1], who proposed a Bayesian method to use the MIPS [2] complexes catalog as gold standard positives and lists of proteins in separate subcellular compartments [3] as gold standard negatives. The various protein features considered in this method include time course mRNA expression fluctuations during the yeast cell cycle [4] and the Rosetta compendium [5], biological function data from the Gene Ontology [6] and the MIPS functional catalog, essentiality data [2], and high-throughput experimental interaction data [7-10]. The MIPS and Gene Ontology functional annotations are used for quantifying the functional similarity between two proteins. The MIPS functional catalog (or GO biological process annotation) can be thought of as a hierarchical tree of functional classes (or a directed acyclic graph (DAG) in the case of GO). Each protein is either a member or not a member of each functional class, such that each protein describes a "subtree" of the overall hierarchical tree of classes (or subgraph of the DAG in the case of GO). Given two proteins, one can compute the intersection tree of the two subtrees associated with these proteins. This intersection tree can be computed for the complete list of protein pairs (where both proteins of each pair are in the functional classification), and thus a distribution of intersection trees is obtained. Then the "functional similarity" between two proteins is defined as the frequency at which the intersection tree of the two proteins occurs in the distribution. Intuitively, the intersection tree gives the functional annotation that two proteins share. The more ubiquitous this shared functional annotation is, the larger is the functional similarity frequency; the more specific the shared functional annotation is, the smaller is the functional similarity frequency. The essentiality data represents a categorical variable that denotes whether zero, one or both proteins in a protein pair are essential. The supplementary online material of [1] provides more details about the quantification of these variables. Their Bayesian method predicts protein-protein interactions genome-wide by probabilistic integration of genomic features that are weakly associated with interactions (mRNA expression, essentiality and localization). The model was used for two separate predictions of probabilistic interactomes (PI), one of which (PIE) is built on four high-throughput experimental interaction data sets, and the other (PIP) on the mRNA expression, Gene Ontology, MIPS functional and coessentiality data. Within the PIP sub-network, different genomic features are assumed to be independent in prior. In addition, this method involved discretizing the raw data into groups and representing the two mRNA expression profiles (cell cycle and Rosetta compendium data) by their first principal component for computational convenience.
Our current study focuses on assessing the contributions of different types of genomic data towards predicting protein-protein interactions. This may help us to understand which genomic features have the closest biological relationship with protein-protein interactions and hence to construct a better prediction model. As prediction rules involving less relevant information may have lower prediction accuracy, our analysis can give us insights into how to construct more parsimonious models with comparable or better prediction accuracy. A potential disadvantage of the Bayesian network approach may be that the data discretization can obscure information contained in the raw genomic data. Thus, in addition to assessing the information content of the data sources, we also propose alternative non-Bayesian models that fully utilize the data without discretization. These methods, such as logistic regression and random forests, do not require prior knowledge, and we can evaluate the importance of the different genomic features in the context of these methods.
Results and discussions
To accurately and quantitatively assess the information contributions of different genomic features, we construct in essence a simplified problem that has some but not all of the elements of the original study. Here, we only look at a subset of the data from [1] comprising the 18 million protein pairs in total and approximately 8,000 gold standard positives and 2.7 million gold standard negatives. This subset (see Additional File 1) contains 2,104 positives and 172,409 negatives. In this subset, we have complete information for each feature and we can thus quantitatively assess the relative contributions of the different features on this set. This data set can be downloaded from . In doing so, we find that some of the features have stronger influence on the overall prediction. While this might be true for the larger problem as well, there are a number of caveats that one has to keep in mind, such as that the features that are present in this subset might not be the strongest in the whole set of 18 million protein pairs.
Alternative models
Here, we construct models for predicting protein-protein interactions that, given the gold standards, are basically dichotomous classifiers. Multiple logistic regression [11] is one commonly used model for such an application [12,13]. An alternative, more sophisticated supervised learning approach that we apply is the random forest algorithm [14]. Note that, although not our focus here, all these methods can be used to compute the estimated probabilities for predicted protein-protein interactions.
Logistic regression
The logistic model has the advantage that it provides an estimated probability that a pair of proteins interact, and is readily available in standard statistical packages. In this paper, the logistic regression analysis was generated using PROC LOGISTIC in SAS/STAT software, Version 9 of the SAS System. Moreover, we can evaluate the importance of different genomic features by variable selections. Among many available schemes, we chose stepwise variable selection that is widely used in standard packages. Stepwise selection is a greedy search algorithm that selects variables with the best marginal prediction power given the current model. To quantify the importance of the predictor variables to the model fitting, we can use the deviance measure
-2(log L1 - log L0),
where L0 is the likelihood of the final model given by the stepwise selection, and L1 is the likelihood of the reduced model by removing all terms that involve the corresponding predictor variable from the final model. However, this measure only considers the prediction power of variables for the training sample but not for any random test samples. Therefore, this measure can be biased due to its dependence on the training sample.
We consider, similarly as in [1], all the main effects and interaction terms among the genomic features in the PIP (indirect evidence for protein-protein interactions) and the PIE (direct experimental protein-protein interaction measurements) respectively. Table 1 presents all the terms remained in the final model and their orders to enter the final model. Table 2 shows the deviance measure of predictor variables. The Gavin data, Gene Ontology and MIPS functional similarity features, and the cell cycle gene expression data are the most important genomic evidences for predicting protein-protein interactions according to the deviance measure, whereas the three other high-throughput experimental data sets are less relevant or even do not have significant effects to be included in the final model. However, the logistic model is restricted by its linear form and may not provide an optimal solution to the prediction problem. And it will be more objective to evaluate the variable importance according to its prediction accuracy for any random test samples. In the following, we present the results from using the random forest, a more sophisticated supervised learning algorithm.
Random forest
The "random forest" method [14] is a supervised learning algorithm that has previously been successfully applied to many genomic studies. It has been implemented in the randomForest package of R [15]. A random forest is an ensemble of many classification trees generated from bootstrap samples of the original data. It is well known that random forests avoid overfitting and usually have better classification accuracy than classification trees. A natural way to evaluate the importance of the feature variables with the random forest algorithm is to measure the increase of the classification error when those variables are permuted. Intuitively, the more important variables will, when permuted, produce larger classification errors. The importance score provided by the random forest is a more accurate estimate of the classification error that considers the situation of random test samples. Therefore, this importance score provides a more objective evaluation of the relative merit of different genomic features on protein-protein interaction prediction. Moreover, the intrinsic tree structure of the random forest easily takes into account the interactions among the different variables and avoids complications caused by missing data that occurred in many other modeling procedures.
We performed our random forest analysis by growing 5,000 trees. Figure 1 shows the importance measures of the genomic evidences used in the random forest algorithm. The result agrees mostly with that of the logistic regression in that the MIPS and Gene Ontology functional similarity features are found to be very important, whereas most of the high-throughput experimental data sets have negligible effects. However, different from the result from logistic regression, the Gavin data set is shown to be less important than MIPS and Gene Ontology functional similarity features after considering the situation of random test samples. These observations motivated us to perform a more thorough information assessment of the genomic evidences considered. We first compared the performance of different classification methods (random forest, logistic regression and Bayesian network), and then evaluated the importance of the different genomic datasets within the framework the best method (the method with the lowest classification error).
Comparison of three methods
We conducted 7-fold cross validations on the subset with complete information (described above) on all the features for random forest, logistic regression and the Bayesian network method. Figure 2 displays their receiver operating characteristic (ROC) curves, where we observe a better performance of the random forest over the other two and similar performances between logistic regression and the Bayesian network.
Information assessment
Information assessment of different genomic data may help us understand their relationship with protein-protein interactions, and form a guideline for future model development.
MIPS and gene ontology functional similarity data
We saw that the MIPS and Gene Ontology functional similarities were the two most important information sources under both the logistic regression and random forests methods. Histograms of the MIPS and GO functional similarity data (Figures 3 and 4) show that they are very different for the gold standard positives and negatives; protein pairs in the gold standard positives are associated with smaller functional similarity values than the gold standard negatives. This pattern explains why the functional similarity features have such a strong impact on classification accuracy in the model fitting, as observed in Figure 2. However, the vast number of protein pairs in the gold standard negatives are likely to be those that have not been thoroughly studied by researchers, and henceforth are observed to belong to large functional categories that actually should be further divided into more specific categories. This conjecture suggests that the information from MIPS and Gene Ontology function data is possibly caused by selection biases other than intrinsic biological relevance. It deserves further investigations of the relationship between the gold standards and the MIPS and Gene Ontology functional similarity data.
In the following paragraphs, we show quantitatively that the MIPS and Gene Ontology functional similarities are the dominating information contributors for predicting protein-protein interactions, while other genomic features have negligible benefit and can not provide credible predictions by themselves. We examine the performance of random forests using three different genomic feature sets: (i) all genomic features included, (ii) MIPS and Gene Ontology functional similarities only, and (iii) genomic features other than the MIPS and Gene Ontology functional similarities. The random forest performance is evaluated with the classification error (Err) defined as follows.
Denote Err1 as the proportion of protein pairs misclassified in the gold standard positives, and Err2 the counterpart for the gold standard negatives. Then we define the classification error as the average of Err1 and Err2.
Err is a balanced error rate across gold standard positives and negatives. Suppose the joint probability density functions of the predictor features X are f1(X) and f2(X) for the gold standard positives and negatives, respectively. Denote a classifier by C(X). Then the classification error can be written as
where I(A) is an indicator function equal to 1 when A is true and 0 otherwise. A minimal classification error Errmin can be computed by minimizing (1) across the space of X. It is easy to see that
is achieved at C(X) = I(f1(X) >f2(X)). With this formula, we can estimate the optimal (minimum) classification error based on any estimates of f1(X) and f2(X). In our study, f1(X) and f2(X) are estimated by their empirical density functions.
Table 3 presents the optimal classification error using the MIPS and Gene Ontology functional similarity data. Using the MIPS and Gene Ontology functional similarity data sets alone results in a highly accurate classification with an optimal error of only 0.28%. Table 3 also shows the effects of the data discretizations that were originally used in the Bayesian network method ("grouped"). The significant discrepancy between optimal classification errors using the raw data and the discretized data ("grouped") suggests that the discretization causes serious loss of information.
Other genomic features
We also estimated the classification errors using the other genomic features within the random forest framework. Table 4 shows that adding the other genomic evidences in the complete-information subset provides only negligible benefit or even reduces the classification accuracy.
Moreover, we compared the ROC curves (Figure 5) of the random forest method using all genomic information, only the MIPS and GO functional similarities, and the genomic information other than MIPS and GO. Figure 5 shows that we barely gain any by considering other genomic information if the MIPS and GO are available; classifications without the MIPS and GO functional similarity data are poor on the complete-information subset. Note, however, that the subset of full interaction data which have the strongest expression correlations is not necessarily the complete-information set considered. Hence, we would expect that expression correlations might be a stronger source of information in other context.
Conclusions
In the restricted problem posed by the complete-information subset, we identified that the MIPS and Gene Ontology functional similarity datasets as the dominating information contributors for predicting the protein-protein interactions under the framework proposed in [1]. Random forests based on the MIPS and GO information alone can give highly accurate classifications. In this particular subset of complete information, adding other genomic data does little for improving predictions. The MIPS and GO information, however, is only available for a small proportion of the ~18M protein pairs.
We considered alternative non-Bayesian methods such as logistic regression and random forest for predicting protein-protein interactions. These existing methods do not require prior information needed for the Bayesian approach, and can fully utilize the raw data without discretization. The logistic model performs similarly as the Bayesian method in terms of classifications and, like the Bayesian method, produces estimated probabilities that two proteins interact. As a dichotomous classifier, the random forest method outperforms the other methods considered and efficiently uses the information, although it is computationally more expensive. In particular, its importance measure provides a more objective assessment of different genomic features on predicting protein-protein interactions than simply considering contributions to model fitting. These findings are motivation to look for other, more sensible data resources and superior models.
We found that the data discretizations used in the Bayesian methods decreased classification performance. We note here that the genomic features datasets investigated here themselves are highly processed versions of the datasets they were derived from and that there may be better ways to take the original data into account.
Another caveat is that the predictions might be just defining groups of proteins that have the same genomic properties as the protein complexes in the MIPS data. This does not necessarily mean that they really represent protein complexes. Rather, they may represent groups of proteins that have the same properties as protein complexes.
In this analysis we have looked at the relative weights of various features in predicting protein-protein interactions based on the previous study in [1]. We looked at a particular subset of the data where we had complete information and we were able to show that, for this particular subset of the full information, we are able to show that the functional classification features in the MIPS functional catalog and Gene Ontology were the most informative and that particular machine learning algorithms, such as random forests were more effective than Bayesian networks. However, one has to keep in mind that in the full problem there is the issue of incomplete information. On data sets with incomplete information Bayesian approaches maybe more effective because they can easily handle the missing information. Further careful studies such as these will be needed to determine what the optimum machine learning method is and the optimum features are in presence of incomplete information. It will be also of great interest to consider other genomic features such as phylogenetic profiles [16] and local clustering information [17]. This is just the first step in that direction.
Methods
Logistic regression
Denote the gold standards by random variable Y and the other genomic features by X1, X2, ..., Xn. Let Y = 1 when two proteins interact, i.e., they are in the same complex, and Y = 0 when not. The logistic model is of the form
where the random vector X consists of X1, X2, ..., Xn and their interaction terms.
Stepwise variable selection
The stepwise selection procedure starts from a null model. At each step, it adds a variable with the most significant score statistics among those not in the model, then sequentially removes the variable with the least score statistic among those in the model whose score statistics are not significant. The process terminates if no further variable can be added to the model or if the variable just entered into the model is the only variable removed in the subsequent elimination. Here, the score statistic measures the significance of the effect of a variable.
ROC curve analysis
Receiving operator characteristic (ROC) curve [18] is a graphical representation used to assess the discriminatory ability of a dichotomous classifier by showing the tradeoffs between sensitivity and specificity. Sensitivity is calculated by dividing the number of true positives (TP) through the number of all positives, which equals the sum of the true positives and the false negatives (FN); specificity is calculated by dividing the number of true negatives (TN) through the number of all negatives, which equals the sum of the true negatives and the false positives (FP).
Sensitivity = TP/(TP + FN), Specificity = TN/(TN + FP).
The ROC curve plot shows 1 - specificity on the X axis and sensitivity on the Y axis. A good classifier has its ROC curve climbing rapidly towards upper left hand corner of the graph. This can also be quantified by measuring the area under the curve. The closer the area is to 1.0, the better the classifier is; and the closer the area is to 0.5, the worse the classifier is.
Authors' contributions
NL and BW conducted the major part of the data analysis, and created all the tables and figures, under the supervision of HZ. RJ and MG provided the data sets for the analysis and contributed to the discussion on the comparisons of different methods. All authors read and approved the final manuscript.
Supplementary Material
Additional File 1
The complete-information subset in ZIP file.
Click here for file
Acknowledgements
This work was supported in part by NSF grant DMS 0241160 and NIH grant GM 59507.
Figures and Tables
Figure 1 Importance measure of genomic features from the random forest algorithm The horizontal axis presents the importance measure whereas the vertical axis denotes the genomic features.
Figure 2 ROC curves of random forest, logistic regression and Bayesian networks using 7-fold cross validations
Figure 3 Histograms of MIPS and Gene Ontology function data for gold standard positives and negatives
Figure 4 Zoom-in histograms of MIPS and Gene Ontology function data for gold standard positives and negatives on the lower end
Figure 5 ROC curves of random forest using different genomic feature sets 'All' – all genomic information; 'MIPS+GO' – only MIPS and Gene Ontology function data; 'ELSE' – genomic features other than MIPS and Gene Ontology function data
Table 1 Order of variables that enter the final model by stepwise selection in logistic regression
Variables Order
Gavin 1
MIPS 2
Rosetta 3
GO 4
cellcycle 5
essentiality 6
Rosetta*cellcycle 7
cellcycle*essentiality 8
Ho 9
GO*essentiality 10
Uetz 11
GO*cellcycle 12
GO*cellcycle*essentiality 13
MIPS*essentiality 14
MIPS*Rosetta 15
Table 2 Deviance of the reduced model from the final model by removing corresponding variables
Variable Deviance
GO 1376.437
MIPS 1333.97
essentiality 579.988
Rosetta 778.493
cellcycle 1271.461
Ho 68.718
Uetz 20.513
Gavin 1839.181
Table 3 Optimal classification errors when using different genomic features
Variables Optimal Classification Error
MIPS 1.69%
GO 2.15%
MIPS+GO 0.28%
MIPS (grouped) 7.31%
GO (grouped) 13.35%
MIPS+GO (grouped) 6.34%
Table 4 Classification errors of the random forest algorithm when using different genomic features
Variables Err1 (positives) Err2 (negatives) Err
MIPS+GO 114/2104 = 5.42% 180/172409 = 0.1% 2.76%
ALL 165/2104 = 7.80% 89/172409 = 0.05% 3.95%
ELSE 1056/2104 = 78.09% 313/172409 = 0.20% 25.20%
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| 15491499 | PMC529436 | CC BY | 2021-01-04 16:02:45 | no | BMC Bioinformatics. 2004 Oct 18; 5:154 | utf-8 | BMC Bioinformatics | 2,004 | 10.1186/1471-2105-5-154 | oa_comm |
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BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-5-1611550424110.1186/1471-2105-5-161Research ArticleImplications for domain fusion protein-protein interactions based on structural information Chia Jer-Ming [email protected] Prasanna R [email protected] The Genome Institute of Singapore, Singapore2004 26 10 2004 5 161 161 29 6 2004 26 10 2004 Copyright © 2004 Chia and Kolatkar; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Several in silico methods exist that were developed to predict protein interactions from the copious amount of genomic and proteomic data. One of these methods is Domain Fusion, which has proven to be effective in predicting functional links between proteins.
Results
Analyzing the structures of multi-domain single-chain peptides, we found that domain pairs located less than 30 residues apart on a chain are almost certain to share a physical interface. The majority of these interactions are also conserved across separate chains. We make use of this observation to improve domain fusion based protein interaction predictions, and demonstrate this by implementing it on a set of Saccharomyces cerevisiae proteins.
Conclusion
We show that existing structural data supports the domain fusion hypothesis. Empirical information from structural data also enables us to refine and assess domain fusion based protein interaction predictions. These interactions can then be integrated with downstream biochemical and genetic assays to generate more reliable protein interaction data sets.
==== Body
Background
Networks of interacting molecules drive every process in biological cells. Proteins dominate these networks, some of which involve transient interactions such as signal transduction cascades and ligand-receptor interactions, while others form more permanent molecular machineries such as ribosomes and polymerases. Unraveling these networks and interactions will not only help us better understand complex cellular processes, but also enable us to make inferences about the function of individual proteins through 'guilt-by-association' [1].
Over the last few years, high-throughput interaction detection assays have been introduced and refined to complement the traditional genetic and biochemical techniques. High-throughput mass spectrometry protein complex identification (reviewed by Pandey and Mann [2]), and yeast two-hybrid systems [3] are examples of these. The success of these techniques is well illustrated in the budding yeast Saccharomyces cerevisiae, in which networks of its interacting proteome where constructed using genome-wide screens. [4-7]
The wealth of genomic and protein sequences, the increase of 3D structures of protein complexes, together with the deluge of microarray expression data, has provided researchers with an overwhelming body of information that can be used to infer both functional as well as interaction linkages. Clearly, bioinformatics and computational biology are necessary tools for delineating this information. In response to the data explosion, several in silico methods have recently been developed to predict associations from these data.
Phylogenetic profiles focus on the co-occurrences of genes across several organisms. By studying the pattern of evolutionary conservation between sets of genes in different organisms (phylogenetic distribution), it has been shown that these phylogenetic profiles can be successfully used to infer both localization as well as functional association between proteins [8-10]. Protein domains that are found fused together within a protein are frequently involved in the same process, and in many examples proven to be physically interacting. This phenomenon is the basis for the domain fusion analysis, which can be used to predict protein interactions in cases where the fused domain pair is found independently across separate protein chains [11,12].
Structural data has also been mined and analyzed for residue patterns within interfaces between pairs of interacting proteins. These are then used to train learning models for ab initio categorization and prediction of protein interactions [13,14]. Jansen and co-workers [15] illustrated how expression profiles from mRNA expression data could be harnessed and used as an effective source for the prediction of protein interactions.
A number of groups have compared and reported on the protein interaction datasets that are emerging from the various genome-scale biochemical, genetic and in silico experiments [16-18]. All of them drew a similar conclusion; high-throughput methods produce little overlapping results, and taken singularly, each technique has a high error rate (false positive and false negative). Each of these methods has their own specific strength and weakness, and covers a separate subset of interactions. Integrating the various result sets together, allows one to piece together a map of the interacting proteome that is more reliable with higher accuracy, and more informative with higher coverage.
The study by von Mering and co-workers [17] showed that in silico methods have higher coverage and higher accuracy than the majority of biochemical/genetics methods, second only to high-throughput mass spectrometry. The use of sensible strategies and filters has allowed in silico analyses to provide better performance. On top of that, these methods are less biased towards abundant proteins. In silico analyses are indispensable, and further improvements of these methods to make them more accurate will provide a cleaner set of data for downstream biochemical/genetic studies.
In this study, we make use of an empirical observation that domain pairs, which lie in close proximity on a protein chain tend to interact, to refine the domain fusion analysis. This way, we aim to improve the accuracy of the domain fusion analysis.
Domain fusion
The basis for domain fusion (or gene fusion) is the observation that certain proteins (termed the Rosetta stones) in a given species are found to consist of a fusion between two separate proteins in another species. Through fusion, the entropy of dissociation between the two proteins is reduced, and it is hypothesized that in all likelihood, these two separate proteins share a functional association, if not a physical interaction [11,12].
Domains have been described as the primary building blocks of proteins [19], recombining in various permutations, resulting in proteins of completely different functions [20]. In our implementation of the domain fusion analysis, we chose the representation of proteins being composed of domains, separated by linkers on a peptide chain.
In this paper, we make use of existing structural data to support the domain fusion hypothesis. We interrogated known 3D structures for evidence of inter-domain physical interactions on the same chain. We investigated and concluded that there was an association between the distances at which domains are spaced apart on the chain, and the propensity for a domain-pair to interact.
We also show that domain pairs, located in close proximity on a protein chain, are likely to interact even when found residing on different chains, hence proving that the domain fusion hypothesis is valid.
Finally, we demonstrate that peptide chains with closely spaced domains are likely to make better Rosetta stones, and we make use of this observation to improve domain fusion based protein interaction predictions.
Results
Supporting the domain fusion hypothesis
The available structural data indicate that intra-chain domain pairs, which lie in close proximity on a peptide chain, tend to physically interact with one another. The mean distances of interacting intra-chain domain pairs are smaller than ones which do not interact; interacting pairs are on the average 50 residues apart, while non-interacting pairs have a mean distance of 166 residues between them.
In order to verify the correlation between distance and interaction, we made use of contingency tables and the chi-squared test statistic. For a set of inter-domain distances ranging from 5 residues to 200 residues, we constructed 2 × 2 contingency tables that classified domain pairs according to two criterions; 1) whether or not they are separated by a distance no greater than a threshold, and 2) whether or not the domain pair is interacting. The chi-squared value of each table was used as a statistic to test the null hypothesis (H0): Domains pairs separated by no greater than a predefined distance and their tendency to interact were independent. The p-values indicate the probability of having the chi-squared test statistic as extreme as, or larger than observed when H0 is true.
We found that the contingency table for domain pairs spaced up to 30 residues apart had the highest chi-squared value, with a statistically significant p-value of less than 0.001, allowing us to confidently reject H0. This trend is noticeable in the chart illustrating the proportion of interacting pairs across various inter-pair distances (figure 1). Domain pairs located less than 30 residues apart are almost certainly (90%) to be in contact with each other, whereas only half (51%) of domain pairs with more than 30 residues separation were categorized as physically interacting. The chi-squared value is also overlaid on the chart in a dotted line, representing the test statistic from each corresponding contingency table.
In order to validate the domain fusion hypothesis, we not only need to show that domain pairs on the same chain tend to interact with each other, but importantly, this same domain pair will tend to be in contact if they are located independently across separate chains of a polypeptide complex. From our data, we noticed that 71% of domain pairs, which lie within 30 residues of each other on the same chain, could be found physically interacting across separate chains of a complex. In contrast only 38% of domain pairs lying greater than 30 residues apart are seen to be in contact within a multi-chain complex. Once again, putting this into a contingency table and evaluating the chi-squared statistic we reject the null hypothesis (p-value <= 0.001). In other words, there is a correlation between domain-pairs spaced less than 30 residues apart on a single peptide chain and their tendency to interact across separate chains of a polypeptide complex.
30 residues criteria applied to Swiss-Prot proteins
We wanted to verify that the 30 residues criteria could be used as a measure to filter and improve predictions made using the domain fusion methodology. A set of proteins for the budding yeast S. cerevisiae was downloaded from Swiss-Prot, and domain fusion based protein interactions were predicted as described in the Methods section. After filtering for promiscuous domains, a total of 9279 protein interactions remained, of which 28% or 2629 were supported by a Rosetta stone with no more than 30 residues between the fused domains.
The functional category assigned to each protein in an interacting pair was used to gauge the plausibility of the interaction; if two different proteins were found physically interacting, one would expect the two proteins to have overlapping functional categories. 62% of the interacting protein pairs, supported by a 30 residue Rosetta stone, have both partners belonging to the same functional category. The same proportion for interacting pairs not supported by a 30 residue Rosetta stone is 48%. This 14% difference is significant with a p-value of less than 0.001, using a two-sample t-test.
Discussion
In silico methods for predicting protein interactions are not only able to match the accuracy of the other genetic, biochemical and biophysical techniques, but also have the added advantage of providing higher coverage [17]. Among the in silico methods, domain fusion is an attractive technique because it enables a functional link to be drawn between two proteins based solely on their primary sequence. Still, large-scale sets of high-throughput protein interaction data available today are spurious, more than half of them proving to be false positives [17], the challenge remains to improve the quality of high throughput protein interaction data sets.
Protein interactions can be classified as either permanent or transient interactions. The data from this study were taken from the PDB, where most of the submitted structures are results from x-ray crystallography experiments. Consequently, we believe that the vast majority of our deduced domain and protein interactions are physical, permanent interactions.
Our study of multi-domain, single and multiple-chain protein structures in the PDB gave us two results. First of all, it supports the domain fusion hypothesis suggested by Marcotte and Enright. Secondly, it allows us to conclude that single chain peptides with closely spaced domain pairs make better Rosetta stones, and hence better predictors of protein interactions.
Evident from the set of PDB structures we studied, is a correlation between the distance separating a pair of domains on a protein chain, and their tendency to physically interact with one another. As described by Marcotte and co-workers when they constructed the domain fusion hypothesis for evolution of protein interactions, affinity between interacting pairs of domains may be enhanced when the domains are fused together on the same chain [11]. Consequently, close proximity of the interacting pair on the same chain increases the effective local concentration of the two domains, facilitating the interaction. The biochemical advantage for such an arrangement would explain the tendency for interacting domains to be found close together on a protein chain. Our observation that domain pairs located less than 30 residues apart are almost certainly to share an interface clearly supports this idea.
Previously, Park and co-workers [21]had observed this figure in an unrelated report. In this study, we adopted a different concept of a protein domain – PFAM categories which are essentially sequence-based annotations. Analyzing a substantial set of structural data from the PDB, we also derive at this similar threshold of 30 residues, and show it to be statistically significant.
Conservation of domain interactions across multi-chain structures
The data from multi-chain PDB structures provide additional support to the domain fusion hypothesis, by showing that most of the intra-chain domain interactions are similarly represented across separate chains of a complex. This provides additional mechanistic evidence that the interaction between the two domains is most probably functional and conserved.
To our knowledge, this is the first time structural data has been used to support the domain fusion hypothesis.
Functional classification of non-interacting domains in close proximity
We tried to uncover a pattern within the set of closely spaced, yet non-interacting domain pairs. We wanted to detect if there was an over-representation of domains from a specific molecular functional category in this non-interacting list. This list is displayed in Table 1. From the Gene Ontology categories of the domains, it is obvious that a good proportion of domains on the list are involved in DNA/RNA processing activities, as well as catalytic functions, but we didn't observe any statistically significant differences when comparing this non-interacting set with the sets of domain pairs which interact. This could be due to the small number of non-interacting domains in close proximity.
Furthermore, since the interactions we can detect from structural data are more likely to be permanent interactions, it is possible that the reason no physical contact is witnessed between these proximal domains in structural data is because the domains form transient interactions that are not captured in the x-ray crystallography data.
Hot loops and interactions
We also looked for a relation between protein disorder and interacting domain pairs. We wanted to see if protein domain pairs which interact on the same chain, tend to be linked by a disordered region. To this effect, we used DisEMBL[22]to do the disorder analysis. However, we were unable to infer any relationship between disorder and interacting domains.
Use of 30 residue criteria to refine domain fusion predictions
Our results from predicting interactions among S. cerevisiae proteins indicate that Rosetta stones with domains separated by less than 30 residues do indeed make better domain interaction (and hence protein interaction) predictors.
The set of protein interactions inferred from these Rosetta stones are enriched with more reliable interactions, as judged by using similar function as a criteria. The total number of interactions is reduced to nearly a quarter when employing this method. This allows us to conclude that the number of false positives is reduced, increasing the accuracy of the prediction. Without needing to employ a hard filter, protein interactions predicted using the domain fusion methodology may be ranked according to the quality of the Rosetta stones each interaction is inferred from, allowing one to identify a much smaller subset of more reliable interactions, and use them for downstream analyses.
Conclusions
We have successfully demonstrated the use of current structural data as a resource for refining current protein interaction predictions, in particular domain fusion predictions. Our data strongly suggests that domain pairs separated by less than 30 residues on a peptide chain are almost certainly to physically interact, and this criterion is useful in accessing protein interactions predicted from Rosetta stone proteins.
Going forward, the availability of a large number of structures through structural genomics programs will facilitate a larger sampling of the domain structure space. New patterns may emerge as use of this data becomes available, allowing better predictions to be made.
Methods
Intra-chain domain interactions
We used domain models from the Protein Family database (PFAM) [23] which were mapped onto structures from the Protein Databank (PDB) [24]. The PFAM to PDB mappings were obtained from PFAM data files, and we only considered PFAM entries that were tagged with the type 'Domain'. There are a total of 4169 peptide chains in the PDB that are annotated with more than one PFAM domain, comprising a total of 504 unique PFAM domains present within the data set. In order to obtain a non-redundant representation of these peptide chains, we took clusters of them based on 50% sequence identity, and selected one representative from each cluster. This left us with a set of 565 3D structures of multi-domain peptide chains, comprising a total of 996 distinct domain pairs, of which 478 are unique pairs.
We used the coordinates within the PDB data files to calculate the distances between domains, and to determine if they are interacting. Two domains are judged to be interacting if they share at least five contacting residue pairs, where contacting residues are residue pairs with less than 6Å between their respective alpha-carbon atoms.
Multi-chain interactions
Using a similar approach to the above, we obtained a set of multi-chain PDB structures in which the previously determined domain-domain interactions can be observed across separate peptide chains within a complex. The ASALIST from the PQS server [25]was used to sift out the biologically significant contacts from the crystal packed structures. Of the 379 domain pairs above, 305 were found on separate chains of a complex, and these were used for the analysis.
GO functional annotation
PFAM domains were categorized into Gene Ontology (GO) molecular functions and cellular processes using the PFAM2GO data provided by the GO consortium [26].
Saccharomyces cerevisiae protein interactions prediction
In order to assess how distance between domains could be used to improve the domain fusion based protein interaction predictions, we predicted interactions between 6918 proteins from the organism Saccharomyces cerevisiae found within the Swiss-Prot database, and gauged the quality of the interactions by looking at the function of each interacting protein.
The steps taken to predict protein interactions based on domain fusion are as follows. Swiss-Prot (release 42.9) and Trembl (release 25.9) [27] protein datasets were first searched for multi-domain proteins, by relying on their PFAM annotations. As above, only PFAM domains of type 'Domain' where considered. These multi-domain proteins were then catalogued as Rosetta Stones. Pairwise domain interactions were inferred by cataloging each distinct domain pair found on every Rosetta Stone protein, together with the number of residues separating the pair. As described by Marcotte and co-workers [11], domain interactions involving the 5% most promiscuous domains were discarded, removing the majority of false positives.
This domain interaction set was then used to predict pairwise protein interactions between the S. cerevisiae proteins, by looking at the complement of PFAM domains between each and every pair of proteins, and seeing if there were any Rosetta stone determined domain interactions between the domains of each protein. The protein interactions were sorted into two groups; one group inferred from domain interactions supported by the existence of a Rosetta stone protein with no more than 30 residues between the domain pair, and the other group with no support from a 30 residue Rosetta stone.
To validate these protein interactions, we mapped the proteins to the MIPS comprehensive yeast genome database [28], and looked for interacting protein partners that share the same MIPS functional category. Interactions between pairs that share the same function are more likely to be true.
All the data was stored in a relational database schema implemented in MySQL, with a set of perl modules written for data transaction and manipulation. The Bioperl bioinformatics tool kit [29] was used to parse Swiss-Prot, Pfam and PDB data, as well as to extract coordinates of each atom from each PDB structure.
Authors' contributions
JMC participated in the design of the study, performed the data and statistical analysis, as well as drafted the manuscript. PRK conceived of the study, participated in its design and coordination, and edited the final draft of the manuscript. Both authors read and approved the manuscript.
Acknowledgements
This work is supported by the Agency for Science, Technology and Research (A*STAR) in Singapore. We would also like to thank Dr Radha Krishna Murthy and Dr Li Yi for their valuable input.
Figures and Tables
Figure 1 Distance between domain pairs on a protein chain and the likelihood that they interact The solid line indicates the percentage of domain pairs, within a distance range apart, which are in contact. The broken line shows the distribution of chi-squared values corresponding to constructed 2 × 2 contingency tables that classified domain pairs according to 2 criterions; 1) whether or not they are separated by a distance no greater than the upper limit of each range, and 2) whether or not the domain pair is interacting. The percentage of interacting domain pairs drop noticeably after 30 residues, and the chi-squared value is also maximum at this threshold.
Table 1 List of domain pairs separated by less than 30 residues but are not interacting
Domain 1 Molecular Function Domain 2 Molecular Function No. of Contacts
2-Hacid_DH oxidoreductase activity 2-Hacid_DH_C oxidoreductase activity 4
CH CH 0
Cytochrome_CIII Cytochrome_CIII 0
dsrm double-stranded RNA binding dsrm double-stranded RNA binding 0
EGF EGF 0
eRF1_l eRFl_2 0
FKBP FKBP 1
fnl fnl 0
fnl fn2 2
fn2 fn2 0
GlutR_NAD_bind glutamyl-tRNA reductase activity GlutR_dimer glutamyl-tRNA reductase activity 1
HTH_9 molybdate ion transporter activity TOBE 0
kazal kazal 0
MHC_II_beta immune response ig 2
myb_DNA-binding DNA binding myb_DNA-binding DNA binding 0
Peptidase_M10 proteolysis and peptidolysis fn2 2
Phe_tRNA-synt_N phenylalanine-tRNA ligase activity tRNA-synt_2d phenylalanine-tRNA ligase activity; 0
resolvase recombinase activity ;DNA recombination HTH_7 recombinase activity;DNA recombination 1
RHD regulation of transcription, DNA-dependent TIG 0
Ribosomal_L9_N structural constituent of ribosome Ribosomal_L9_C structural constituent of ribosome 0
RNase_PH_C RNA binding;RNA processing KH nucleic acid binding 0
Rotamase isomerase activity Rotamase rrm isomerase activity 0
rrm rrm 4
rve DNA binding ;DNA recombination integrase integrase activity 0
SH2 intracellular signaling cascade SH3 0
sushi sushi 4
TPP_enzymes_N TPP_enzymes 2
WW WW 0
zf-Sec23_Sec24 Sec23_trunk 0
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Ho Y Gruhler A Heilbut A Bader GD Moore L Adams SL Millar A Taylor P Bennett K Boutilier K Yang L Wolting C Donaldson I Schandorff S Shewnarane J Vo M Taggart J Goudreault M Muskat B Alfarano C Dewar D Lin Z Michalickova K Willems AR Sassi H Nielsen PA Rasmussen KJ Andersen JR Johansen LE Hansen LH Jespersen H Podtelejnikov A Nielsen E Crawford J Poulsen V Sorensen BD Matthiesen J Hendrickson RC Gleeson F Pawson T Moran MF Durocher D Mann M Hogue CW Figeys D Tyers M Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry Nature 2002 415 180 183 11805837 10.1038/415180a
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| 15504241 | PMC529437 | CC BY | 2021-01-04 16:02:41 | no | BMC Bioinformatics. 2004 Oct 26; 5:161 | utf-8 | BMC Bioinformatics | 2,004 | 10.1186/1471-2105-5-161 | oa_comm |
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BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-5-1631550713610.1186/1471-2105-5-163SoftwareTETRA: a web-service and a stand-alone program for the analysis and comparison of tetranucleotide usage patterns in DNA sequences Teeling Hanno [email protected] Jost [email protected] Thierry [email protected] Margarete [email protected]öckner Frank Oliver [email protected] Microbial Genomics Group, Max Planck Institute for Marine Microbiology, D-28359 Bremen, Germany2 International University Bremen, D-28759 Bremen, Germany2004 26 10 2004 5 163 163 17 8 2004 26 10 2004 Copyright © 2004 Teeling et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
In the emerging field of environmental genomics, direct cloning and sequencing of genomic fragments from complex microbial communities has proven to be a valuable source of new enzymes, expanding the knowledge of basic biological processes. The central problem of this so called metagenome-approach is that the cloned fragments often lack suitable phylogenetic marker genes, rendering the identification of clones that are likely to originate from the same genome difficult or impossible. In such cases, the analysis of intrinsic DNA-signatures like tetranucleotide frequencies can provide valuable hints on fragment affiliation. With this application in mind, the TETRA web-service and the TETRA stand-alone program have been developed, both of which automate the task of comparative tetranucleotide frequency analysis.
Availability:
Results
TETRA provides a statistical analysis of tetranucleotide usage patterns in genomic fragments, either via a web-service or a stand-alone program. With respect to discriminatory power, such an analysis outperforms the assignment of genomic fragments based on the (G+C)-content, which is a widely-used sequence-based measure for assessing fragment relatedness. While the web-service is restricted to the calculation of correlation coefficients between tetranucleotide usage patterns of submitted DNA sequences, the stand-alone program generates a much more detailed output, comprising all raw data and graphical plots. The stand-alone program is controlled via a graphical user interface and can batch-process a multitude of sequences. Furthermore, it comes with pre-computed tetranucleotide usage patterns for 166 prokaryote chromosomes, providing a useful reference dataset and source for data-mining.
Conclusions
Up to now, the analysis of skewed oligonucleotide distributions within DNA sequences is not a commonly used tool within metagenomics. With the TETRA web-service and stand-alone program, the method is now accessible in an easy to use manner for a broad audience. This will hopefully facilitate the interrelation of genomic fragments from metagenome libraries, ultimately leading to new insights into the genetic potentials of yet uncultured microorganisms.
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Background
At present, a majority of the microbes from natural microbial communities cannot be transferred into pure cultures, either because proper cultivation conditions have yet to be found, or due to currently unidentified fundamental obstacles [1]. For decades, this has limited our understanding of the functioning of microbes within their natural habitats, such as their metabolic roles, interactions and dependencies.
The metagenome-approach allows for the first time to circumvent the restrictions imposed by the limited culturability of environmental microorganisms [2]. Now, DNA fragments of 40 – 150 kb of uncultured microorganisms can be cloned directly from the environment. This delivers insights into the microorganisms' genetic potentials, sometimes even allowing the reconstruction of entire genomes. Prominent example include the unexpected finding of bacteriorhodopsin in marine Gammaproteobacteria [3-6], and the almost complete reconstruction of two bacterial genomes from an acid mine drainage microbial biofilm [7], respectively.
However, the metagenome-approach is not without its limitations and problems. A major constraint is that especially small genomic fragments, as are obtained from libraries that have been constructed with fosmids or cosmids as cloning vectors, often lack suitable phylogenetic marker genes. This leads to the problem that fragments belonging to the same organism cannot be reliably identified as such unless they overlap. In order to nonetheless interrelate such genomic fragments, measures such as the (G+C)-content or BLAST hits and codon usage of the fragment's coding regions are commonly used to assess whether two unlinked fragments from a metagenome library belong to the same organism. These measures, however, can produce ambiguous or even misleading results, and should be supplemented by additional tools that assess the relatedness of the genomic fragments [8].
Since numerous studies have shown that oligonucleotide frequencies within DNA sequences exhibit species-specific patterns [9-18], comparative analysis of such oligonucleotide frequencies is a promising approach to this problem. For tetranucleotides, it has even been demonstrated that their frequencies carry an innate but weak phylogenetic signal [19]. Comparative analysis of tetranucleotide usage patterns also provides a good balance between computational requirements and attainable resolution. This makes the method particularly well-suited for use as a high-throughput method that can assist in tackling the fragment identification problem in metagenomics [8].
In order to automate and facilitate such an analysis, the TETRA software suite was developed, comprising both, a web-service and a stand-alone program.
Implementation
The algorithms that are used within TETRA have been described elsewhere [8]. In brief, DNA sequences are extended by their reverse-complements to compensate for different patterns of tetranucleotide over- and underrepresentation between the leading and the lagging strand. Then, the frequencies of all 256 possible tetranucleotides are counted and the corresponding expected frequencies are calculated by means of a maximal-order Markov model from the sequences' di- and trinucleotide composition. In order to evaluate the significance of the level of over- or underrepresentation for each tetranucleotide, the divergence between the observed and expected tetranucleotide frequencies is then transferred into z-scores using an approximation published by Schbath [20,21]. Finally, all DNA sequences are compared in pairs by computing the Pearson's correlation coefficient of their z-scores. Details on the method, its applicability and its limits are given in Teeling et al. (2004) and the TETRA online manual.
The TETRA web-service [22] has been implemented as set of PERL CGI scripts. Access is free to all users. A multi-headed FASTA file with DNA sequence data can be uploaded (actual file size limit: 2 Mb) and after having entered a valid e-mail address, the calculation can be started (Figure 1). Results are sent to the respective e-mail address as a tab-delimited crosstabulation of correlation coefficients in plain text format.
The TETRA stand-alone program can be downloaded for free from the TETRA website [23]. The current release has been implemented in REALbasic ( REAL Software Inc., Austin, Texas) and is available for Mac OS X. Versions for Linux and Windows are also available, but differ in details regarding their implementation and features. The counting of the tetranucleotides in the current version of TETRA stand-alone program is done by ocount – a self-written C program that has been integrated into the program.
Results and discussion
TETRA web-service
The TETRA web-service computes correlation coefficients between tetranucleotide usage patterns of DNA sequences, which can be used as an indicator of sequence relatedness. Details on the in- and output formats is available in the comprehensive online documentation [22].
TETRA stand-alone program
The stand-alone version of TETRA has many additional features that are not available via the TETRA web-service. Firstly, it comes with pre-computed tetranucleotide usage patterns of all 166 prokaryote chromosomes that were publicly available by June 2004 (Figure 2). These patterns have been incorporated into the program to provide the user with reference data that can also be used to get familiar with the program. With a few mouse clicks, correlation coefficients for the tetranucleotide usage patterns of all genomes can be computed and exported into PHYLIP format [24]. While not being well-suited for phylogenetic reconstruction, the resolution boundaries of the method can be easily evaluated by looking at the resulting whole genome trees.
Secondly, besides calculating correlation coefficients for tetranucleotide usage patterns, the TETRA stand-alone program allows the user to investigate the raw data (Figure 2) and can produce plots for a more detailed analysis of tetranucleotide over- and underrepresentations (Figure 3). This allows for hints into possible restriction sites by the examination of significantly underrepresented tetranucleotides. Tetranucleotide usage patterns for user-provided sequences can be generated in two ways. Single sequences shorter than 100 kb can be pasted into the so called 'Single Sequence Window'. From there, a sequence can be extended by its reverse complement and its tetranucleotide usage pattern can be calculated. Additionally, the sequence's base composition and GC-content can be computed. Sequences longer than 100 kb or files with multiple sequences can be imported by the 'Batch Mode'. The 'Batch Mode' reads a multi-headed FASTA file and computes the tetranucleotide usage patterns of all sequences within this file in a fully automated manner.
The tetranucleotide usage patterns of an average-sized genome (4 Mb) is computed in less than 10 minutes on a dual 1.8 GHz G5 (IBM PPC 970) computer. Newly computed tetranucleotide usage patterns are displayed within the 'Navigator' window, which is the central place for data management, access to the raw data and the calculation of plots and correlation coefficients (Figure 2). Raw data and correlation coefficients that have been computed for multiple patterns can be saved as tab-delimited tables in plain-text format and the graphical output (2D-plots) can be saved in JPEG-format.
A detailed documentation of the TETRA stand-alone program and its functions is available via the program's online help system.
Applicability
As has been demonstrated in a previous study [8], the analysis of tetranucleotide usage patterns is often (but not always) a much more reliable measure of sequence relatedness than the (G+C)-content. However, as a sequence-based measure it is affected by local changes in sequence composition. For example, large stretches of horizontally acquired genes will blur the resolution. Likewise, resolution is a function of sequence-length, i.e. the shorter the sequence, the less meaningful a tetranucleotide frequency analysis will be.
While the method works quite well for sequences in the range of 40 kb, it is certainly not suited for the analysis of single-read end-sequences, which are usually shorter than 1 kb. Since the phylogenetic signal within tetranucleotide usage patterns is faint, the method performs weakly for whole genome phylogenetic tree reconstructions. In a whole-genome tree calculated from the pre-computed 166 prokaryotic chromosomes (data not shown), organisms are mostly grouped at the species level and at the level of genera, when these are closely related (i.e. Escherichia sp., Shigella sp., Yersinia sp. or Mesorhizobium sp., Sinorhizobium sp., Bradyrhizobium sp.). However, more distantly related genera or even species with larger evolutionary distances are often not correctly clustered (e.g. Prochlorococcus sp.).
Therefore, the analysis of tetranucleotide usage patterns should not be regarded as a tool to deduce phylogenetic relationships, but rather as a fingerprinting technique for genomic fragment correlation. For example, assignment of fosmid-sized genomic fragments from metagenome libraries of a microbial consortia that mediates the anaerobic oxidation of methane was possible using tetranucleotide frequency analysis, and was shown to be in perfect agreement with 16S rRNA sequence analysis [8].
Conclusions
With the worldwide ongoing programs to sequence and analyze natural communities, new approaches for sequence correlation beyond G+C content, read densities and codon usage have to be developed and made available to the users. The easy to use TETRA software will facilitate this task and provide additional decision support for, e.g., fragment assignment also when complete genomes have to be assembled in environmental sequencing projects.
Availability and requirements
• Project name: TETRA
• Project home page:
• Operating system(s): Platform independent (web-service); Mac OS X (stand-alone program)
• Programming language: REALbasic
• Other requirements: none
• License: none
• Any restrictions to use by non-academics: none
List of abbreviations
BLAST – basic local alignment search tool
megx – marine environmental genomics
Authors' contributions
TETRA was implemented by HT and JW, TL contributed to the TETRA web-service, MB and FOG contributed important ideas regarding implementation, features and tested the programs.
Acknowledgements
We thank Christian Quast for helping with programming details and for making the TETRA web-service HTML 4.0.1 compliant and Melissa Duhaime for carefully reading the manuscript. This work was supported by the Max Planck Society.
Figures and Tables
Figure 1 Data-entry page of the TETRA web-service.
Figure 2 (left) Navigator window of the TETRA stand-alone program showing a subset of the pre-computed 166 prokaryote chromosomes for selection. (right) Data window with a subset of the tetranucleotide frequencies of the Aeropyrum pernix K1 chromosome.
Figure 3 Dotplot of Z-scores for the tetranucleotide frequencies of two Bacilli. A regression line is calculated as well as the Pearson correlation coefficient (0.823). Hovering with the mouse over a dot shows a small information window with the tetranucleotide(s) the spot is referring to (as shown for GATC in this case).
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TETRA web-service
TETRA standalone program
PHYLIP homepage
| 15507136 | PMC529438 | CC BY | 2021-01-04 16:02:45 | no | BMC Bioinformatics. 2004 Oct 26; 5:163 | utf-8 | BMC Bioinformatics | 2,004 | 10.1186/1471-2105-5-163 | oa_comm |
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BMC MicrobiolBMC Microbiology1471-2180BioMed Central London 1471-2180-4-411550423210.1186/1471-2180-4-41Research ArticleA touchdown nucleic acid amplification protocol as an alternative to culture backup for immunofluorescence in the routine diagnosis of acute viral respiratory tract infections Coyle Peter V [email protected] Grace M [email protected]'Neill Hugh J [email protected] Conall [email protected] Ornellas Dennis [email protected] Frederick [email protected] Suzanne J [email protected] Susan A [email protected] Dorothy E [email protected] Marian [email protected] Joanne [email protected] Regional Virus Laboratory, Royal Hospitals Trust, Belfast, BT12 6BA, UK2 Bacteriology, Royal Hospitals Trust, Belfast, BT12 6BA, UK3 Enteric, Respiratory and Neurological Virus Laboratory, Health Promotion Agency, 61 Colindale Avenue, London NW9 5HT, UK2004 25 10 2004 4 41 41 22 6 2004 25 10 2004 Copyright © 2004 Coyle et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Immunofluorescence and virus culture are the main methods used to diagnose acute respiratory virus infections. Diagnosing these infections using nucleic acid amplification presents technical challenges, one of which is facilitating the different optimal annealing temperatures needed for each virus. To overcome this problem we developed a diagnostic molecular strip which combined a generic nested touchdown protocol with in-house primer master-mixes that could recognise 12 common respiratory viruses.
Results
Over an 18 month period a total of 222 specimens were tested by both immunofluorescence and the molecular strip. The specimens came from 103 males (median age 3.5 y), 80 females (median age 9 y) and 5 quality assurance scheme specimens. Viruses were recovered from a number of specimen types including broncho-alveolar lavage, nasopharyngeal secretions, sputa, post-mortem lung tissue and combined throat and nasal swabs. Viral detection by IF was poor in sputa and respiratory swabs. A total of 99 viruses were detected in the study from 79 patients and 4 quality control specimens: 31 by immunofluorescence and 99 using the molecular strip. The strip consistently out-performed immunofluorescence with no loss of diagnostic specificity.
Conclusions
The touchdown protocol with pre-dispensed primer master-mixes was suitable for replacing virus culture for the diagnosis of respiratory viruses which were negative by immunofluorescence. Results by immunofluorescence were available after an average of 4–12 hours while molecular strip results were available within 24 hours, considerably faster than viral culture. The combined strip and touchdown protocol proved to be a convenient and reliable method of testing for multiple viruses in a routine setting.
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Background
Acute respiratory tract infections are major causes of morbidity and mortality. In 2000, lower respiratory tract infections were globally the number one infectious cause of disability adjusted life-years [1]. The commonest respiratory viruses that cause acute upper and lower respiratory tract infections and which are routinely tested for in most virus diagnostic laboratories are: influenza A virus (FLA); influenza B virus (FLB); respiratory syncytial virus (RSV); parainfluenza virus type 1 (PF1); parainfluenza virus type 2 (PF2); parainfluenza virus type 3 (PF3) and adenovirus (ADV). Additionally, human rhinoviruses (HRV) and coronavirus 229E (CoV-229E) are also linked to acute respiratory infection but less commonly included in laboratory reports; human metapneumovirus (hMPV) is not yet part of most United Kingdom virus laboratory test repertoires (personal feed-back from the United Kingdom Clinical Virology Network).
As part of service development it was necessary to provide an alternative to virus culture for testing immunofluorescence negative respiratory specimens. Historically and indeed currently immunofluorescence [2] and virus culture [3,4] are the main methods used to diagnose acute respiratory virus infections. Culture is accepted as more sensitive than immunofluorescence but slower and therefore less useful for direct patient management decisions. Using a standard culture technique [4] for the culture of respiratory viruses our median reporting times for culture positive and culture negative specimens were 6 days (based on 407 specimens) and 7 days (based on 2159 specimens) respectively; virus identification by this technique required the use of monoclonal antibody staining of the cell monolayer in addition to observation for viral cytopathic effect. We therefore wished to develop a test capable of reporting on immunofluorescence negative specimens within a 24 hour period.
Increasingly however, the sensitivity of nucleic acid amplification techniques for diagnosis has become recognised [5-10]. However widespread concerns about contamination issues [11,12] and perceived cost [13] have slowed their widespread adoption. An added problem for acute respiratory tract infections is the relatively large number of viruses that need to be accounted for, a problem which presents specific technical challenges.
One such challenge is the different optimal annealing temperatures of the primer sets for each prospective virus target. The ABI PRISM 7000 real-time facility from Applied Biosystems addresses this by using bundled software to design primer/probe combinations that use a common amplification protocol. However this approach is compromised by the inability of software to allow for target heterogeneity. In addition it does not allow users to adopt clinically validated primer sets from the literature.
To address these problems we adopted an alternative approach through the development of a generic touchdown amplification protocol. Touchdown protocols involve a pre-designed stepped reduction in the annealing temperature used for primer-to-template binding, which introduces a competitive advantage for specific base-pair priming over non-specific priming [14]. A detailed knowledge of the optimum annealing temperature is therefore not required. The study protocol was empirically constructed and proved robust when applied to a large range of respiratory viral and bacterial targets, without compromising individual test sensitivity. It was designed for use with in-house primer master-mixes that recognise 12 common respiratory viruses.
Before deciding on the layout of the molecular strip, as described in the methods, we undertook a wide range of preliminary validation steps for each primer set. The complexity of the strip makes it impossible to fully evaluate using the classical approach of applying an individual gold standard to each virus type. Classically this approach works well where a single target is under investigation [15]. However although the strip is putatively designed to identify 12 viruses, the actual number of individual types targeted is over one hundred and sixty because of the inclusion of generic primer sets for HRV [16] and ADV [17] respectively. The classical approach is further compounded for viruses (a) that cannot be grown or grown easily; (b) for which commercial IF sera are not available; (c) for which specimen panels are not available. We therefore adopted a phased validation, culminating in the present study. Sensitivity was ascribed by undertaking copy number determination on cloned targets and these ranged form 6 × 103 copies per ml for human rhinovirus type 1b to 4.2 × 103 copies/ml for RSV-A. Specificity was ascribed through reproducibility, i.e. specimens which were repeatedly positive, following our standard clinical reporting algorithm [6], were regarded as true positives; a similar approach was recently described for hMPV [18]. In addition amplicon sequencing was used as an initial specificity check. The primers sets were tested on clinical respiratory specimens arising from a number of ethically approved studies. These included respiratory specimens from patients: (a) with chronic obstructive pulmonary disease; (b) with acute asthma; (c) on assisted ventilation in intensive care. They were also tested on respiratory specimens collected as part of an influenza spotter program as well as on laboratory specimens of known virus reactivity.
To test the feasibility of its routine use we needed to clinically validate its performance in a routine setting on specimens tested in parallel with our standard immunofluorescence protocol for the diagnosis of acute virus respiratory infections. Although the routine immunofluoresence panel lacked capacity for the detection of rhinoviruses, human metapneumovirus and CoV-229E, these were included on the strip for clinical reasons during the period of the study. These findings and their implications are reported.
Results
Patients and specimens
A total of 99 viruses were detected in 84/222 specimens from a total of 79/183 patients and 4/5 National External Quality Assurance Scheme (NEQAS) controls; immunofluorescence did not detect the parainfluenza virus type 2 virus in one of the NEQAS specimens. Viruses were detected in all of the specimen types processed. The molecular strip detected virus in: 16/36 (44.4%) broncho-alveolar lavages, 62/120 (51.6%) nasopharyngeal secretions, 11/35 (31.4%) sputa and 10/31 (32.2%) combined throat and nasal swabs. Immunofluorescence detected virus in: 6/36 (16.6%) broncho-alveolar lavages, 23/120 (19.1%) nasopharyngeal secretions, 1/35 (2.8%) sputa and 1/31 (3.2%) combined throat and nasal swabs.
The median age of male and female patients where virus was detected was 3 y (range 2 weeks – 79 years) and 4 y (5 weeks – 81 years) respectively. Sixteen viruses were detected in 14/27 (51.8%) specimens, confirming a respiratory virus in 12 out of 24 (50%) patients investigated in general practice. Seventy-nine viruses were detected in 70/191 (36.6%) specimens, confirming a respiratory virus in 67 out of 159 (42.1%) patients investigated in hospital. Of the 16 viruses detected in specimens from the community, PCR detected all 16 in contrast to a single identification, influenza A (H3), by immunofluorescence.
Nested PCR
PCR identified one or more viruses in specimens from 84 of the 183 patients and the 4 NEQAS positive specimens, detecting a total of 99 viruses as shown in Table 1. The viruses detected were: influenza A (H3) virus (17); influenza A (H1) virus (4); influenza B virus (2); human rhinovirus (39); adenovirus (22); parainfluenza virus type 2 (1); parainfluenza virus type 3 (10); respiratory syncytial virus type A (2); respiratory syncytial virus type B (2);. No parainfluenza virus type 1, coronavirus 229E or human metapneumovirus were detected.
Dual infections were detected in 11/79 (13.9%) patients. The dual infections were: influenza A (H3) and adenovirus (4); influenza A (H3) virus and rhinovirus (2); influenza A (H1) and adenovirus (1); adenovirus and rhinovirus (3); respiratory syncytial virus type B and rhinovirus (1).
Nine patients had more than one specimen taken on the same day in which a virus was detected in at least one specimen by PCR. For 5 of the patients the same virus was detected in each of the 2 specimens. The viruses identified were rhinovirus (3), adenovirus (1) and parainfluenza type 3 (1); the latter was also immunofluorescence positive. In 2 cases a rhinovirus was detected in only one of the specimens. As part of a separate rhinovirus validation protocol one of these specimens was subjected to retesting coupled with limited sequencing of the 5' non-coding region amplicon which confirmed the presence of a rhinovirus sequence. Additionally, premature twin boys admitted to intensive care on the same day with severe bronchiolitis, both had evidence of acute rhinovirus infection by PCR. Limited sequencing of the 5' non-coding region of these viruses as part of the rhinovirus validation protocol confirmed the presence of an identical sequence of rhinovirus in both specimens.
Immunofluorescence
Immunofluorescence identified a virus in specimens from 28 of the 183 patients and 3/4 NEQAS positive specimens (16.4%), detecting a total of 31 viruses as shown in Table 1. The viruses detected were: influenza A virus (15); influenza B virus (1); parainfluenza virus type 3 (8); respiratory syncytial virus (4); adenovirus (3). No parainfluenza virus types 1 or 2 were detected including a NEQAS mock parainfluenza virus type 2 infection which was recorded as negative. No dual infections were detected. One patient had 2 specimens taken on the same day in which the same virus, parainfluenza type 3, was detected.
Discussion
Although touchdown PCR has been used successfully to help overcome some of the uncertainties associated with the thermal amplification of microbial nucleic acid targets [19-22], its use in this study has extended its role further and in so doing brought closer the goal of undertaking molecular diagnostics in a routine setting. Previously its main impact has been seen where multiplexing [23,24] or degenerate primers have been needed [25-27] and where the problems of choosing correct annealing temperatures are at their most difficult.
In this study the large number of targets is the main problem encountered. Using an empirical approach a series of amplification steps linked to a stepped reduction in annealing temperature from 55°C to 46°C was constructed. This proved extremely resilient when used with a wide range of primer sets and included the apparent anomaly of putting adenovirus through an initial reverse transcription step to stream line all of the targets on to a single strip; we have previously reported this approach for testing group F adenovirus alongside norovirus, astrovirus and rotavirus [28]. The touchdown surprisingly out-performed individual amplification protocols optimised for annealing temperature and thus proved suitable for use on the diverse range of respiratory viruses addressed in the study.
Where multiple viral targets are sought in clinical practice, we believe that it is only feasible to correlate the performance of the new assay in a head-to-head comparison with that already in routine use. Unfortunately for many clinical laboratories there is an elusion of testing for a wider range of viruses than is the case, by the inoculation of cell lines with a theoretical ability to grow the respective viruses. The annual reports of most clinical laboratories of one of the commonest human respiratory viruses, human rhinovirus, is an example of this; using the touchdown protocol we now report approximately 450 HRV infections per annum. The under reporting of adenovirus by standard methods [17] and the paucity of hMPV reporting, further underlines this assertion.
The ability to simultaneously validate the performance of multiple molecular primer sets in a routine clinical setting is a major accomplishment of the current methodological approach. The results demonstrated that a range of primers from both the medical literature and from in-house development could be amplified with a single generic touchdown protocol. It therefore confirmed the feasibility of directly incorporating primer sets into a standard operating procedure without the necessity for the individual optimisation of cycling parameters. As such the study results should facilitate primer selection and formal critical evaluation as here described. As an example of this enhanced flexibility we have recently replaced the primer sets for influenza A H1 and H3 (with respective copy number sensitivities of 8 × 103 and 2 × 103 copies per ml) with a generic matrix set (copy number sensitivity of 1 × 103 copies per ml).
The use of strips containing pre-dispensed mastermixes facilitates their use in a routine setting where laboratory personnel have only to thaw the strip and add the specimen extract. We make and aliquot for routine use a large range of multi-reaction mastermixes which are repeatedly subjected to freeze-thaw cycles as required on a daily basis. Provided the mixes are handled on ice, they remain extremely stable, over many months if so required. However the strip is designed for a single use only and thus only goes through a single freeze-thaw cycle. Mix stability is not a problem and the single positive control is used only to confirm that the touchdown amplification cycle has run successfully.
Because the technique of using nested amplification followed by running agarose gel electrophoresis is relatively cumbersome, it was important to evaluate how the complete protocol, inclusive of report generation, would perform when introduced into a routine line-managed diagnostic setting. Over the 18 months of the study the technique fitted in well to the demands of routine service. Central to this was the use of pre-dispensed and quality checked primer master-mixes which allowed the molecular strip to be adapted for use in a routine laboratory. The study confirmed that a broad based molecular approach was feasible as an alternative to virus culture to support immunofluorescence in the diagnosis of respiratory viruses. The overall superior performance of the strip and the missed NEQAS specimen by immunofluorescence underlines the need for a more sensitive back-up for negative specimens.
While nested protocols must be regarded as a pragmatic, interim solution until perfected single round systems are available, the format of the strip reduces the concern most attached to nested formats, i.e. false positive results. In our experience there is little evidence to support contamination arising from environmental sources and that the two major points of contamination in a nested system are (a) cross-contamination during manual extraction and (b) contamination of second round adjacent wells with product from a first round positive amplification. The use of the QIAGEN BioRobot for the extraction of all specimens reduced the former while the nature of the strip prevents the latter, since all the wells have separate mixes (Table 2). With both nested and non-nested assays the most critical requirements for reliable results are the use of well trained, appropriately skilled and knowledgeable staff, operating in a managed environment. As with any service, test performance must stand up to both external and internal quality assurance and in this regard we welcome the new respiratory quality control panel soon to be made available from Quality Control for Molecular Diagnostics (QCMD), Glasgow.
The results obtained were very encouraging. Although the strip was constructed to detect a wider range of viruses than immunofluorescence, over the period of validation it almost doubled (59 versus 31) the number of viruses that could have been detected by immunofluorescence, including a positive NEQAS specimen which was missed by immunofluorescence. Of this group of viruses the detection of adenovirus showed the most dramatic increase, an observation we have also previously made in a separate study [17] and which we continue to see both in routine respiratory specimens and in a number of respiratory studies. Similar to HRV viruses we believe these common infections are underdiagnosed by the standard techniques of immunofluorescence and culture. They are the second commonest virus, after HRV, that we observe in mixed infections and it is self-evident that these additional infections are at a level below the detection thresholds of standard methods. Their clinical significance when detected at these lower copy numbers remains to be determined.
As mentioned in the introduction a factor which often impacts negatively on a laboratory's decision to use molecular diagnostics is one of cost. It is worth considering that no matter which assay is chosen for use, it will attract the same overheads needed to provide the infrastructure of a laboratory set-up i.e. building, utilities, staff and equipment. In this regard there are no cheap tests and to use reagent costs as the sole factor in determining which assay to use is somewhat perverse. While the reagent costs of the strip are higher that commercial immunofluorescence reagents by a factor of 3, including extraction, this would undoubtedly narrow if immunofluorescence were capable of closing the pathogen gaps that currently exist e.g. HRV, hMPV. Currently using this approach, we have been able to replace both immunofluorescence and viral culture and this ability makes molecular diagnostics a more cost effective method for diagnosing viral infections. Taking into account the superior range, sensitivity, ability to quantify and speed of molecular techniques it is incredible how little they are used in routine laboratories. With the advent of SARS and the threat of avian influenza, this deficit is now beginning to disturb health care planners at the highest level.
Because specimen sampling was not contiguous seasonal peaks were not detected, accounting for the small numbers of respiratory syncytial virus detected and the lack of detection of human metapneumoviruses, parainfluenza virus type 1 and coronavirus 229E; subsequent (unpublished) data from the routine use of the molecular strip support an important role for human metapneumovirus in acute respiratory infections and the sporadic nature of infections caused by parainfluenza type 1 and coronavirus 229E.
Several interesting observations need highlighting. First, for immunofluorescence to perform reliably it was essential that a good nasopharyngeal specimen was available. The use of throat and/or nasal swabs with immunofluorescence alone is inappropriate. Second, immunofluorescence was very poor at detecting viruses from patients in the community, again almost certainly because of the universal use of swabs in that setting. Third, the rapid results of immunofluorescence were complemented by the touchdown protocol which can report definitive results within 24 hours, considerably faster than culture. Fourth, the molecular strip was better at detecting multiple infections. Even allowing for the inability of immunofluorescence to detect rhinoviruses, it should have detected the mixed adenovirus and influenza virus infections. Although immunofluorescence is capable of diagnosing dual infections, its routine use along with culture probably grossly underestimates their prevalence. The most plausible explanation is that the molecular technique detects infections where one of the viruses is below the detection threshold of immunofluorescence. These low level viruses are either just starting or more likely reaching the end of an infectious episode (latency is less likely) and this raises the previously unaddressed question of their role in viral respiratory pathogenesis. Fifth, the extent of rhinovirus infections was very significant. Their clinical significance ranged from acting as a definitive respiratory pathogen to a less certain role when acting as the most frequently detected co-pathogen in mixed infections.
Conclusions
In conclusion the use of the touchdown protocol with pre-dispensed and quality checked primer master-mixes was suitable for replacing virus culture for the diagnosis of respiratory viruses for immunofluorescence negative specimens. Immunofluorescence results were available after an average of 4–12 hours while molecular strip results were available within 24 hours, considerably faster than viral culture. The combined strip and touchdown protocol is a convenient and reliable method of testing for multiple viruses in a routine setting. Its generic nature makes it especially useful for introducing test repertoire modifications e.g. incorporating primers for the newly identified coronaviruses SARS-CoV and HCoV-NL63.
Methods
Patients and specimens
A total of 222 specimens were included in the validation between January 2002 and June 2003, including 14 from an influenza surveillance scheme. The specimens were collected from 183 patients including: 103 male, median age 3.5 y (7 m – 84 y); 80 female patients, median age 9 y (7 m – 84 y); both male and female ages were skewed towards the lower age ranges, and 5 national external quality assurance scheme (NEQAS) specimens (4 positive, 1 negative). One hundred and fifty-nine patients were in hospital and 24 were in the community at the time of sampling. Specimens tested consisted of a wide range of specimens including: broncho-alveolar lavage (36), nasopharyngeal secretions (120), sputum (35) and combined throat and nasal swabs (31).
Immunofluorescence
Nasopharyngeal secretions, broncho-alveolar lavage and sputum specimens were received in dry sterile containers at ambient temperature. Upon receipt they were re-suspended in 2 ml of virus transport medium (VTM) consisting of phosphate buffered saline pH 7.1, bovine serum albumin 7.5 μg/ml, penicillin G sodium 1000 units/ml, streptomycin sulphate 1000 μg/ml and amphotericin B 2.5 μg/ml. Throat and nasal swabs were received in 2 ml of VTM and vortexed on arrival to release cells attached to the fibres of the swab. An aliquot of 410 μl was taken off for extraction after which the specimens were centrifuged at 2600 g for 5 min and the resulting cell deposits air-dried on glass multi-well slides and fixed in acetone prior to testing. Immunofluorescence was set up on the respiratory specimens using commercial reagents according to the manufacturer's instructions, and was able to detect: influenza A, influenza B, respiratory syncytial virus, adenovirus, parainfluenza type 1, parainfluenza type 2 and parainfluenza type 3 (Dako diagnostics, Ely, UK).
Specimen extraction
A volume of 200 μl of the respiratory specimen suspension was extracted on a QIAGEN BioRobot 9604 using the Blood and Body Fluid Vacuum Protocol of the QIAamp DNA Blood Kit (Qiagen Ltd., Crawley, England, U.K). This protocol allows the co-extraction of both RNA and DNA simultaneously.
Nested PCR
Simultaneous amplification of all targets was facilitated by using a standard 8 well multi-well PCR strip to which all mixes were pre-dispensed and stored frozen; this format is referred to in the paper as the "respiratory strip" because of the respiratory nature of the targets. The respiratory strip targeted the following 12 common respiratory viruses: influenza A (H3), influenza A (H1), influenza B, respiratory syncytial virus type A, respiratory syncytial virus type B, adenovirus, coronavirus 229E, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, human rhinovirus and human metapneumovirus. The final configuration of the single and multiplex primer mixes in the 8 well strip are shown in Table 2. The primer sets used were taken mainly from published studies [16,29-31] but also included primer sets validated in-house after modification or de-novo design, including those for influenza A (H1), influenza A (H3) and the generic adenovirus primers [17]. The primers, gene targets and expected product sizes following amplification are shown in Table 3.
Each primer master-mix was made-up and titrated against a known positive control before being aliquoted and dispensed into its respective well of the 8-well microtube strip. The strips were stored frozen at -20°C until used. A positive control was also aliquoted and stored separately at -20°C until used. For the duration of the study the positive control was the cloned target of parainfluenza virus type 1; a negative control was not deemed necessary.
First round volumes were made-up in Access RT-PCR buffer (Promega, Southampton, England, U.K) and in the final 10 μl volume contained the following reagent amounts: 1.5 mM MgSO4, 1 unit AMV reverse transcriptase, 1 unit Tfl DNA polymerase, 0.2 mM each deoxynucleoside triphosphate (dATP, dCTP, dGTP, dTTP) and 1 μM outer primers.
Second round volumes were made-up in Taq Buffer B (Promega) and in the final 10 μl volume contained the following final amounts: 10 mM Tris-HCl (pH 9.0), 3.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 0.2 mM of each deoxynucleoside triphosphate (dATP, dCTP, dGTP, dTTP), 0.25 units of Taq DNA polymerase (Promega) and 0.2 μM inner primers.
First round amplification was performed on 2 μl of extract added to 8 μl of first round primer master-mix per well. Second round amplification was performed on 0.2 μl of the first round reaction added to 9.8 μl of second round primer master-mix per well; a multi-channel pipette facilitated the transfer of the 8 volumes in one step. The positive control was run on the eighth well of each strip. The second round products were run on ethidium bromide stained 2% agarose gels and photographed. Specimens were reported positive when respectively the correct size bands and the positive control bands were present.
Touchdown amplification protocol
Amplification was carried out on a range of thermal cyclers including the Applied Biosystems GeneAmp 2400 and 9700 series and a DNA Engine Tetrad PTC 225 (MJ Research, USA). The first and second round amplification protocols consisted of 36 identical cycles with the exception that (a) a reverse transcription step of 48°C-10 min preceded the first round and (b) a hot-start preceded the second round by transferring the strip directly from ice to a thermal cycler held at 94°C. The touchdown protocol consisted of 6 steps as follows: (a) initial denaturation (94°C-2 min); (b) 3 cycles of denaturation (94°C-30 s), annealing (55°C-30 s) and extension (72°C-30 s); (c) 3 cycles of denaturation (94°C-30 s), annealing (52°C-30 s) and extension (72°C-30 s); (d) 20 cycles of denaturation (94°C-30 s), annealing (49°C-30 s) and extension (72°C-30 s); (e) 10 cycles of denaturation (94°C-30 s), annealing (46°C-30 s) and extension (72°C-30 s); (f) 72°C for 5 mins.
Authors' contributions
PVC: Touchdown and molecular strip design and manuscript preparation.
GMO: Early application of touchdown cycling to respiratory samples.
HJO'N: Study protocol and manuscript preparation.
CMcC: Study protocol and manuscript preparation.
DDEO: Routine application of touchdown protocol during study.
FM: Routine application of touchdown protocol during study.
SJM: Routine application of touchdown protocol during study.
SAF: Primer-mastermix manufacture and quality control.
DEW: Early application of touchdown cycling to respiratory samples and manuscript preparation.
MF: Routine application of immunofluorescence protocol during study.
JS: Design and validation of primers for human metapneumonavirus detection.
Acknowledgements
We wish to thank the Northern Ireland Chest, Heart and Stroke Foundation for funding that contributed to the development of the molecular strip.
Figures and Tables
Table 1 Viruses detected by the molecular strip and immunofluorescence.
Virus Molecular Strip Immunofluorescence
Influenza A Virus 21* 15
Influenza B Virus 2 1
Human Rhinovirus 39 Not Tested
Adenovirus 22 3
Parainfluenza Virus Type 2 1 0
Parainfluenza Virus Type 3 10 8
Respiratory Syncytial Virus 4§ 4
Total Positive 99 31
Total Tested 222 222
* Included 17 influenza A H3 and 4 influenza A H1.
§ Included 2 respectively of respiratory syncytial virus type A and type B.
Table 2 Template for position of primer master-mixes on the molecular strip.
Well Number Virus
1 Parainfluenza Virus Types 1,2,3
2 Human metapneumovirus
3 Influenza A H1 & oronavirus 229E
4 Influenza A H3 & Human Rhinovirus
5 Influenza B Virus
6 Respiratory Syncytial Virus Types A & B
7 Adenovirus
8 Positive Control
Table 3 Primer sequences, gene targets and expected product sizes for viruses on the molecular strip.
Name Sequence (5' to 3') Gene Target 1st/2nd Size (bp)
PF1 1A CCT TAA ATT CAG ATA TGT AT HN1 1 478
PF1 1B GAT AAA TAA TTA TTG ATA CG
PF1 1C CCG GTA ATT TCT CAT ACC TAT G 2 317
PF1 1D CCT TGG AGC CGA GTT GTT AAG
PF2 1A AAC AAT CTG CTG CAG CAT TT HN1 1 508
PF2 1B ATG TCA GAC AAT GGG CAA AT
PF2 1C CCA TTT ACC TAA GTG ATG GAA T 2 204
PF2 1D GCC CTG TTG TAT TTG GAA GAG A
PF3 2A CTTG TAA ACT CAG ACT TGG TA HN1 1 478
PF3 2B TTT AAG CCC TTG TCA ACA AC
PF3 2C ACT CCC AAA GTT GAT GAA AGA T 2 103
PF3 2D TAA ATC TTG TTG TTG AGA TTG
hMPV 1A GCG GCA ATT TTC AGA CAA CG Fusion 1 696
hMPV 1B ACA TGC TGT TCG CCT TCA AC
hMPV 1C CAG CAG CAG GAA TCA ATG TT 2 288
hMPV 1D TCG CCT TCA ACT TTG CTT AG
CoV229E 1A GGT ACT CCT AAG CCT TCT C G Nucleocapsid 1 450
CoV229E 1B TGC ACT AGG GTT AAG AAG AGG
CoV229E 1C TTT GGA AGT GCA GGT GTT GTG G 2 100
CoV229E 1D GAC TAT CAA ACA GCA TAG CAG C
ENT 2A TCC TCC GGC CCC TGA ATG 5' Non-coding 1 120
ENT 2B GAA ACA CGG ACA CCC AAA GTA
ENT 1C GGC CCC TGA ATG CGG CTA AT 2 90
HRV 1B GGT CCC RTC CCG CAA TT
FLA 3A (H1)2 GAA ATT TGC TAT GGC TGA C Haemagglutinin 1 482
FLA 3B (H1)2 ATA TTT TGG GCA CTC TCC TAT
FLA 3C (H1)2 GTC TCT GTA GTG TCT TCA CAT TAT 2 197
FLA 3D (H1)2 CCG GAC CCA AAC CCT CTA CTC
FLB 1A GTG ACT GGT GTG ATA CCA CT Haemagglutinin 1 900
FLB 1B TGT TTT CAC CCA TAT TGG GC
FLB 1C CAT TTT GCA AAT CTC AAA GG 2 767
FLB 1D TGG AGG CAA TCT GCT TCA CC
FLA 2C (H3)2 AGC AAA GCT TTC AGC AAC TG Haemagglutinin 1 591
FLA 2D (H3)2 GCT TCC ATT TGG AGT GAT GC-3
FLA 2E (H3)2 AGT GCT GAA CGT GAC TAT GC 2 149
FLA 2F (H3)2 TTT GCT GGC TTC TCT TGG T
RSV 2A GTC TTA CAG CCG TGA TTA GG Nucleoprotein 1 838
RSV 2B GGG CTT TCT TTG GTT ACT TC
RSVA 2C GAT GTT ACG GTG GGG AGT CT 2 334
RSVA 2D GTA CAC TGT AGT TAA TCA CA
RSVB 2C AAT GCT AAG ATG GGG AGTTC 2 183
RSVB 2D GAA ATT GAG TTA ATG ACA GC
ADV 2A2 GCCGCAGTGGTCTTACATGCACATC Hexon 1 301
ADV 2B2 CAGCACGCCGCGGATGTCAAAGT
ADV 2C32 GACGCCTCGGAGTACCTSWSYCC 2 185
ADV 2DD2 TACGAGTACGTGGTGTCCTCKCGRTC
1 HN = Haemagglutinin-neuraminidase gene; 2 Primer sets designed for this study. N.B. 1 = 1st round primer set, 2 = 2nd round primer set; ENT 2A 2B, 1C, HRV 1B primer set = Rhinovirus.
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| 15504232 | PMC529439 | CC BY | 2021-01-04 16:03:38 | no | BMC Microbiol. 2004 Oct 25; 4:41 | utf-8 | BMC Microbiol | 2,004 | 10.1186/1471-2180-4-41 | oa_comm |
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BMC MicrobiolBMC Microbiology1471-2180BioMed Central London 1471-2180-4-421551626410.1186/1471-2180-4-42Research ArticleGel shift analysis of the empA promoter region in Vibrio anguillarum Denkin Steven M [email protected] Pedja [email protected] David R [email protected] Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI 02881, USA2 Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, 615 N. Wolfe Street, Baltimore, MD 21205, USA3 Department of Medicine, University of Massachusetts Medical School, LRB 360D, 364 Plantation Street, Worcester, MA 01605, USA2004 29 10 2004 4 42 42 20 7 2004 29 10 2004 Copyright © 2004 Denkin et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The induction of metalloprotease encoded by empA in Vibrio anguillarum occurs at high cell density in salmon intestinal mucus. Previously we have shown that there are significant differences in empA expression in two strains of V. anguillarum, M93Sm and NB10. It is hypothesized that differences in empA regulation are due to differences in binding of regulatory elements.
Results
Two strains of V. anguillarum, M93Sm and NB10, were examined and compared for the presence of DNA regulatory proteins that bind to and control the empA promoter region. Gel mobility shift assays, using a digoxigenin (DIG)-labeled oligomer containing a lux box-like element and the promoter for empA, were done to demonstrate the presence of a DNA-binding protein. Protein extracts from NB10 cells incubated in Luria Bertani broth + 2% NaCl (LB20), nine salts solution + 200 μg/ml mucus (NSSM), 3M (marine minimal medium), or NSS resulted in a gel mobility shift. No gel mobility shift was seen when protein extracts from either LB20- or NSSM-grown M93Sm cells were mixed with the DIG-labeled empA oligomer. The azocasein assay detected protease activity in all incubation conditions for NB10 culture supernatants. In contrast, protease activity was detected in M93Sm culture supernatants only when incubated in NSSM. Since the luxR homologue in V. anguillarum, vanT, has been cloned, sequenced, and shown to be required for protease activity, we wanted to determine if vanT mutants of NB10 exhibit the same gel shift observed in the wild-type. Site-directed mutagenesis was used to create vanT mutants in V. anguillarum M93Sm and NB10 to test whether VanT is involved with the gel mobility shift. Both vanT mutants, M02 and NB02, did not produce protease activity in any conditions. However, protein extracts from NB02 incubated in each condition still exhibited a gel shift when mixed with the DIG-labeled empA oligomer.
Conclusions
The data demonstrate that protein extracts of V. anguillarum NB10 cells contain a protein that binds to a 50 bp oligomer containing the empA promoter-lux box-like region. NB10 cells express empA during stationary phase in all growth conditions. The DNA binding protein is not present in M93Sm extracts. M93Sm cells express protease activity only when incubated at high cell density in fish gastrointestinal mucus. The gel shift observed with NB10 cells is not due to VanT binding. The data also suggest that the DNA binding protein is responsible for the less restrictive expression of empA in NB10 compared to M93Sm.
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Background
Vibrio anguillarum is the causative agent of vibriosis, one of the major bacterial diseases affecting fish, bivalves, and crustaceans [1-3]. Vibriosis has been a major problem for the aquaculture industry around the world. Large economic losses due to this fish pathogen are sustained by the fish farming industry. Annual losses of cultured fish species in Japan alone exceed $30 million [1]. Vibriosis in fish is observed as a hemorrhagic septicemia. Infected fish display skin discoloration and erythema around the base of the fins, vent, and mouth. Necrotic lesions form in the abdominal muscle. Mortalities within affected fish farm stocks range from 30–100% [1,4,5].
Extracellular metalloproteases are important virulence factors for many pathogenic bacteria including Vibrio cholerae [6,7] and Vibrio vulnificus [8-10]. The EmpA metalloprotease genes of V. anguillarum strains NB10 and M93Sm have been cloned and sequenced by Milton et al [11] and Denkin and Nelson [12], respectively. The EmpA metalloprotease of V. anguillarum shares significant sequence homology with other known proteases. Although the role of the EmpA metalloprotease is not completely understood, we showed previously that V. anguillarum empA mutants are either avirulent or attenuated for virulence in Atlantic salmon depending on the route of inoculation [12]. It can be hypothesized that EmpA causes tissue damage during pathogenesis.
Recently, the Vibrio harveyi luxR (LuxRVh) homologue in V. anguillarum, vanT, was cloned, sequenced, and shown to be required for protease activity [13]. LuxRVh dependent regulation of luminescence in V. harveyi occurs at the promoter of the lux operon [14,15]. While the amino acid sequence of LuxRVh in V. harveyi is not similar to the LuxRVf protein in V. fischeri which also regulates luminescence [16], it is thought that LuxRVh binds to the lux box and activates the expression of the luciferase genes. Since LuxRVh functions as a transcriptional activator in V. harveyi and VanT is required for protease expression in V. anguillarum, we wanted to determine whether any proteins bind to the regulatory regions (e.g. the lux box-like element and promoter) immediately upstream of empA.
We have previously shown that V. anguillarum cells grow rapidly in Atlantic salmon intestinal mucus [17,18] and that empA is strongly induced when cells are incubated at high density in mucus [17]. Further, we have demonstrated that empA transcription is RpoS-dependent [12]. Our findings also suggest that quorum sensing and undescribed autoinducer(s) help to regulate the expression of empA in V. anguillarum [12]. Additionally, Milton et al [19] previously identified a lux box-like element about 70 bp upstream of the translational start of empA, which we subsequently showed to span the transcriptional start site [12]. In this study, we examine, compare, and contrast the expression of metalloprotease encoded by empA in two strains of V. anguillarum (M93Sm and NB10). Differences in empA expression were investigated with regards to various growth conditions, the presence or absence of DNA-binding proteins, and putative regulatory proteins.
Results
Gel shift analysis of the empA promoter
The possibility of DNA-binding proteins interacting with regulatory sequences of empA, were examined using a digoxigenin (DIG) labeled oligomer containing the lux box-like element and empA promoter region. The location of double stranded oligomer relative to the promoter and coding sequences is shown in Fig. 1. Protein extracts from V. anguillarum M93Sm and NB10 cells incubated in LB20 and NSSM were mixed with the DIG-labeled oligomer (Fig. 2). NB10 and M93Sm cells were incubated in LB20 and NSSM for 3 h and protein extracts from cells at 0 and 3 h were prepared. DNA binding was observed in protein extracts from NB10 incubated in LB20 and NSSM (Fig. 2A and 2B) at both 0 and 3 h. A 3.7-fold increase (from T = 0 h) in the amount of DNA binding was observed by 3 h in extracts from NSSM-grown NB10 cells (Fig. 2B, NB10 lane 2). An additional 1.4-fold increase in DNA binding was observed when the amount of protein was increased from 5 μg to 7.5 μg (Fig. 2B, NB10 lane 3). Although only a minor change in the amount of DNA binding (1.4-fold increase) was detectable by 3 h in NB10 extract from LB20-grown cells, a 4.7-fold decrease (from T = 0 h) in free DIG-labeled oligomer was observed (Fig. 2A, NB10 lane 2). In addition, a 8.9-fold decrease in free DIG-labeled oligomer was detected when 7.5 μg protein was added (Fig. 2A, NB10 lane 3). Specificity of protein binding to the DIG-labeled oligomer was tested by competitive inhibition of binding by the addition of excess amounts of an identical unlabeled oligomer. When excess unlabeled lux box-empA promoter oligomer was added to protein extracts from LB20- and NSSM-grown NB10 cells binding to the DIG-labeled oligomer was abolished. Further, the addition of unlabeled oct2A (a non-competitive oligomer) to protein extracts did not affect the gel mobility shift of the DIG-labeled oligomer caused by protein extracts from NB10 cells incubated either in LB20 or NSSM. In contrast to protein extracts from NB10 cells grown in LB20 or NSSM causing a gel shift, protein extracts from M93Sm cells incubated in either condition did not show any binding to the DIG-labeled oligomer (Fig. 2A and 2B). Even when 7.5 μg of protein was used, a band shift was not observed. In a separate experiment, protein extracts from NB10 cells incubated in 3M or starved in NSS also caused a similar gel mobility shift for the DIG-labeled oligomer (Fig. 3). Additionally, even when 5 μg of protein extract from exponentially growing NB10 cells in LB20 was added to the DIG-labeled oligomer, a band shift was observed. Further, in this gel shift experiment, M93Sm protein extract was added as a negative control to the labeled DIG-labeled oligomer and no shift was observed.
Protease activity in LB20, NSSM, 3M, and NSS
Expression of empA in NB10 occurs in both LB20 and NSSM, while M93Sm exhibits empA expression only in NSSM [12]. Since a gel mobility shift was observed in NB10 protein extracts in all conditions of growth and during starvation, these cells were tested for protease activity in 3M and NSS in addition to LB20 and NSSM (Fig. 4). Protease activity was detected in NB10 cells after 1 and 3 h of incubation in NSSM or LB20, respectively (Fig. 4A). Maximum protease activity was observed at 4 h for both conditions of incubation. In addition, protease activity was observed in NB10 cells incubated in 3M or starved in NSS after 1 h and continued to increase during the next hour of the 4 h experiment. Throughout the incubation, NB10 cells in 3M produced 3–4 fold greater amounts of protease activity than cells in NSS. The amount of protease measured in NSS at 4 h was only 14 and 17% of the amount produced in LB20 and NSSM, respectively. In contrast, protease activity was observed in M93Sm cells only when incubated in NSSM (Fig. 4B). M93Sm cells did not produce protease activity in any of the other conditions examined.
Effects of vanT mutations on protease activity and gel mobility shift
Since vanT of V. anguillarum NB10 was cloned, sequenced and shown to be required for protease activity [13], vanT mutants of both M93Sm and NB10 were created by site directed mutagenesis and tested for protease activity in LB20 and NSSM. When the mutants (NB02 and M02) were incubated in NSSM for 3 h, a small amount of protease activity was detected (5 and 13% of the wild-type levels, respectively) (Fig. 5). No protease activity was observed in M93Sm, M02, or NB02 incubated in LB20.
The vanT mutant strains (M02 and NB02) and their parental wild-types (M93Sm and NB10, respectively) were also examined for vanT transcription by RT-PCR (Fig. 6). Neither vanT mutant produced vanT transcripts. Interestingly, both wild-type strains produced vanT mRNA in both LB20 and NSSM.
To determine if VanT was binding to the lux box-empA promoter, protein extracts from NB02 were tested in the gel shift assay (Fig. 7). Protein extracts from NB02 cells incubated in NSSM and LB20 exhibited binding to the DIG-labeled oligomer similar to that observed with protein extracts from wild-type NB10 cells (compare to Fig. 2). These data show that VanT was not responsible for the gel mobility shift of the lux box – empA promoter oligomer.
Discussion
It has been demonstrated that the transcriptional activator, LuxRVf, in Vibrio fischeri, activates expression of the lux operon via interaction with the lux box [15,16,20,21]. Although there is incomplete understanding of LuxRVf interaction with the lux box, good evidence is available that shows a specific lux box sequence required for efficient LuxRVf binding [21]. LuxRVh from V. harveyi and LuxRVf from V. fischeri are not the same transcriptional activator proteins [22]. These proteins differ in their activation of the lux operon. In V. fischeri, LuxRVf interacts with an acylhomoserine lactone autoinducer and then binds to the lux box to activate transcription of the lux genes [23]. In V. harveyi, the regulation of luminescence occurs via a phosphorelay signal transduction system, which involves LuxRVh binding to the lux box independent of an autoinducer [24]. Additionally, a central response regulator, LuxO, has been shown to regulate the expression of luxR [24] in V. harveyi. Recently, the LuxRVh homologue of V. fischeri was identified as LitR [25]. However, it is not known if V. fischeri uses a phosphorelay cascade to control LitR interaction with the lux operon.
In this study, two strains of V. anguillarum (NB10 and M93Sm) were examined for a empA promoter region binding protein. In all conditions, incubation with NB10 protein extracts resulted in a gel mobility shift to the DIG-labeled oligomer. In contrast, M93Sm extracts did not show any protein binding to the labeled DNA in any conditions tested. This was interesting because NB10 protease production occurs in all conditions, while M93Sm protease activity is detected only when the cells are incubated in mucus. Recently, VanT, the LuxRVh homolog in V. anguillarum, was identified by Milton et al [13] and shown to positively regulate EmpA metalloprotease and biofilm production. We created vanT mutants in both M93Sm and NB10 and tested them for vanT expression, protease activity, and DNA binding. These two mutants, M02 and NB02, did not produce detectable vanT mRNA. However, both exhibited low levels of protease activity in mucus - about 5% and 13% of wild type activity in mucus, respectively. For NB02, no protease activity in LB20 was observed. These results demonstrate that vanT is required for protease activity in a non-mucus containing medium (V. anguillarum NB10 in LB20), confirming the result reported by Milton et al [13]. Our results also show that in NSSM, vanT mutants still exhibit some protease activity, but VanT is required for full protease activity in both M02 and NB02 cells.
We tested a vanT mutant of V. anguillarum for the possibility of VanT binding to and activation of empA expression. Since the V. anguillarum NB10 strain expresses empA during stationary phase in all conditions tested and protein extracts from these conditions show DNA binding to the empA promoter region, it was hypothesized that VanT binds to this region. The vanT mutant, NB02, was analyzed for gel shift using protein extracts from cells incubated in LB20 and NSSM. Protein extracts from NB02 cells incubated in each condition still caused a band shift. This result suggests that VanT, the LuxRVh homolog in V. anguillarum, either does not bind to the empA promoter region containing the lux box-like element or perhaps VanT binds to a second protein that binds to this DNA sequence. The former possibility is more likely since the gel shift observed in NB02 is identical to that observed in NB10 extracts. Further, since the M93Sm vanT mutant, M02 still exhibits about 13% of the wild type protease activity in NSSM and does not exhibit protein binding in the gel shift analysis, it is probable that VanT does not bind to the empA promoter-lux box region. However, Croxatto et al. [26] demonstrated recently that purified VanT protein binds to the V. anguillarum NB10 promoter regions for empA, vanOU, and vanT. Examination of their data shows that VanT binds more strongly to the promoters for vanOU and vanT (binding observed with 0.3 μg purified VanT per 2 ng DIG-labeled DNA) than to the empA promoter (binding observed with 2.5 μg VanT per 2 ng DIG-labeled DNA). It should be noted that in our study only 5 μg of total protein extract was used in the gel shift analysis. That binding to the empA promoter is still observed in vanT mutants strongly suggests that an unidentified protein binds to the promoter-lux box region.
Previously, we have shown that empA in V. anguillarum M93Sm is positively regulated by at least two components of salmon gastrointestinal mucus [17]. In this study we show that NB10 cells exhibit protease activity under all nutritional conditions including starvation in NSS when at high density, whereas M93Sm only induces protease activity in mucus. RpoS is required for expression of empA in both M93Sm and NB10 [12]. This was demonstrated by the observation that rpoS mutants of M93Sm (M03) and NB10 (NB03) exhibit no protease activity when cells are incubated in either LB20 or mucus during stationary phase. Extracellular factors present in conditioned media showed both positive and negative effects on empA expression [12]. LB20 conditioned supernatants from M93Sm cause a more rapid and increased production of protease activity (in NB10 and M93Sm cells). However, conditioned LB20 from the luxS mutant, M01added to wild-type cells results in a more rapid induction of protease activity, which suggests that AI-2 may act as a negative regulator of empA.
In this study we confirmed that VanT is required for empA expression in NB10 when incubated in LB20; however, when NB10 or M93Sm cells are incubated in NSSM, a small percentage of protease activity remains in the vanT mutants. Our data support the observation by Croxatto et al [13] that VanT positively regulates empA expression. Further, the role of VanT as a transcriptional activator of empA via the lux box-like element has not been established. Our data show that VanT does not bind to the empA promoter lux box-like region, but is required for maximum protease activity. In addition, we show that in NB10 an unknown protein binds to the empA promoter-lux box region that may increase protease activity in all conditions. In contrast, M93Sm protein binding to this region is not observed and protease activity is detected only when cells are incubated in mucus. In conclusion, the expression of the empA virulence gene in V. anguillarum is regulated transcriptionally by the alternative σ-factor RpoS (σS) and the transcriptional activator VanT (a LuxRVh homologue). Additionally, an unknown DNA binding protein binds specifically to the empA promoter lux box-like region in NB10 cells, but not in M93Sm cells. We hypothesize that this protein permits empA expression in the absence of the inducing factors in fish gastrointestinal mucus. A better understanding of how and why these two wild-type strains of V. anguillarum exhibit different levels of empA expression will further reveal how virulence genes are regulated in this fish pathogen.
Conclusions
Differences in empA expression between the two strains of V. anguillarum, M93Sm and NB10, correspond with the production of an unknown regulatory protein that binds to a 50 bp oligomer identical to the promoter-lux box region of empA. For NB10, protein binding to the oligomer correlated with the expression of empA in all incubation conditions. However, M93Sm, which expresses empA only in mucus, did not exhibit protein binding in this condition. Additionally, a null mutation in vanT (luxR homologue) results in severely decreased or no empA expression, but does not abolish protein binding (in NB10) to the oligomer. Our data suggest that while empA expression in both V. anguillarum M93Sm and NB10 is dependent upon RpoS and VanT, the two strains differ in production of an unknown protein that binds to the promoter-lux box-like region of empA. We also hypothesize that the presence of this DNA binding protein permits NB10 cells to express empA in the absence of fish gastrointestinal mucus.
Methods
Bacterial strains, plasmids, and growth conditions
All bacterial strains and plasmids used in this report are listed in Table 1. V. anguillarum strains were routinely grown in Luria-Bertani broth + 2% NaCl (LB20) [18,27] supplemented with the appropriate antibiotic, on a rotary shaker at 27°C. Escherichia coli SM10 was grown in LB10 (LB + 1% NaCl) on a rotary shaker at 37°C. Experimental media included: LB20, 3M (marine minimal media) [18,28], nine salt solution (NSS, a carbon-, nitrogen-, and phosphorus-free salt solution) and NSS plus 200 μg mucus protein/ml (NSSM) [18]. Gastrointestinal mucus was harvested from Atlantic salmon as previously described by Garcia et al. [18]. Overnight cultures of V. anguillarum were grown in LB20, centrifuged (9,000 × g, 10 min), and pelleted cells washed twice with NSS [18]. Washed cells were resuspended to appropriate cell densities in experimental media. Specific conditions are described in the text for each experiment. Cell densities were determined by serial dilution and plating on LB20 agar plates or by measuring optical density at 600 nm (OD600). Antibiotics were used at the following concentrations: streptomycin (Sm), 200 μg/ml and chloramphenicol (Cm), 5 μg/ml.
Bacterial matings
Plasmids were introduced into V. anguillarum M93Sm and NB10 from E. coli SM10 by conjugation using the procedure described by Milton et al. [11,29,30]. Briefly, overnight cultures of V. anguillarum M93Sm or NB10 and E. coli SM10 containing the pNQ705-1 vanT clone, pNQVanT, were prepared and mixed using ratios of 1:1 or 3:1 (recipient: donor) in NSS plus 10 mM MgSO4. The cell suspension was vacuum filtered onto a 0.22 μm nylon membrane, which was placed on an LB15 (LB + 1.5% NaCl) agar plate and allowed to incubate overnight at 27°C. Following incubation, the cells were removed from the filter by vigorous mixing in NSS plus 10 mM MgSO4. The cell suspension (100 μl) was plated on LB20 Sm200 Cm5 (for M93Sm mutants) or on TCBS Cm5 (for NB10 mutants) and allowed to incubate at 27°C until V. anguillarum colonies were observed (usually 16–24 h).
Site-directed mutagenesis of vanT
Site-directed mutagenesis was used to create gene interruptions within the structural gene of vanT. Primers (Table 2) were generated based on the vanT sequence for V. anguillarum NB10 (accession # AF457643). A 320 bp region from vanT was PCR amplified using Qiagen Taq DNA polymerase and cloned into the suicide vector, pNQ705, using SacI and XbaI restriction endonucleases to yield pNQVanT. The presence of the 320 bp empA-derived insert was confirmed by both PCR amplification and restriction analysis using SacI and XbaI. The mobilizable suicide vector, pNQVanT, was transferred into V. anguillarum by conjugation with E. coli SM10. E. coli SM10 contains the λ pir protein that is required for replication of pNQ705. Chloramphenicol resistant colonies were selected and screened for insertion within vanT. PCR and Southern blot analysis were used to confirm the incorporation of pNQVanT. For PCR analysis, a primer described previously by Milton et al. [11] complementary to the pNQ705 vector was utilized (Table 2). The forward primer, SD vanT-Forward (Table 2) is complementary to a region upstream of the insertion. PCR products were analyzed by electrophoresis through a 1.0 % agarose gel in Tris-acetate EDTA (TAE) [31] buffer containing 0.2 μg/ml ethidium bromide. The gene was interrupted within the 320 bp region of vanT rendering the mutants resistant to chloramphenicol at 5 μg/ml. The resulting V. anguillarum vanT mutants were designated M02 (derived from M93Sm) and NB02 (derived from NB10) (Table 1).
Gel mobility shift assay
Protein extracts were prepared from 2 ml of cells using the Sigma CelLytic™ B bacterial cell lysis/extraction reagent containing 0.2 mg/ml lysozyme, 1 mM DTT, 2 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride (PMSF). Briefly, the cells were pelleted from the experimental conditions by centrifugation at 9,000 × g for 10 min, 4°C. In addition, cells were lysed by multiple freeze-thaw cycles in crushed dry ice or in liquid nitrogen and then heated at 37°C. Insoluble cell debris was centrifuged at 12,000 × g for 10 min, 4°C. Protein concentration was determined using the Bradford assay (Bio-Rad).
The digoxigenin (DIG) gel shift kit for 3'-end labeling of oligonucleotides (Roche Applied Science, Indianapolis, IN) was used for protein-DNA binding assays. The 50 bp oligomers (Table 2) used contain the empA promoter region and putative lux box. These oligomers were synthesized and purified by HPLC (Integrated DNA Technologies, Coralville, IA). The oligomers were annealed, labeled, and used in the gel shift reactions according to the manufacturer's instructions (Roche). An 8% native polyacrylamide gel in 0.25 × TBE (Tris-borate-EDTA buffer) was prepared and used for electrophoresis (in 0.5 × TBE) of the gel shift reactions. Blotting was performed using a Biorad electro-blotting system (model Trans blot) according to the manufacturer's instructions. Chemiluminescence detection of DIG-labeled DNA-protein complexes on the nylon membranes was detected using Hyperfilm ECL (Amersham Pharmacia). Gel mobility shift fluorograms were examined quantitatively using a densitometer (Molecular Dynamics, Personal Densitometer, SI). All gel mobility shift experiments were repeated three times and the data presented are representative examples of each experiment.
Detection and quantification of protease activity
Culture supernatants were assayed for proteolytic activity using our previously described modification of the method by Windle and Kelleher [17,32]. Briefly, culture supernatant was incubated with azocasein (5 mg/ml) dissolved in Tris-HCl (50 mM, pH 8.0) containing 0.04% NaN3. Culture supernatant was prepared by centrifuging 1 ml of cells at 12,000 × g (10 min, 20°C). Supernatant was removed and filtered through a 0.22 μm pore size cellulose-acetate filter. Filtered supernatant (100 μl) was incubated at 30°C with 100 μl of azocasein solution. Incubation time of 30 min was sufficient for assays of supernatants from cell suspensions of ≥ 5 × 108 cells/ml. Reactions were terminated by addition of trichloroacetic acid (TCA) (10% wt/vol) to a final concentration of 6.7% (wt/vol). The mixture was allowed to stand for 1 to 2 min and centrifuged (12,000 × g, 4 min) to remove unreacted azocasein, and supernatant containing azopeptides was suspended in 700 μl of 525 mM NaOH [32]. Absorbance of the azopeptide supernatant was measured at 442 nm using a Pharmacia Ultrospec 4000 spectrophotometer. A blank control was prepared by boiling V. anguillarum M93Sm supernatant (100°C, 10 min). TCA (final concentration 10%) was added to the blank control supernatant immediately after the addition of azocasein. The mucus used was also boiled (10 min) to destroy any inherent protease activity. Protease activity units were calculated using the following equation: 1 protease activity unit = [1000 (OD442)/CFU] × (1 × 109)] [17].
RNA isolation and RT-PCR conditions
Total RNA was isolated from V. anguillarum cells as previously described [12] using the RNeasy purification kit (QIAGEN). Reverse transcriptase (RT) reactions were performed using the OneStep RT-PCR system from QIAGEN. Prior to the RT reaction, RNA samples (2 μg) were treated with RQ1 (RNase-free) DNase (1 U/μl) according to the manufacturer's specifications (Promega, Madison, WI.). Primers used for RT-PCR reactions were vanT-F2 and vanT-R2 (Table 2). PCR amplification without the addition of RT was performed on an equal amount of RNA to demonstrate the absence of DNA. Amplification products were analyzed by agarose gel electrophoresis and sizes were determined using Kodak digital imaging software (Kodak 1D image analysis software, version 3.0.2; Eastman Kodak Co., Rochester, N. Y.).
DNA sequencing
DNA sequencing was performed by the University of Rhode Island Genomics and Sequencing Center (Kingston, RI). Sequencing was performed on a Beckman-Coulter CEQ 8000. The Dye Terminator Cycle Sequencing (DTCS) quick start kit was used for the sequence reactions that were prepared according to the manufacturer's specifications and run in a thermal cycling program. DNA samples were mixed with the appropriate primer (Table 2) and then submitted for sequencing.
Authors' contributions
S.M.D. carried out the experimental part of the study and drafted the manuscript. D.R.N. conceived of the study, participated in its design and coordination, and edited the manuscript. P.S. participated in the experiment design and assisted with gel shift analysis. All authors have read and approved the final manuscript.
Acknowledgements
This research was supported by USDA NRICGP grant 2002-35204-12252 awarded to D.R.N.
Figures and Tables
Figure 1 Nucleotide sequence of the promoter region of empA. The 5'-coding sequence and immediate upstream/promoter sequence of empA is presented. The base-paired sequence indicates the annealed primers empA3W and empA3C (Table 2). The transcriptional start site is indicated by +1, the rpoS-dependent promoter regions are indicated as -10 and -35 with the bases in italics [12], and the lux box-like element [19] is underlined. Additionally, the start translation site (ATG) is in bold with the translated product below and the ribosome binding site (RBS) is marked above (*).
Figure 2 Gel shift analysis using a DIG-labeled 50 bp oligomer (0.8 ng/lane) containing the empA promoter region and putative lux box. Protein extracts were prepared from V. anguillarum M93Sm (left) and NB10 (right) incubated in (A) LB20 or (B) NSSM and added to DIG-labeled DNA. DIG-labeled DNA alone was added to the lane marked with an asterisk (*). Lane 1, 5 μg of protein extract from cells at time 0 h; lane 2, 5 μg protein extract from cells at time 3 h; lane 3, 7.5 μg protein extract from cells at time 3 h; lane 4, 5 μg protein extract from cells at time 3 h plus the unlabeled lux box-empA promoter containing oligomer; and lane 5, 5 μg protein extract from cells at time 3 h plus non-competitive oct2A (from E. coli) unlabeled 39 bp oligomer. The data presented are representative of three replicate experiments.
Figure 3 Gel shift analysis of proteins from NB10 cells incubated in 3M and NSS. Cells grown overnight in LB20 were washed twice in NSS and resuspended at 2 × 109 cfu/ml in the appropriate medium. Protein extract (5 μg) from cells incubated for 3 h in each condition was added to the gel shift reaction with the DIG-labeled oligomer. DIG-labeled DNA alone was added to the lane marked with an asterisk (*). Reactions containing protein extracts prepared from each condition was loaded onto the gel: lane 1, NSSM; lane 2, NSSM + unlabeled oligomer; lane 3, LB20; lane 4, 3M; lane 5, NSS; lane 6, exponential phase cells in LB20; and lane 7, M93Sm in NSSM. The data presented are representative of three replicate experiments.
Figure 4 Induction of protease activity in V. anguillarum NB10 (A) and M93Sm (B) cells incubated in LB20 (●), NSSM (■), 3M (▲) or NSS (▼). Cells were grown for 16 h in LB20 at 27°C, harvested by centrifugation, washed twice in NSS, and resuspended at 2 × 109 CFU/ml in each condition. Samples were taken at the indicated times and cell-free supernatant was tested for protease activity using the azocasein assay. Both of the experiments shown represent a single experiment, although each experiment was repeated at least three times with similar results.
Figure 5 Induction of protease activity in V. anguillarum wild type (circles) and vanT mutant strains (squares). Cells were grown for 16 h in LB20, harvested by centrifugation, washed twice in NSS, and resuspended at 2 × 109 CFU/ml in either LB20 (open symbols) or NSSM (solid symbols). (A) NB10 and vanT mutant, NB02. (B) M93Sm and vanT mutant, M02. Both of the experiments shown represent a single experiment, although each experiment was repeated at least three times with similar results.
Figure 6 RT-PCR analysis of vanT expression in wild-type (M93Sm and NB10) and vanT mutant (M02 and NB02) strains of V. anguillarum. Cells were grown overnight in LB20, washed twice in NSS, and resuspended in either LB20 or NSSM at 2 × 109 CFU/ml. RNA from LB20- (L) and NSSM- (M) grown cells was prepared and used in RT-PCR reactions. RNA was extracted from cells at 0 h and 3 h after resuspension. As a positive control, PCR was performed on DNA from M93Sm (lane A) and NB10 (lane B). As a negative control, RT was omitted from the reaction containing RNA samples; a representative result is shown in lane C. The PCR and RT-PCR products were visualized on a 1% agarose TAE gel containing ethidium bromide. Molecular weight standard (indicated in kilobase pairs) are in lane S. The data presented are representative of three replicate experiments.
Figure 7 Gel shift analysis of the V. anguillarum vanT mutant, NB02 in NSSM and LB20. The same labeled oligomer used in Figures 1 and 2 was added to protein extracts from NB02 cells incubated in NSSM and LB20. DIG-labeled DNA alone was added to the lane marked with an asterisk (*). Lane 1, 5 μg of protein extract from cells at time 0 h; lane 2, 5 μg protein extract from cells at time 3 h; lane 3, 5 μg protein extract from cells at time 3 h plus unlabeled lux box-empA promoter; and lane 4, 5 μg protein extract from cells at time 3 h plus the oct2A (from E. coli) unlabeled oligomer. The data presented are representative of three replicate experiments.
Table 1 Strains and plasmids used in this study
Strain or plasmid Genotype and description Source or reference
Strains
Vibrio anguillarum
M93Sm Spontaneous SmR mutant of WT M93 (serotype J-O-1) Denkin & Nelson [17]
M02 SmR CmR; M93Sm vanT mutant This study
NB10 WT (serotype O1) Milton et al. [11]
NB02 CmR; NB10 vanT mutant This study
Escherichia coli
SM10 thi thr leu tonA lacY supE recA Milton et al. [11]
RP4-2-Tc::Mu::Km (λ pir)
Plasmids
pNQ705-1 CmR; suicide vector with R6K origin Milton et al. [11]
pNQVanT CmR; 320 bp vanT in pNQ705-1 This study
Table 2 Oligomers used in this study
Oligomer Sequencea
pNQ705-R 5'-GCGTAACGGCAAAAGCACCGCCGGACATCA-3'
SDvanT-F 5'-GGTGAGCTCCTGATGGAAATCGCTCTTGAAG-3'
SDvanT-R 5'-GGTTCTAGAGTGGCCACACTTCTTCGCG-3'
vanT-F2 5'-GGAAACATCGATAGAAAAACG-3'
vanT-R2 5'-GATGCAGAGCATGCTGAGG-3'
empA3W 5'-TGGTTTTCTTGATCCATTTTCAATGAGCGAATCATTCGCAGTCACGTCGC-3'
empA3C 5'-ACCAGCGACGTGACTGCGAATGATTCGCTCATTGAAAATGGATCAAGAAA-3'
a Restriction sites SacI (GAGCTC) and XbaI (TCTAGA) are underlined.
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| 15516264 | PMC529440 | CC BY | 2021-01-04 16:03:38 | no | BMC Microbiol. 2004 Oct 29; 4:42 | utf-8 | BMC Microbiol | 2,004 | 10.1186/1471-2180-4-42 | oa_comm |
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BMC Mol BiolBMC Molecular Biology1471-2199BioMed Central 1471-2199-5-191550714410.1186/1471-2199-5-19Methodology ArticleReal-time PCR quantitation of glucocorticoid receptor alpha isoform Melo Murilo R [email protected] Cláudia DC [email protected] Keli C [email protected]ças Nancy A [email protected] Carlos A [email protected] Pediatric Endocrinology Unit, Irmandade da Santa Casa de Misericórdia de São Paulo, Brazil2 Department of Physiology, Biomedical Sciences Institute, University of São Paulo, Brazil2004 26 10 2004 5 19 19 15 6 2004 26 10 2004 Copyright © 2004 Melo et al; licensee BioMed Central Ltd.2004Melo et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The expression of glucocorticoid-receptor (GR) seems to be a key mechanism in the regulation of glucocorticoid (GC) sensitivity and is potentially involved in cases of GC resistance or hypersensitivity. The aim of this study is to describe a method for quantitation of GR alpha isoform (GRα) expression using real-time PCR (qrt-PCR) with analytical capabilities to monitor patients, offering standard-curve reproducibility as well as intra- and inter-assay precision.
Results
Standard-curves were constructed by employing standardized Jurkat cell culture procedures, both for GRα and BCR (breakpoint cluster region), as a normalizing gene. We evaluated standard-curves using five different sets of cell culture passages, RNA extraction, reverse transcription, and qrt-PCR quantification. Intra-assay precision was evaluated using 12 replicates of each gene, for 2 patients, in a single experiment. Inter-assay precision was evaluated on 8 experiments, using duplicate tests of each gene for two patients. Standard-curves were reproducible, with CV (coefficient of variation) of less than 11%, and Pearson correlation coefficients above 0,990 for most comparisons. Intra-assay and inter-assay were 2% and 7%, respectively.
Conclusion
This is the first method for quantitation of GRα expression with technical characteristics that permit patient monitoring, in a fast, simple and robust way.
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Background
Glucocorticoids (GC) are a vital class of steroidal hormones that mediate profound and diverse physiological effects in vertebrates. GC are key hormones in the regulation of glucose homeostasis, but other essential functions are assigned to GC as well, such as regulatory roles in development and other metabolic pathways, stress and immune responses, neurobiology, and programmed cell death. In addition, corticosteroids are among the most widely prescribed class of drugs, primarily for their anti-inflammatory and immunosuppressive roles. They are also used in many chemotherapy regimens for leukemias and other cancers due to their critical capability to induce apoptosis.
Cortisol and its synthetic derivatives act upon the glucocorticoid receptor (GR), a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors. Prior to ligand binding, GR is primarily localized within the cytoplasm as an oligomeric complex composed of one receptor polypeptide, two molecules of heat shock protein 90 (hsp90), one molecule each of hsp 70, hsp 56 (immunophilin) and hsp23. When the hormone binds to the receptor, the GC-GR complex undergoes conformational changes, followed by dissociation from the hsp complex and dimerization of the GR molecules. The activated GR dimer is translocated into the nucleus and because of its high DNA affinity is able to bind to a specific DNA sequence named glucocorticoid response element (GRE), which is located in the vicinity of the target-regulated gene. The GR-GRE complex interacts with other components of the transcription apparatus to either enhance or repress the expression of the targeted gene [1-3].
There are several molecular mechanisms involved in glucocorticoid resistance or hypersensitivity (reviewed by Yudt, 2002) and GR expression seems to be a key one. These mechanisms are important for the regulation of cell and tissue-specific GC sensitivity, but they can be pathologically modified in clinical conditions such as AIDS, glucocorticoid-resistant asthma, rheumatoid arthritis and familial glucocorticoid resistance, among others [4-7].
Evaluation of GR expression in these conditions presents critical restrictions and has been limited to research protocols, partly due to analytical difficulties. Methods employed so far include ligand-binding assays, northern and western-blots and PCR. These methods could only provide qualitative or semi-quantitative information and a truly quantitative and reproducible evaluation of GR expression was still needed. In this study, we describe a quantitative real-time PCR (qrt-PCR) for GR alpha isoform (GRα) expression that is suitable for patient monitoring and can be easily reproduced.
Results
GRα and BCR (breakpoint cluster region) standard-curves were very stable using five different standard preparations, with maximum coefficient of variation (CV) of 10.3% observed for GRα most concentrated standard-point. GRα standard-curves presented CVs of 10.3%, 7.8%, 9.1%, 5.5% and 3.6% for the cycle-thresholds (Ct) obtained with each standard (6 to 2 logs of Jurkat cells). On both genes, standard-curves CV were greater at the most concentrated standard-points. BCR CV was smaller than those observed for GRα CV in all standard-points and all standard-curves. Standards of 6 to 1 log of Jurkat cells presented, respectively, CV of their Ct of 4.5%, 2.7%, 3.1%, 2.7%, 1.9% and 0.9%. BCR standard-curves had greater analytical sensitivity than GRα, with 10 EC/mL versus 100 EC/mL.
Linear regression analysis of pairs of standard-curves demonstrated strong correlation for both genes. The smallest Pearson correlation coefficient on GRα curves was 0.982, but most of them were higher than 0.990. Two pairs of BCR curves showed Pearson correlation coefficient of 1.000 (Table 1). Standard-curves slopes presented a CV of 7.3% for GRα and 3.7% for BCR (mean slopes of -0.279 and -0.271, respectively). Using the t-test, we found this difference in GRα and BCR slopes not significant (t = -0.819 with 8 degrees of freedom, P = 0.437).
Table 1 Evaluation of the standard-curve stability on five different sets of standard constructs.
A B C D E
A 1.000 0.993 0.996 0.994 0.993
1.000 1.000 0.994 0.997 0.996
B 0.993 1.000 0.987 0.981 1.000
1.000 1.000 0.994 0.997 0.996
C 0.996 0.987 1.000 0.999 0.989
0.994 0.994 1.000 0.998 0.997
D 0.994 0.981 0.999 1.000 0.982
0.997 0.997 0.998 1.000 1.000
E 0.993 1.000 0.989 0.982 1.000
0.996 0.996 0.997 1.000 1.000
Pearson's coefficients of correlation of GRα and BCR standard-curves for the same pair of experiments are expressed in the upper and lower rows, respectively. A to E refer to different sets of standard constructs, from Jurkat cell culture, viable cell count, RNA extraction, cDNA synthesis, standard dilution and qrt-PCR for GRα and BCR with calculations.
Intra-assay precision was around 2% for both evaluated controls. Cases 1 and 2 presented, respectively, mean GRα-EU of 1.406 and 1.443, with SD of 0.030 and 0.027 and CV of 2.1% and 1.9%.
Inter-assay precision was approximately 7% for both cases. Cases 1 and 3 presented, respectively, mean GRα-EU of 1.427 and 1.333, with SD of 0.088 and 0.104 and CV of 6.2% and 7.8%.
The median GRα-EU values of case 1 were similar both in intra and inter-assay evaluations, respectively, 1.402 and 1.414 (figure 1). This difference was not considered significant when we applied the Mann-Whitney Rank Sum test (p = 0.847).
Figure 1 Intra (A) and Inter-assay (B) precision evaluation of GRα expression Case 1 was evaluated in both situations. Bars indicate median values of GRα-EU (expression units). Intra-assay CV was 2.1% and 1.9% for case 1 and 2. Inter-assay CV was 6.2% and 7.8% (cases 1 and 3).
Discussion
Glucocorticoid resistance and hypersensitivity are determined by a number of factors such as intra-cellular hormone concentration, GR expression levels, GRα/GRβ heterodimerization, GR gene polymorphisms or mutations and GC-GR-protein interaction, among others [1,3,8,9]. One of the major factors affecting GC sensitivity seems to be the expression of its receptor. However, there were methodological difficulties regarding absolute quantitation of GR.
Binding assays were initially used to evaluate GR levels, but this assay is labor-intensive (and therefore, more error-prone), it assess only GR ligand-binding properties in the cytoplasm, providing no information regarding GR in the nucleus and requires the use of radioactive materials. Western blot assays were developed to assess GR protein levels. Despite numerous efforts to make well-characterized and specific antibodies, to date there are no highly specific human monoclonal antibodies targeted to GR isoforms, preventing the development of true quantitative methods, such as ELISA [2,10]. Quantitative PCR approaches were difficult until qrt-PCR was developed. Quantitative real-time PCR assays are based on cycle-by-cycle fluorescence monitoring of detectable PCR products (e.g., using TaqMan probes) and analysis of amplification during the exponential phase of PCR (for a review in qrt-PCR, see Mocellin, 2003 [11]).
Three groups recently described qrt-PCR techniques for GRα evaluation. DeRijk et al. developed Taqman evaluation of GRα and GRβ, however they did not describe the use of standard-curves or gene normalization [12]. Since their group was investigating GRα/GRβ ratio in the hippocampus, controls were produced using mixtures of PCR products of each isoform quantified by agarose gel densitometry. They found GRα/GRβ ratio of approximately 8300 in leukocytes and 14500 in hippocampal cells suggesting a minor physiological role for GRβ in normal cells from these tissues.
Boullu-Ciocca et al. evaluated GRα and GRβ expression using real-time PCR in obese and control cases [13]. They used 18S mRNA as a normalizing gene, but no standard-curves or precision characteristics were described. Mononuclear cells from control individuals showed GRα/GRβ ratio of 32:1, much higher than that observed by DeRijk and co-workers, and 9.2 and 2.6 in patients with gluteofemoral and visceral obesity, respectively. The lack of information about test precision makes interpreting the difference observed in the GRα/GRβ ratio among the two groups very difficult.
Pedersen and Vedeckis evaluated two cell lines using real-time PCR for seven different isoforms of GR (GRα, GRβ, and exon 1 splicing variants: 1A1, 1A2, 1A3, 1B, 1C), total GR (using exons 5–6) and 18S mRNA as normalizing gene [14]. Standards were plasmid constructs for each isoform. They observed a 1000-fold difference of GRα to GRβ expression levels and that the major exon 1 splicing transcripts are 1A3, 1B and 1C. Although elegantly designed, the authors conclude that their technique presents excessive variability for routine use. They estimated intra-assay CV to be 13%, inter-assay to be ca. 4%, inter-standard to be ca. 8% and overall CV of 15%. The test performance data was presented in a very succinct way and it was not possible to establish how inter-assay CV was calculated neither how inter-assay CV was smaller than intra-assay.
The aim of this study was to describe and validate the reproducibility for clinical monitoring of absolute quantitation of glucocorticoid receptor alpha isoform using real-time PCR. Our approach used a standardized procedure for Jurkat cell culture to make cDNA standards for both GRα and BCR. Standard-curves were reproducible, with less than 11% CV for all standards used and Pearson correlation coefficient above 0.990 for most comparisons. Intra-assay and inter-assay precision were 2% and 7%, respectively.
The BCR gene, derived from normal chromosome 22, was chosen as an appropriate endogenous control. The gene is ubiquitously expressed, has no reported pseudogenes and is a stringent control for the detection of degradation of RNA [15,16]. We found our approach to standard-curve construction to be an advantage of our method, since GRα and BCR were equally affected during all phases of standard construction. Another probable factor for achieving the precision reported is the similar expression levels and amplification rates of GRα and BCR.
Conclusions
In conclusion, we described for the first time a method for quantitation of GRα expression with technical characteristics that permit patient monitoring, in a fast, simple and robust way.
Methods
Standards
We developed standards for real-time quantitation of GRα expression by employing standardized Jurkat (E6-1 clone, ATCC) cell culture. Jurkat cells were grown in RPMI 1640 (Gibco) supplemented with 10% FBS, 1% penicillin-streptomycin (Gibco), 5% CO2 at 37°C. Culture medium was changed every 72 h, by centrifugation at 500 rpm for 5 min and 10 mL of fresh medium was added. Viable cells were counted in a hemocytometer (Neubauer chamber) using Trypan Blue (Sigma) and re-suspended to obtain a final density of 105 cells per milliliter. This procedure was repeated three times and 24 hours after the third medium exchange, cells were re-suspended to a final density of a 106 cells per milliliter. This strict protocol is necessary to ensure RNA extraction on the log phase of cell growing phase in order to minimize the variation of mRNA expression.
Controls
Three normal individuals participated in this study, in accordance with the guidelines proposed in The Declaration of Helsinki and approved by the ethics committee of our institution. Cases 1 and 2 are 30 and 32 year-old females, respectively, and case 3 is a 9 year-old boy. Heparinized blood was collected by venipuncture. After collection of 20 mL blood, mononuclear cells were separated using Histopaque-1077 (Sigma) by density gradient separation after centrifugation at 1750 rpm for 30 minutes. Cells were washed and re-suspended in PBS and 10% DMSO (Sigma) was added before freezing at -80°C. Prior to RNA extraction, DMSO was removed by PBS washing and re-suspension.
RNA isolation and cDNA synthesis
Total RNA was isolated from cells using guanidinium thiocyanate-chloroform extraction (Trizol, Gibco), according to the manufacture's recommendations. RNA was dissolved in DNAse-RNAse-free water (Gibco), after which the OD 260/280 was confirmed as >1.7, allowing the estimation of RNA concentration. RNA was stored at -80°C when the RT step was not immediate. cDNA was synthesized from 1 μg of total RNA, using TaqMan Reverse Transcription reagents (N808-0234, Applied Biosystems) with 1 × buffer, 5.5 mM MgCl2, 500 mM each dNTP, 2.5 mM random primers, 1.25 U/mL reverse transcriptase (Multiscribe reverse transcriptase) and DNAse-RNAse-free water as needed. After 10 minutes at 25°C, reaction was carried at 48°C for 30 minutes, followed by enzyme inactivation at 95°C for 5 minutes.
Standard preparation
Jurkat cell cDNA was serially diluted in 1:10 ratio with DNAse-RNAse-free water in order to obtain 6 logs of standards from one million cells per millimeter to ten cells per milliliter. They were numbered accordingly to their log number (6 to 1).
Real-Time PCR quantitation
GRα primers and probes were designed with Primer Express v.1.5 (Applied Biosystems) and both GRα and BCR (a normalizing gene) primers sets target two different exons, in order to prevent genomic amplification. BCR primers were previously used by Branford et al and our group [16,17]. Primers and probes used are indicated in figure 2.
Figure 2 Primer and probe hybridization sites for each transcript. The 5' end of the TaqMan probe is labeled with the reporter dye and the 3' end with the quencher dye. GRα stands for glucocorticoid receptor alpha isoform and BCR for breakpoint cluster region, used as a normalizing gene.
PCR conditions were equal for both genes, using TaqMan PCR Core kit (N808-0228, Applied Biosystems). Briefly, 1 × TaqMan buffer A, 500 μM each dNTP, 4.5 mM MgCl2, 200 nM of each primer, 100 nM of probe, 0.025 U/μL of AmpliTaq Gold, 5 μL of cDNA and water were incubated in a total volume of 25 μL. Cycle conditions on an ABI 7700 (Applied Biosystems) were: 95°C for 10 minutes (AmpliTaq Gold activation) followed by 45 cycles of 95°C for 15 seconds (denaturation) and 60°C for 90 seconds (annealing and extension). No template controls and duplicates of each standard, for both genes, were used in each run. Results were exported to MS-Excel to perform linear regression analysis for each gene, determining a standard-curve for Log of EC (equivalent of cells) based on the average Ct of duplicates (fig. 3).
Figure 3 Standard-curves for GRα (A) and BCR (B) in a typical experiment. In the upper part of the graph, we show the linear regression analysis equation and coefficient of correlation for log of Jurkat cell equivalents of expression and real-time PCR Ct (cycle-threshold), for each gene.
The average Ct for each sample was employed in the linear regression equation in order to obtain the EC, for each gene. Then, GRα-EC was divided by BCR-EC in order to establish the GRα-EU (Expression Units).
Standard-curve evaluation
In order to evaluate standard-curve stability with this process, we used five different sets of cell culture passages, trypan blue viable cell counting, RNA extraction, cDNA synthesis and qrt-PCR. These standard-curves sets were named A to E. We calculated the mean, SD and CV for each standard, the CV for standard-curves slopes, as well as Pearson correlation coefficient for each pair of curves.
Precision evaluation
Intra-assay precision was evaluated in a single experiment that included standard-curves for each gene and 12 replicates for each gene, in samples obtained from two normal controls (1 and 2). In order to calculate GRα-EU, pairs of samples (GRα and BCR) were established according to their position on the thermocycler.
Inter-assay precision was evaluated over eight different experiments, each one with its own standard-curve and duplicates of each gene, with samples obtained from two normal controls (1 and 3). Duplicates were averaged for calculation of GRα-EU.
Statistical analysis
Standard-curves and GRα-EU were calculated on MS-Excel 2000 for Windows. Linear regression of standard-curves pairs was calculated using SPSS 10.0 (SPSS, Chicago). For comparison of intra and inter-assay results of case 1, we employed Mann-Whitney Rank Sum test on SigmaStat v.2.03 (SPSS, Chicago), which was also used to perform the t-test. Differences of p < 0.05 were considered statistically significant.
Authors' contributions
MRM planned, performed and analyzed real-time PCR reactions and wrote the manuscript; CDCF did sample collection, cell culture optimization, RNA extraction and cDNA synthesis; KCM did statistical calculations and prepared figures and manuscript; NAR provided real-time PCR support; CAL planned the study, revised experimental data and the manuscript.
Acknowledgements
This work was supported by FAP – Fundação de Amparo à Pesquisa da Santa Casa de São Paulo.
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| 15507144 | PMC529441 | CC BY | 2021-01-04 16:48:01 | no | BMC Mol Biol. 2004 Oct 26; 5:19 | utf-8 | BMC Mol Biol | 2,004 | 10.1186/1471-2199-5-19 | oa_comm |
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BMC Mol BiolBMC Molecular Biology1471-2199BioMed Central 1471-2199-5-201551626510.1186/1471-2199-5-20Research ArticleThe Drosophila methyl-DNA binding protein MBD2/3 interacts with the NuRD complex via p55 and MI-2 Marhold Joachim [email protected] Alexander [email protected] Katja [email protected] Research Group Epigenetics, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany2 Adolf-Butenandt-Institut, Ludwig-Maximilians-Universität, Schillerstrasse 44, 80336 München, Germany2004 29 10 2004 5 20 20 2 7 2004 29 10 2004 Copyright © 2004 Marhold et al; licensee BioMed Central Ltd.2004Marhold et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Methyl-DNA binding proteins help to translate epigenetic information encoded by DNA methylation into covalent histone modifications. MBD2/3 is the only candidate gene in the Drosophila genome with extended homologies to mammalian MBD2 and MBD3 proteins, which represent a co-repressor and an integral component of the Nucleosome Remodelling and Deacetylase (NuRD) complex, respectively. An association of Drosophila MBD2/3 with the Drosophila NuRD complex has been suggested previously. We have now analyzed the molecular interactions between MBD2/3 and the NuRD complex in greater detail.
Results
The two MBD2/3 isoforms precisely cofractionated with NuRD proteins during gel filtration of extracts derived from early and late embryos. In addition, we demonstrate that MBD2/3 forms multimers, and engages in specific interactions with the p55 and MI-2 subunits of the Drosophila NuRD complex.
Conclusion
Our data provide novel insights into the association between Drosophila MBD2/3 and NuRD proteins. Additionally, this work provides a first analysis of the architecture of the Drosophila NuRD complex.
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Background
Methyl-DNA binding proteins are connecting DNA methylation to transcriptional silencing [1-4]. Up to now, six methyl-DNA binding proteins could be identified in vertebrates [5]. MeCP2, MBD2 and MBD3 can be found in large chromatin complexes containing histone deacetylase activity [1,6,4,3] whereas MBD4 is involved in DNA mismatch-repair [7]. MBD1 has been shown to repress transcription in cell culture [8] and recruits the histone H3-K9 methyltransferase SETDB1 to the chromatin assembly factor CAF-1 during S phase [9]. MBD2, which can bind methylated DNA [6], is a transcriptional repressor recruiting a Nucleosome Remodelling and Deacetylase complex (NuRD) to methylated CpG dinucleotides [6,3], whereas mammalian MBD3, which is not able to bind methylated DNA [10] is an integral component of NuRD [3]. Kaiso, a transcriptional repressor protein, can bind directly to CpG methylated DNA even though it lacks a conserved methyl-DNA binding domain [11]. Kaiso is a component of a subpopulation of MeCP1 complexes that lack MBD2 [11].
The Drosophila gene MBD2/3 encodes a protein, which shares high homology to mammalian MBD2 and MBD3 [12,4]. Due to differential splicing, Drosophila MBD2/3 is expressed in two isoforms, the smaller one is lacking part of the putative methyl-DNA binding domain [12-15]. The large isoform is expressed during early development, whereas the small isoform can only be detected during late embryogenesis [14,15]. In insect cells expressing only the small MBD2/3 isoform, this protein was found to be associated with components of the Drosophila NuRD complex [12-14]. Moreover, this protein could repress transcription effectively in transfected Drosophila cells [13,14].
The NuRD complexes of vertebrates are a heterogeneous group of complexes containing both histone deacetylase and nucleosome remodelling activities [3]. NuRD complexes comprise at least seven proteins. The ATP- dependent nucleosome-remodelling activity is mediated by MI-2, which contains a SWI2/SNF2-type helicase/ATPase domain, two chromodomains and two PHD fingers [16]. The related MTA1, MTA2 and MTA3 proteins have been found in various complex preparations [17,4,3,18]. MTA1 was originally identified as being overexpressed in metastatic carcinomas [19]. The histone deacetylases HDAC1 and HDAC2 and the two histone binding proteins RbAp46 and RbAp48 form the histone deacetylase core of NuRD complexes. Finally, as mentioned above, mammalian MBD3 is an integral component of at least some NuRD complexes [3].
Strikingly, the Drosophila genome contains clear homologues for all verterbrate NuRD proteins. Recombinant Drosophila MI-2 was shown to have ATPase and nucleosome mobilization properties [20]. In Drosophila the HDAC gene Rpd3 is important for segmentation of the embryo [21]. Drosophila p55, a WD-40 protein, is homologous to the histone deacetylase-associated proteins RbAp46 and RbAp48 [22]. Finally, Drosophila MTA-like displays extensive homology to the vertebrate MTA proteins [23].
The strong conservation between vertebrate NuRD complexes and the Drosophila NuRD complex implies a conserved function during the animal development. In previous studies cell lines were analysed that express only the small isoform of MBD2/3, lacking part of the putative methyl-DNA binding domain and a Drosophila specific domain. In order to analyse isoform-specific differences of MBD2/3 and their ability to bind NuRD proteins, we now extend our analysis to both isoforms in Drosophila embryos.
Results
MBD2/3 is associated with Drosophila NuRD proteins
An association of Drosophila MBD2/3 with the NuRD complex has been suggested previously [14,12]. However, these experiments were done in vitro or with protein extracts from tissue culture cells that express only the small isoform of MBD2/3. In addition, only a limited number of NuRD proteins were analysed. To confirm the association between MBD2/3 and NuRD in Drosophila embryos we size-fractionated nuclear protein extracts from 2–4 h or 0–12 h wild type embryos, respectively, by Superose 6 gel filtration chromatography. Proteins from individual fractions were then separated by standard SDS-PAGE and analysed by Western blotting using antibodies directed against known homologous subunits of the NuRD complex. MI-2 and MTA-like, which are homologues of subunits that are specific for vertebrate NuRD complexes [24,3] cofractionated with an apparent molecular weight of 1 MDa when extracts derived from early embryos (2–4 h) were applied (Fig. 1A, fractions 19 to 21). When extracts of late embryos (0–12 h) were analysed, MI-2 and MTA-like were detected in fractions 15 to 19, which correspond to an apparent molecular weight of approx. 2 MDa (Fig. 1B). This result indicates that the formation of NuRD is developmentally regulated during embryogenesis. Additionally, we found a precise cofractionation of the large isoform of MBD2/3 (Fig. 1, MBD2/3li) with MI-2 and MTA-like in both cases. The small MBD2/3 isoform (Fig. 1B, MBD2/3si) present in extracts derived from late embryos likewise cofractionated with MI-2 and MTA-like. This suggests a close association of both MBD2/3 isoforms with the Drosophila NuRD complex. The RPD3 and p55 proteins also cofractionated with MBD2/3 isoforms, but they were also found in fractions of lower molecular weight (Fig. 1). This is in agreement with the fact that both RPD3 and p55 are also components of other chromatin related complexes [25,26].
Figure 1 Cofractionation of Drosophila NuRD homologues in embryonic protein extracts Nuclear protein extracts were prepared from (A) 2–4 h and (B) 0–12 h old embryos, respectively and size-fractionated by FPLC using a Superose 6 column. Proteins from selected fractions were then separated by SDS gel electrophoresis and analysed by Western blotting. (A) During early embryogenesis the long isoform of MBD2/3 (MBD2/3li) cofractionated with MI-2 and MTA-like in fractions 19 to 21 (indicated in red), which correspond to a molecular weight of approx. 1 MDa. The small isoform of MBD2/3 is not expressed at this stage of embryogenesis. (B) During later stages of embryogenesis both isoforms of MBD2/3 cofractionated with MI-2 and MTA-like in fractions 15 to 19 (indicated in red), which correspond to a molecular weight of approx. 2 MDa. The size of marker proteins is shown left and on top, IN indicates input protein.
Interactions between MBD2/3 and NuRD homologues
To analyse the association between MBD2/3 and the NuRD complex in more detail and to identify direct NuRD interaction partner(s) of MBD2/3 we first used a GST-pulldown assay. In order to eliminate unspecific interactions with NuRD proteins, we established highly stringent assay conditions. This was achieved by assessing the interactions between radioactively labeled SV40 large T antigen (Fig. 2A, large T) and a number of control GST fusion proteins. When we used an incubation and washing buffer with high salt and detergent concentrations, significant amounts of SV40 large T were precipitated by p53 only (Fig. 2A), which is known to be a strong interactor of the large T antigen [27]. Residual binding detected with some of the other proteins (Fig. 2A) was considered background.
Figure 2 Interactions between MBD2/3 and NuRD homologues in a GST-pulldown assay (A) Analysis of interactions between radioactively labelled SV40 large T antigen (large T) and a number of control GST fusion proteins under stringent buffer conditions. Significant amounts of SV40 large T were only precipitated by p53. The faint bands seen with some of the other proteins were considered background. (B) GST-MBD2/3 fusion proteins for long and small isoforms (MBD2/3li, MBD2/3si, respectively) efficiently precipitated radioactively labelled MBD2/3 long isoform and p55 (solid arrowhead). Similarly, MI-2 protein could be precipitated by the small GST-MBD2/3 isoform (solid arrowhead). No interaction could be observed between long MBD2/3 or small MBD2/3 isoforms and MTA-like or RPD3 (open arrowheads). (C) A MBD2/3-specific antibody immunoprecipitates p55, but not GAGA factor from embryonic nuclear extracts. No proteins were detectable in control immunoprecipitations with a myc-specific antibody. (D) Size-fractionation of baculovirus-expressed MBD2/3 long isoform and RPD3. Baculovirus-expressed MBD2/3li elutes in fractions that significantly exceed the calculated molecular weight of the protein (36 kDa), thus indicating that MBD2/3li efficiently multimerizes in solution. This effect appeared to be specific for MBD2/3 and was not observed with baculovirus-expressed RPD3 protein (58kDa). The size of marker proteins is shown left and on top, IN indicates input protein.
We then performed the MBD2/3-NuRD interaction assay using these conditions and observed strong interactions only between the long and small MBD2/3 isoforms (Fig. 2B, MBD2/3li and MBD2/3si, respectively) and p55. To confirm the association between MBD2/3 and p55 in Drosophila embryos we immunoprecipitated nuclear extracts with MBD2/3-specific antibodies and with myc-specific control antibodies. Precipitates were analysed by Western blot for the presence of p55 and GAGA factor. This revealed a specific association of MBD2/3 with the Drosophila NuRD homologue p55, but not with the unrelated GAGA factor (Fig. 2C).
A weaker interaction could also be detected between the small MBD2/3 isoform and MI-2 (Fig. 2B). Neither RPD3 nor MTA-like binding exceeded the background level defined by our pilot experiment with SV40 large T protein (Fig. 2B). Our results thus identified p55 and MI-2 as the direct interaction partners of MBD2/3 in the NuRD complex. In addition, our results from the GST-pulldown assay indicate that Drosophila MBD2/3 forms dimers or multimers, similar to mammalian MBD2 and MBD3 [28]. To discriminate between the latter two possibilities we expressed a recombinant long MBD2/3 isoform and RPD3 in insect cells using a baculovirus expression system. Extracts from infected cells were subjected to Superdex 200 gel filtration and fractions were analysed by Western blot. The 58 kDa RPD3 protein eluted in fractions corresponding to molecular weights ranging from 66 kDa to 158 kDa, which suggested that RPD3 exists as monomers or dimers in solution (Fig. 2D). In contrast, the 36 kDa MBD2/3 isoform showed a strikingly different elution profile (Fig. 2D). The long MBD2/3 isoform eluted in a broad peak ranging from more than 158 kDa up to high molecular weight fractions (>400 kDa). No MBD2/3 was detected in fractions corresponding to the expected size of MBD2/3 monomers or dimers. This result is consistent with our earlier observation that MBD2/3 forms distinct aggregates in embryonic nuclei [15] and suggests that the protein efficiently oligomerises to form high-molecular weight complexes in solution.
To confirm these interactions in an independent set of experiments we also analysed the association between MBD2/3 isoforms and the NuRD proteins in a yeast two-hybrid assay. In agreement with our previous results, growth on highly selective (Fig. 3A, panel H) plates was only observed upon co-expression of the MBD2/3 small isoform and either MI-2, MBD2/3 long isoform or p55, and upon co-expression of both MBD2/3 isoforms. These interactions were confirmed by X-Gal staining of filter-lifted yeast colonies (Fig. 3A, panel X). In these assays the two MBD2/3 isoforms differed only with regard to MI-2 binding (Fig. 3A). However, differential binding could be eliminated by a slight reduction in the stringency of the assay (Fig. 3A, panel M), which suggested that both isoforms of MBD2/3 are capable of interacting with MI-2. Consistent with our GST-pulldown assays no interaction could be detected between the MBD2/3 long isoform and RPD3 or MTA-like (Fig. 3A).
Figure 3 Interactions between MBD2/3 and NuRD homologues in a yeast two-hybrid assay (A) An MBD2/3 long isoform bait efficiently transactivated reporter gene transcription in yeast expressing p55 or MBD2/3 small isoform prey constructs, respectively. A weaker transactivation could be seen upon expression of a MI-2 prey construct (panel M). An MBD2/3 small isoform bait transactivated reporter gene transcription co-expressing a MI-2 prey also under high-stringent conditions (panel H). No transactivation could be observed with MTA-like or RPD3 preys. N shows growth on non-selective plates, M on medium-stringency plates and H on high-stringency plates. X indicates X-gal staining of colonies grown on non-selective plates. (B and C) Delineation of interaction domains in a yeast two-hybrid assay. The methyl-DNA binding domain (MBD) is highlighted in red, the Drosophila specific domain (DSD) in green and the C-terminal coiled-coil domain (cc) in yellow. (B) The C-terminal region of MBD2/3 interacts with p55. The full-length p55 construct was used as a bait and various deletion mutants of MBD2/3 were used as preys. This identified the region between amino acids 178 and 305 of the MBD2/3 long isoform as the p55 interaction domain. (C) Deletion of the coiled-coil domain abolishes interactions between MBD2/3 small isoform and MI-2 under high stringent conditions.
Next we sought to delineate the domains that mediate the association between the MBD2/3 isoforms and their interacting proteins. To this end, we generated several MBD2/3 deletion mutants and tested their interaction with other proteins in a yeast two-hybrid assay. In a first series of experiments we tested the MBD2/3 mutants for their ability to interact with p55. This identified the region between residues 178 and 305 of the MBD2/3 long isoform as the p55 interaction domain (Fig. 3B). We also delineated the domain that mediates the interaction with MI-2. Our experiments revealed a strongly reduced interaction between a MBD2/3 small isoform derivative lacking the coiled-coil domain and MI-2 (Fig. 3C). It has been shown previously that vertebrate MBD2 and MBD3 form homo- and heterodimers via their N-terminal MBD domain and their C-terminal coiled-coil like sequences [28]. Our findings might reflect a direct interaction between the coiled-coil domain and MI-2 or a requirement for efficient MBD2/3 multimerization for MI-2 interaction. In conclusion, our results identify distinct regions in the MBD2/3 protein that mediate protein-protein interactions with other NuRD proteins.
In order to analyse the architecture of the NuRD complex, we performed a detailed yeast two-hybrid analysis including additional bait and prey constructs for NuRD proteins. Bait constructs for MI-2, RPD3, p55 and both MBD2/3 isoforms and prey constructs for MTA-like, RPD3, p55, and both MBD2/3 isoforms were co-transformed in all possible combinations and transformation was confirmed by growth on non-selective plates (Fig. 4A). Colonies were then replicated onto highly selective plates that allow growth only upon interaction between the two expressed proteins (Fig. 4B). Finally, we performed a filter lift assay followed by X-Gal staining to confirm the interactions (Fig. 4C). In addition to the results presented above, we found strong interactions between p55 and all NuRD proteins. We also observed a homotypic interaction for p55 but no homotypic interaction for RPD3. To confirm the specificity of the interactions, we tested most of the constructs reciprocally by changing the bait and prey vectors for expression of NuRD proteins and MBD2/3 isoforms. This data allows the establishment of a more detailed model of the NuRD complex. Fig. 4D summarizes the data from all experiments. p55 interacted with MI-2, MTA-like, RPD3, and both MBD2/3 isoforms. The small isoform of MBD2/3 interacted with MI-2 as well as with the large MBD2/3 isoform. Homodimerization of p55 and MBD2/3 is not shown in the model.
Figure 4 Analysis of the architecure of NuRD (A) Yeast colonies growing on non-selective plates carrying bait (left panel) and prey (top panel) NuRD and MBD2/3 constructs. (B) Replica plate with high-selective medium. Only yeast colonies with constructs encoding interacting proteins are able to grow. MBD2/3liΔ59-339 represents a truncated MBD2/3li construct that was used to confirm the specificity of the interaction between p55 and MBD2/3. (C) X-Gal staining of yeast colonies to confirm interactions. (D) Schematic illustration of protein-protein interactions in the NuRD complex. Homodimerization of p55 and MBD2/3 are not shown.
Discussion
It has been previously suggested that MBD2/3 is associated with the Drosophila NuRD complex [14]. This study determined that the small isoform MBD2/3 coelutes with some putative Drosophila NuRD subunits during fractionation of extracts derived from a Drosophila cell line. We have now extended this analysis to show that both isoforms of MBD2/3 coelute with NuRD homologues during fractionation of embryonic extracts. This data provides further evidence for a direct interaction between MBD2/3 and the NuRD complex.
Using several independent assays, we have demonstrated that MBD2/3 engages in homotypic interactions to form multimers. This effect is consistent with the formation of foci in embryonic nuclei [15] and also reminiscent of the interactions described for vertebrate MBD2 and MBD3 [28].
In addition, our data provides new insights into the association between MBD2/3 and NuRD. For example, we have shown that p55 appears to be the primary interaction partner of MBD2/3. We also observed a strong interaction between the small isoform MBD2/3 and MI-2, but not between MBD2/3 small isoform and MTA-like or RPD3. Additionally, we found interactions between p55 and all NuRD proteins, as well as a p55 homotypic interaction. The last finding is consistent with the fact that in the vertebrate NuRD complex the two p55 homologues RbAp46 and RbAp48 were identified as integral components [3]. We note that the vertebrate NuRD complex also contains the two RPD3 homologues HDAC1 and HDAC2 [3]; unexpectedly, we were not able to detect any homotypic interaction of RPD3 in the yeast two-hybrid assay. One explanation could be that the dose of the two co-expressed RPD3 bait and prey proteins could interfere with the reporter gene activity due to their inherent histone deacetylation activity. However, the gel filtration assay revealed that the 58 kDa RPD3 protein eluted in fractions corresponding to molecular weights ranging from 66 kDa to 158 kDa, which suggested that RPD3 can interact homotypically (Fig. 3C).
It is possible that more complex interactions are involved in the assembly of the NuRD complex but they might not have been detectable under the stringent conditions of our assays. The interaction between MBD2/3 and MI-2 detected in our assays could also contribute to the specific association between MBD2/3 and the NuRD complex.
The interaction between MBD2/3 and p55 could promote the assembly of specialized chromatin structures in the fly. p55 is a WD-40 repeat protein that is involved in many aspects of chromatin organization [29]. For example, p55 has been shown to be a component of the Drosophila CAF-1 complex that promotes nucleosome assembly [22]. In addition, p55 is also contained in the NURF chromatin remodelling complex [30] and in the E(Z) complex that regulates homeotic gene expression [25,26]. A mutant Drosophila allele for p55 is not available, but results obtained from Arabidopsis mutants with decreased levels of a p55 homologue indicate that the protein plays an important role in stabilizing epigenetic chromatin structures [31].
Conclusions
The goal of this study was to identify the interacting partners of the Drosophila methyl-DNA binding protein MBD2/3 within the Drosophila NuRD complex. We identified p55 and MI-2 as the primary interacting partners. We also found homo- and heterotypic interactions of the MBD2/3 isoforms, similar to vertebrate MBD2 and MBD3. Additionally, yeast two-hybrid assays revealed that p55 is able to specifically interact with all other NuRD proteins and can form homotypic interactions. Our data provides for the first time information about the architecture of the Drosophila NuRD complex. This allows us to develop a structural model of the NuRD complex.
Methods
DNA constructs
Constructs for the yeast two-hybrid assay were generated by PCR amplification from cDNA clones [32] using specific primers (Tab. 1) with an attached restriction endonuclease target site for cloning. The PCR products were subsequently cloned into pGBKT7 or pGADT7 (Clontech) using standard procedures. All constructs were sequenced and in vitro translated to confirm the expression of corresponding proteins. Additionally, the bait constructs were expressed in yeast and expression of the fusion proteins was confirmed by Western analysis.
Antibodies
The following antibodies have been described previously: rabbit anti-MI-2 [20], rabbit anti-MTA1 [19,4], rabbit anti-RPD3 [20], rabbit anti-p55 [22], rabbit anti-MBD2/3 [14] and rabbit anti-GAGA [33].
Immunoprecipitations
Immunoprecipitations were caried out in 300 mM KCl supplemented with 0.2 % NP-40. Rabbit anti-MBD2/3 or mouse anti-myc (Clontech) antibodies were added to 75 μl of nuclear extracts prepared as mentioned below and incubated for 12 h at 4°C. Protein G beads (Amersham) were blocked in 3 mg/ml BSA for 20 min at room temperature, washed three times with 300 mM KCl, 0.2 % NP-40 and added to the samples. Incubation was carried out for an additional 1 h at 4°C. The beads were collected by centrifugation and washed four times in 1 ml 300 mM KCl, 0.2 % NP-40. The beads were resuspended in loading buffer for SDS-PAGE and vigorously vortexed for 15 sec. The immunoprecipitates were separated by SDS-PAGE, without futher boiling, transferred to a PVDF membrane and probed with antibodies against RPD3, p55 and GAGA factor, respectively.
Preparation of protein extracts and gel filtration
For baculovirus expression, the MBD2/3 cDNA [32] was subcloned into the pVL1392 baculovirus transfer vector. Transfer vector and linearised baculovirus DNA were cotransfected into Sf9 cells using the Bac'n'Blue transfection kit (Invitrogen) and recombinant virus was amplified according to the manufacturer's instructions. Whole cell extracts of infected Sf9 cells were generated by resuspending cell pellets in lysis buffer (20 mM Hepes pH 7.6, 200 mM KCl, 0.1 % NP40), incubation on ice for 10 min, three freeze/thaw cycles and sonication. Nuclear extracts from Drosophila embryos were prepared as described previously [20]. Extracts were cleared by centrifugation and passaged through a 0.2 μm filter. 200 μl of cleared extract was applied to Superdex 200 HR 10/30 or Superose 6 HR 10/30 gel filtration columns (Amersham Pharmacia) and resolved in 20 mM Hepes pH 7.6 and 300 mM KCl on an Äkta Purifier system (Amersham Pharmacia) according to the manufacturer's instructions.
GST-pulldown assays
35S-methionine-labelled proteins were generated by in vitro trancription/translation of pGADT7_T, pGADT7_Mi-2, pGADT7_MTA-like, pGADT7_Rpd3, pGADT7_p55, pGADT7_MBD2/3 long isoform and pGADT7_MBD2/3 small isoform using the TNT coupled reticulocyte lysate system (Promega) according to the manufacturer's protocol. GST-MBD2/3 long isoform and GST-MBD2/3 small isoform fusionproteins were obtained by cloning the coding region of the two isoforms in pGEX4T1 (Amersham) and subsequent expressing of the constructs in BL-21 bacteria according to the manufacturer's protocol. GST-MBD2a was obtained from Hidetoshi Fujita [34]. GST-pull downs were performed under the following conditions: As incubation and washing buffer we used 20 mM HEPES, pH 7.8, 300 mM NaCl, 0.1 % desoxycholate, 0.1 % IGEPAL, 10 % glycerol. The radioactively labelled proteins were incubated in incubation buffer for 3 h at room temperature with GST fusion proteins coupled to Sepharose 4B (Amersham) or with GST alone coupled to Sepharose 4B. Beads were then washed five times for 10 min at room temperature. After the last washing step, beads were boiled in SDS loading buffer for 10 min and loaded onto standard SDS-polyacrylamide gels. After separation by SDS-PAGE, proteins were blotted onto a PVDF membrane, which was stained with Ponceau S, dried and exposed on X-ray films.
Yeast two-hybrid assays
The Matchmaker Two-Hybrid system 3 (Clontech) was used for all experiments, according to the manufacturer's instructions. The AH109 strain was used as host and transformed with various constructs (see above) using standard procedures. Transformants were selected on SD/-Ade/-His/-Leu/-Trp plates.
Authors' contributions
KK and JM carried out the molecular interaction studies. AB carried out the gel filtration experiments. JM conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.
Table 1 PCR primers used for the cloning of recombinant plasmids
MBD-BD-F: gga att cat gca aat gaa ccc gag cgt c
MBD-BD-R: tcc ccc ggg tgt ctt gag tgc atc ctg cag
Mi2-BD-F: cgc cat atg atg gca tcg gag gaa gag aat gac
Mi2-BD-R: gcg gcc tcc atg gcc gac gcc gga att att cga tag c
MTA-AD-F: ccg gaa ttc atg gcc aca aat atg tat cga gtc gg
MTA-AD-R: tcc ggg ccc ggt gac act ata gaa ctc gag
Rpd3-BD-F: atg gcc atg gat gca gtc tca aca gc
Rpd3-BD-R: ggc cgc tgc aga atg ttg ttc tcc ttg gcg
Rpd3-AD-F: cgc tca tat gat gca gtc tca cag c
Rpd3-AD-R: gca gct cga gaa tgt tgt tct cct tgg cg
p55-BD-R: ggc tca atc ttt ggt tat ggc gaa ttg gat ccg cg
p55-AD-F: ccg gaa ttc atg gtg gat cgc agc g
p55-AD-R: ggc gag ctc tta agc ggt att ggt ttc taa ctc gg
MBD-AD-F: gga att cat gca aat gaa ccc gag cgt c
MBD-AD-R: ccg ctc gag tgt ctt gag tgc atc ctg cag
MBD-AD-Δ59-339: ccg ctc gag cca cct tgt tat tgt tgt tgt tgc
BD designates primers used for the binding domain-constructs, AD designates primers used for the activation domain-constructs. F indicates forward primers, R indicates reverse primers.
Acknowledgements
We would like to thank Bodo Brueckner and Frank Lyko for critical revision of the paper and helpful guidance. We would also like to thank Jürg Müller and Jessica Tyler for antibodies, and Angelika Mitterweger and Michael Korenjak for help with protein extract preparation.
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| 15516265 | PMC529442 | CC BY | 2021-01-04 16:48:01 | no | BMC Mol Biol. 2004 Oct 29; 5:20 | utf-8 | BMC Mol Biol | 2,004 | 10.1186/1471-2199-5-20 | oa_comm |
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BMC NeurosciBMC Neuroscience1471-2202BioMed Central London 1471-2202-5-401550069810.1186/1471-2202-5-40Research ArticleMapping the brain's orchestration during speech comprehension: task-specific facilitation of regional synchrony in neural networks Härle Markus [email protected] Brigitte S [email protected] Andreas [email protected] Christian [email protected] Thomas R [email protected] Department of Psychology, University of Konstanz, Germany2004 24 10 2004 5 40 40 23 2 2004 24 10 2004 Copyright © 2004 Härle et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
How does the brain convert sounds and phonemes into comprehensible speech? In the present magnetoencephalographic study we examined the hypothesis that the coherence of electromagnetic oscillatory activity within and across brain areas indicates neurophysiological processes linked to speech comprehension.
Results
Amplitude-modulated (sinusoidal 41.5 Hz) auditory verbal and nonverbal stimuli served to drive steady-state oscillations in neural networks involved in speech comprehension. Stimuli were presented to 12 subjects in the following conditions (a) an incomprehensible string of words, (b) the same string of words after being introduced as a comprehensible sentence by proper articulation, and (c) nonverbal stimulations that included a 600-Hz tone, a scale, and a melody. Coherence, defined as correlated activation of magnetic steady state fields across brain areas and measured as simultaneous activation of current dipoles in source space (Minimum-Norm-Estimates), increased within left- temporal-posterior areas when the sound string was perceived as a comprehensible sentence. Intra-hemispheric coherence was larger within the left than the right hemisphere for the sentence (condition (b) relative to all other conditions), and tended to be larger within the right than the left hemisphere for nonverbal stimuli (condition (c), tone and melody relative to the other conditions), leading to a more pronounced hemispheric asymmetry for nonverbal than verbal material.
Conclusions
We conclude that coherent neuronal network activity may index encoding of verbal information on the sentence level and can be used as a tool to investigate auditory speech comprehension.
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Background
One key function of the cerebral cortex involves the integration of elements into a percept that separates them from the background. In this process, changes in cortical networks are formed and modified by experience through the simultaneous excitation of groups of neurons [1-3]. These "long-range connections formed by excitatory cortical neurons" [[4] p.3] are considered the anatomical substrate of this integrative capability. This integration has been modeled in detail for the visual system [e.g., [4]] and similar principles should also describe other sensory functions such as auditory speech perception and comprehension. This assumption was tested in the present study by probing patterns of co-activation within and across hemispheres during the processing of verbal and nonverbal acoustic material. Intra-hemispheric co-activation was taken as a large-scale measure of functional network activation, and coherence of oscillatory electromagnetic activity served as measure of co-activation in time. Coherence is defined as the correlated activity between two locations within a distinct frequency range.
Event-related brain responses, traditionally used in the study of cognitive processes, have been found to result from regional perturbations in ongoing brain activities in a self-organizing system rather than constituting a response set from an otherwise silent system. For example, Makeig and coworkers [5-7] showed that event-related potentials (ERP) must be viewed as perturbations in the oscillatory dynamics of the ongoing EEG. The response of successively activated groups of neurons is governed by an attractor, which means that different neuron groups, one after the other, contribute to large-scale changes in the magnetic field that move across brain areas, indicating spatio-temporal changes on a macroscopic level. The basin of attraction guarantees robustness of the propagating synchrony. Therefore, the activation of functional cortical networks may best be determined by examining the pattern of dynamic co-activation of groups of neurons [8,9]. As such, whenever neuronal cell assemblies fire 'in phase' the amplitude of oscillatory activity will increase.
On a macroscopic level, oscillatory coupling between large neuronal populations can be examined by externally driving the nervous system using oscillatory stimulation and then measuring the regional coherence of the electromagnetic activity [10]. Amplitude modulation of the stimuli induces the oscillatory pattern of the Steady-State-Response (SSR). For auditory stimuli the SSR is most prominent at modulation frequencies around 40 Hz [11]. Patel & Balaban [12] assessed the synchronization of the magnetoencephalographic SSR at this frequency over time (i.e., coherence) in order to investigate neural correlates of musical comprehension. When the stimulus sequences formed a percept (a melody relative to random sequence), coherence increased between left posterior and right frontal nodes. Similarly, Srinivasan et al [13] found increased inter- and intra-hemispheric coherence in the visual SSR when subjects consciously recognized visual stimuli in their field of view. Coherence measures have also been employed in the investigation of complex networks involved in the processing of nouns [14,15], music [16], the perception of Necker cube reversals [17], and in the acquisition of contingencies in a conditioning paradigm [18].
The present study investigated coherence patterns of the auditory evoked magnetic Steady-State-Field (SSF), specifically coherence among SSF-generators within and across hemispheres, as a measure of neural networks involved in speech comprehension. If, as we hypothesized, the comprehension of speech was related to the activation of neuronal assemblies in the left hemisphere, then we should see increased coherence in this region with the recognition of a meaningful sentence as compared to an incomprehensible string of sounds. We further hypothesized that meaningful verbal stimuli should be processed differently from musical melodies. That is to say, verbal material should affect the coherence of electromagnetic signals more in the left than in the right hemisphere whereas listening to a nonverbal complement of a meaningful sentence like a melody will activate more right- than left-hemispheric neuronal networks and influence coherence patterns involving the right hemisphere. Given that language and music share components, we assumed only a relative dominance in the interconnection of networks toward left- or right-hemispheric activity.
Results and discussion
The present study studied co-activated cortical networks involved in speech comprehension by using auditory steady-state (41.5-Hz amplitude modulated) stimuli and measuring the coherence of generator activity of the magnetic steady state response. Steady-state stimulus modulation were used for a sentence, which – following a German play-of-words – was first presented as an incomprehensible string of sounds, but became a comprehensible sentence after the sentence's meaning was explained to the subjects and was properly articulated. In addition to verbal stimuli, non-verbal stimuli were also studied which included a 600-Hz tone, a scale, and a melody-like combination of the scales' tones. The present analysis of SSF coherence in the source space (see methods) extended previous approaches [12], which employed SSR in the signal space to disclose networks involved in auditory perception.
Figure 1 (lower part) gives an example of the evoked magnetic 41.5-Hz SSF, averaged for the tone condition at the 148 sensors across the 12 subject. The sinusoidal 41.5 Hz oscillation is evident at all 148 sensors and a change in polarity over temporal areas suggests generator sources in the temporal cortices of each hemisphere. The Fourier Transform confirms the peak at the modulation frequency of 41.5 Hz for all stimulus-conditions in the sensor space (Fig. 1, upper left graph) and in the source space (mid-right graph in Fig. 1; illustrated for a selected dipole in the expected generator structure of the SSF, as indicated by the filled circle). No such peak was observed during the baseline. A comparison of the grand averages of the power spectra in sensor and source space (see Fig. 2A) demonstrates that conversion using the Minimum Norm Estimate (see methods) preserves the basic profile across conditions.
As expected for acoustic stimulation, overall MNE amplitudes were most pronounced in auditory areas of both hemispheres, with a varying degree of laterality. For the Laterality Index (see methods and Fig. 2B) an interaction of CONDITION × HEMISPHERE (F(4,44) = 3.06, p < 0.05, ε = 0.69) verified that nonverbal conditions as compared to the verbal ones induced a more pronounced asymmetry with more activity in the right compared to the left hemispheres (for the main effect of HEMISPHERE, F(1, 11) = 3.33, p < 0.1, and for the main effect of CONDITION, F(4,44) = 12.65, p < 0.0001, ε = 0.57). Planned comparisons confirmed significant effects of HEMISPHERE only for the nonverbal conditions (tone, t(1,11) = 4.5, p < 0.0001, scale, t(1,11) = 4.3, p < 0.000, and melody-like tone sequence, t(1,11) = 3.8, p < 0.0005).
Intra-hemispheric coherence was specifically affected by conditions (CONDITION × HEMISPHERE, F(4, 44) = 3.72, p < 0.05, ε = 0.46): As illustrated in Fig. 3A for the Laterality Index, higher intra-hemispheric coherence in the left than in the right hemisphere was induced when the string of words became a comprehensible sentence (planned comparison: t(1, 11) = 2.7, p < 0.01), whereas the tone induced higher intra-hemispheric coherences in the right as compared to the left hemisphere (t(1, 11) = 2.3, p < 0.05). The main effect of CONDITION was significant for intra-hemispheric coherence (F(4,44) = 8.35, p < 0.001, ε = 0.62) and inter-hemispheric coherence (F(4,44) = 10.79, p < .001, ε = 0.61) indicating higher coherence was induced by nonverbal than by verbal conditions. Since inter-hemispheric coherence may depend on the different generator strength, which was higher in the right than in the left hemisphere, the coherence measures were normalized in order to compensate for an effect of the signal to noise ratio. For normalization, the inter-hemispheric coherence measures were divided by the intra-hemispheric coherence measure of each condition. Still, a main effect CONDITION (F(4,44) = 12.1, p < 0.0001, epsilon = 0.76) indicates that coherence was larger for nonverbal than for verbal conditions.
Given that the major goal was to depict network signatures specifically involved in sentence comprehension, we applied an ANOVA to compare the coherence measure of the two verbal conditions. These were identical with respect to the physical stimulation, but differed in meaningful comprehension. For intra-hemispheric coherence a significant interaction involving CONDITION × HEMISPHERE × GRADIENT (F(1, 11)= 7.37, p < 0.05) reflected a relatively higher coherence in the left-posterior area after the string of words had been made comprehensible by explaining the sentence's meaning as opposed to the higher coherence in the right-posterior area for the incomprehensible word string. Profiles of intra- and inter-hemispheric coherence were similar, thereby resulting in similar statistical power for the CONDITION effect. This cannot be explained simply by a reduced signal-to-noise ratio in the verbal conditions, because normalized values show the same effect. We rather assume that increased laterality varies with decreased inter-hemispheric communication (coherence).
Inter-hemispheric coherence between dipoles located in the left (left cortical input) and right (right cortical input) auditory cortex and the remaining dipole sites are characterized (Fig. 3B) by larger coherence of activity across areas including the left auditory, occipital and right-posterior regions in response to the comprehensible sentence relative to the incomprehensible word string.
Considering coherent activity, i.e., synchronized oscillations between spatially distributed maps, as the representation of a percept, we followed Makeig et al. [6,7] who discuss evoked activity in terms of oscillatory perturbations, i.e., alteration of synchrony in ongoing activity. The comparison of two conditions with identical physical stimulation but different degrees of integration into a percept revealed that the synchronicity of auditory SSF increased among areas in the posterior left-temporal and right-occipital cortex when a sentence was comprehensible compared to the same material being incomprehensible. This suggests that a network was activated when an intelligible sentence was being processed. This assumption is in line with previous research in which a left-posterior activity focus was found during semantic processing [19-23], a left lateralized auditory-conceptual interface was localized at the temporal-parietal-occipital junction [24], and an occipital focus of oscillatory activity found for the processing of (visually presented) content words relative to verbs [25].
Whereas Scott et al. [26] reported an increase in regional cerebral blood-flow in the anterior part of the left superior temporal sulcus for intelligible sentences compared to acoustically equivalent non-intelligible sentences, the present results indicated such a pattern – enhanced left-anterior coherence – to be induced by the incomprehensible string of words (see Fig. 3B). At this point, hypotheses to resolve this discrepancy must remain provisional. However, it seems possible, that the speech-like – though incomprehensible – stimuli activated syntactical processing which has been associated with frontal activity [27]. In addition, the attempt to determine a syntactical structure has been found to activate the right temporal area [39] which would be in line with the right temporal coherence found for the present condition of incomprehensible word string processing (see Fig. 3B). Patel and Balaban [12] discussed increased coherence between the left posterior and right frontal areas for melody-like stimuli as a correlate of integrative processing of local and global pitch information. Thus, it seems possible that in our study the condition of incomprehensible word string similarly activated pitch processing.
Finally, there is the possibility that the order of stimulus presentations may have affected the results. While counterbalancing was not possible for the specific verbal stimulus condition (see methods), we would not have expected order effects to be large since similar temporal dynamics were not observed for the nonverbal conditions. However, an effect of time cannot be ruled out as steady state responses and their generator activity were largest for a simple 600-Hz tone which was presented first.
SSF were larger for the nonverbal conditions (tone, scale, melody) than for the verbal material, particularly in the right hemisphere. While right-hemispheric processing of tonal perception has frequently been reported [28-31], the general dominance of right-hemispheric SSF remains to be explained. As mentioned before, it seems possible that it reflects a carry-over effect from the sequence of conditions which invariably started with the tone. It may also reflect bilateral processing of verbal material which has been indicated by various imaging approaches [19]. The combination of verbal and nonverbal conditions within one experimental session may have blurred rather than elucidated the co-activation of material-specific networks.
Still, greater right- over left-hemispheric generator activity asymmetry was found in the nonverbal conditions and less asymmetry found in the verbal conditions. Moreover, intra-hemispheric coherence patterns showed distinct, hemisphere-specific patterns for verbal (more pronounced left-hemispheric) and nonverbal (more pronounced right-hemispheric coherence) processing. When lateralized coherence patterns were examined by a laterality index, the clearest left-hemispheric coherence focus emerged for the comprehensible sentence and the clearest right-hemispheric coherence focus emerged for the tone. While we had expected a melody induced dominant right-hemispheric activation, a more bilateral activation was found for the melody-like tone sequence. For the scale, there was a shift towards left-hemispheric asymmetry of coherence. An explanation for this finding might be that the 'melody' was constructed to include the tones of the scale which may have resulted in a melody-like tone sequence even though it did not resemble common melodies or songs. This processing of an unfamiliar 'melody' might have activated temporal (left) and spectral (right) processing, as suggested by [28,29], resulting in a more bilateral activation. While a simple tone contains only spectral information, a melody also contains temporal information.
Conclusions
In sum, the present study demonstrates that the analysis of the synchronization of evoked magnetic steady-state fields in the source space can map neuronal networks (co-)activated during speech comprehension. Our techniques add spatial information to evidence on left-hemispheric areas involved in language processing, and support co-activation or synchronization within complex neuronal networks as a cortical substrate of integration in perception – like speech comprehension.
Methods
Subjects
Data of twelve German native speaking subjects (7 female, mean age 25.3 ± 6.3 years) were included in the analysis. (From the 14 subjects, who had participated in the study, data from one subject had to be discarded because of frequent movement artifacts and from another one, who recognized the play-of-words, see below.) It was ascertained by interview that the subjects did not suffer from any language, audiological or neurological dysfunction. Right-handedness was assessed by a modified version of the Edinburgh handedness questionnaire [32] to be 97.1 ± 4.3. Moreover, all subjects reported having first-degree right-handed relatives. None of the subjects reported to be a professional musician and none reported to be particularly involved in hearing or practicing music. Prior to the experimental session, subjects were informed about the procedure and given informed consent forms. After the experiment, each subject received a financial bonus of 15€.
Material and design
All stimuli were amplitude modulated at 41.5 Hz (sinusoidal amplitude) with a modulation depth of 90%. Verbal stimuli consisted of words composing a sentence. Nonverbal stimuli consisted of tones forming a scale or a tune or a simple tone. A German play-on-words served as the template for the two verbal conditions. In the first case a sentence is spoken without spacing between words and without accents which creates an incomprehensible word string. The German sentence 'Mähn Äbte Heu? Heu mähn Äbte nie! Äbte mähn Gras' means in English 'Do abbots cut hay? Abbots never cut hay, abbots mow lawns'. If pronounced as a string 'MähnÄbteHeuHeumähnÄbtenieÄbtemähnGras' this utterance, due to a lack of non-phonetic context [33], sounds like speech although meaning cannot be inferred. When the sentence is properly pronounced in the second case, the meaning becomes clear and can be used to parse the information at subsequent trials, allowing a listener to comprehend the sound string as a sentence. For the present study, the incomprehensible string-of-word-version was generated synthetically (software: MBROLA) with a female voice and a fundamental frequency of 200 Hz. None of the 12 subjects included in the data analyses knew the play-of-words and were unable to recognize the meaning of the sentence before it was properly articulated and explained.
The three nonverbal conditions comprised of a 600 Hz sinusoidal tone, a descending major scale (C6 B5 A5 G5 F5 E5 D5 C5, 1034 – 517 Hz), and an arrangement of the same tones (C5 E5 G5 C6 A5 F5 D5 B5). All stimuli of all conditions were adjusted to the length of the sentence and lasted for 4419 ms (sample-rate of 16 kHz/16 bit, mono), and each of the five conditions comprised 15 repetitions that were separated by inter-stimulus intervals of 4419 ms. This long inter-stimulus interval allowed the same signal-to-noise ratio for the baseline and the stimulus conditions which should prevent habituation effects on the SSF. Stimuli were adjusted to have the same average loudness by normalizing to root-mean square (RMS) and were presented at 50 dB above the individually assessed hearing threshold balanced for both ears. In each subject, the hearing threshold was assessed by presenting short 600 Hz beeps with ascending and descending intensity. For each subject and ear the mean hearing threshold was determined from the ascending and descending sequence.
Task and procedure
During the experiment, which lasted about 45 minutes, the subject was seated in a supine position. Subjects were asked to listen carefully to the stimuli, while fixating a point at the ceiling of the chamber in order to avoid head and eye movements. They were further informed that they would be asked questions about the stimuli during the experimental session, and that they should reply by saying 'yes' or 'no'.
All stimuli were presented in blocks with 15 repetitions. Conditions were separated by breaks of about 5 min each. For every subject the experimental session started with the 600-Hz sinus tone (15 repetitions, condition 1), followed by the word string (condition 2). After 5 repetitions, the subject was asked whether he/she understood what he/she was hearing and could reproduce the meaning of the speech. (None of the subjects could.) Subsequently, the stimulus presentation was continued, and the subject was asked again after the 10th and the 15th presentation, whether he/she understood the meaning of the speech (None of them could).
Then, the experimenter entered the room and pronounced the sentence properly and slowly, so that its meaning became clear. Each subject was asked to reproduce the sentence, in order to ascertain that it was properly understood. After the experimenter had left the subject chamber, the experiment continued with condition 3, which comprised the identical physical stimulation as condition 2 differing only in that the subject now listened to the string of words knowing its meaning, Again, the subjects were asked after 5 repetitions, if they could reproduce the meaning of the sentence, which now they all could. Given that once the sentence's meaning is obvious, one can easily grasp the sentence, the sequence of condition 2 and 3 could not be reversed and thus, the sequence of presentation could not be randomized across subjects.
Condition 4 (scale) and 5 (melody-like tone sequence) were arranged in a similar way, in that the subject was asked after 5 repetitions each, whether or not s/he perceived the sequence of tones as a melody. Eleven of the twelve subjects indicated that the tone sequence sounded like a melody and one was not sure about it. None of them perceived the scale or the simple tone as melodic.
Data acquisition and analysis
The magnetoencephalogram (MEG) was recorded with a 148-channel whole head system (MAGNES® 2500WH, 4D Neuroimaging, San Diego, USA) installed in a magnetically shielded room (Vaccumschmelze, Hanau, Germany). Data were recorded continuously with a sampling-rate of 1017.25 Hz and a 0.1–100 Hz band-pass filter. The electrooculogram (EOG) and the electrocardiogram (EKG) were recorded and stored together with the MEG-data for offline artifact control. Silver-silverchloride electrodes were placed on the outer canthi for the monitoring of horizontal eye movements, and above and below the right eye for vertical eye movements. EKG electrodes were placed on the right collarbone and below left costal arch.
Prior to data analysis, the trials for each condition were submitted to a noise-reduction procedure that subtracted the external noise recorded by MEG reference channels. These noise-corrected data were then bandpass filtered (28–60 Hz, 48 db/Oct, zerophase) and averaged across epochs separately for each condition (epoch-length: 8838 ms, 4419 ms pre-stimulus baseline). Epochs were visually inspected for EOG and EKG artifacts and epochs with magnetic fields greater than 5 pT were rejected. A minimum 13 (of the total 15) epochs per subject were available for further analyses.
The steady state field (SSF) in response to the 41.5-Hz amplitude modulated stimuli was extracted using a moving average procedure. A window of 5 cycles (120.5 ms) of the 41.5 Hz Steady-State signal was shifted 179 times cycle-by-cycle (24.5 ms) across averaged epochs (separately for the 4419-ms baseline and the 4419-ms stimulus duration, the moving average procedure starting 144.5 ms post stimulus). The resulting moving-average epoch was detrended. Figure 1 illustrates that a SSF was successfully induced by the stimulation.
The generators of the SSF were determined in the source space for each epoch using the minimum norm estimate, MNE [34-37] using an algorithm implemented in MATLAB-based in-house software developed by Hauk [35,36].
The MNE is an inverse method reconstructing the primary current that underlies an extracranially recorded time-locked magnetic field. The procedure is based on the assumption that the data vector d, which contains the recorded magnetic activity at given sensor sites, can be described as the product of the leadfield matrix L, which specifies the sensor's sensitivity to the sources, the source current vector j [34] and a noise component ε. Since L and d are known, and ε is treated as if estimated with an accuracy of ~.05, the MNE for j is the mathematically unique solution of the equation which minimizes the squared current density (j2 = min). This solution is obtained by multiplying the pseudo-inverse of the leadfield matrix L with the data. Given the high number of sensors and the presence of noise, spatial regularization is performed with the factor λ. This algorithm allows sources to be omitted, if they do not contribute to the measured magnetic field. A priori information about the number or locations of cortical sources is not required. Following Hauk et al. [35,36], who evaluated the dependence of the accuracy of inverse solutions on the depth of the source for concentric shells, solutions for a shell at 60% radius were determined as a compromise between blurring and depth sensitivity (ca. average radius of cortex, 77 equidistant dipole locations, covering the lateral surface of the brain, were chosen). That is, voltage data were projected to a source space consisting of 350 evenly distributed dipoles with three orthogonal orientations at each dipole location. For every location two tangentially orientated dipole-components were included in further analysis. The mean MNE amplitude, corresponding to the dipole strength in nAm/cm2, was determined as mean vector length of both tangentially orientated dipole-components across 5 cycles.
Co-activation of generators was evaluated by all possible pair-wise combinations of the MNE at all dipole locations, according to the algorithm (Matlab, Mathworks):
Spectral coherence is a function of frequency with values between 0 and 1 that indicate how well the input x (in the present study MNE at dipole location x) corresponds to the output y (MNE at dipole location y) as a function of frequency (in the present study 41.5 Hz). This algorithm estimates the coherence of two vectors x and y by computing the ratio of the squared cross power spectra (Pxy), divided by the product of the power spectra for each vector (Pxx.*Pyy), where Pxy(f) is the cross power spectrum estimate, Pxx(f) is the power spectrum of the time series at location x, Pyy(f) is the power spectrum estimate of time series at location y and f is the frequency index.
The vectors of 4495 points in length were subdivided into 8 overlapping segments of 613 points (603 ms), each of which was submitted to Hanning windowing. For each vector, the power spectra were obtained as the product of the Discrete Fourier Transforms and its complex conjugate, scaled by the number of points used for x and y, and averaged across segments [38]. For cross spectra the products of discrete Fourier Transforms for vectors x and y were averaged across segments. This algorithm was applied to four pairs of dipole orientations (dpo), normalized (Fisher Z-transformation) and averaged to result in one coherence measure for every pair of locations (dpx -dpy). As a measure of co-activation or coherence, the first order coherence between a region of interest (ROI) covering 7 locations over Heschl's gyrus and all other 77 locations was determined.
Effects of the five conditions on the distribution of MNE amplitudes and on the coherence measure were evaluated by means of repeated measurement analyses of variance (ANOVA) with the factors CONDITION, HEMISPHERE (comparing all left and all right dipole-locations, excluding midline locations), and GRADIENT (comparing left- and right-anterior versus left- and right- posterior dipole-locations, excluding midline locations). For inspection of the hemispheric asymmetry of MNE, the ANOVA was performed on the Laterality Index (LI: left- minus right-hemispheric MNE divided by their sum, resulting in an index without units). For the evaluation of intra-hemispheric coherence, the first order coherence between the respective left- or right-hemispheric ROI and the other 34 locations of the respective hemisphere entered the ANOVA (comparing conditions), for the evaluation of inter-hemispheric coherence, coherence between the left-hemispheric ROI and all other 34 locations of the right hemisphere and between the right-hemispheric ROI and all other 34 locations of the left hemisphere was submitted to the ANOVA comparing conditions. A separate ANOVA of the two verbal conditions with the factors CONDITION, HEMISPHERE and GRADIENT probed the hypothesis of a change in coherence-topography induced by sentence comprehension. Where appropriate, significance levels are reported with Greenhouse-Geisser correction adjusted degrees of freedom. Interactions were verified by planned posthoc comparisons (two-tailed paired t-tests), and displayed in t-maps without additional alpha correction.
List of abbreviations
ANOVA: Analysis of variance
EEG: Electroencephalogram
MEG: Magnetoencephalogram
MNE: Minimum Norm Estimate
RMS: Root-mean square
SSF: Steady-State- (magnetic) Field
SSR: Steady-State-Response
Authors' contributions
MH developed the experimental design, carried out the experimental study and developed and accomplished the data analyses, BR supervised the study and composed the paper, AK advised and assisted the SS-design and SSF analysis, CW supervised the MEG measurements and advised the coherence analyses, TE provided the experimental idea, advised the experimental design, the MEG methods and analyses.
Acknowledgment
Research was supported by the Deutsche Forschungsgemeinschaft and the Volkswagen-Stiftung. We are grateful to Dr. William J. Ray for correcting the revision.
Figures and Tables
Figure 1 Spectral power and topography of the Steady-State-Field (averages of 12 subjects). Bottom graph: topography of the magnetic SSF evoked by the 600 Hz tone (top view, nose up). Upper left graph (green box): spectral power of the magnetic SSF at a selected sensor (left anterior, indicated by green frame) for all conditions and the baseline. Upper right graph: (blue box): spectral power of the magnetic SSF in the source space (MNE) for one selected dipole location. This dipole is approximately located in the area of the left auditory cortex and is indicated by a blue filled circle. Conditions: Tone = 600 Hz tone, NoComp = incomprehensible word string, Comp = word string after comprehension as sentence, T.seq = melody-like sequence of tones, Scale = tone scale).
Figure 2 A: Topographical distributions of the SSF in source space (averages of 12 subjects) separately for the five conditions; Orthographical top view, spherical spline interpolation, nose indicated by a small triangle. Each contour-line represents 0.000125 nAm/cm2 (rounded values at scale). Dark grey indicates higher values of activity. B: Asymmetry of activity in the source space as indicated by the Laterality Index (all left- minus all right-hemispheric MNE amplitude values divided by their sum), averaged across subjects. Positive values indicate left lateralized activity and negative values indicate right-lateralized activity. T-bars represent the standard error of the mean). Tone = 600 Hz tone, NoComp = incomprehensible word string, Comp = word string after comprehension as sentence, T.seq= melody-like sequence of tones, Scale = tone scale.
Figure 3 Topography of coherence measure in source space (averages of 12 subjects). A: Asymmetry of intra-hemispheric coherence measures as indicated by the Laterality Index (all left- minus all right-intra-hemispheric coherence values divided by their sum) for the five conditions. Positive values indicate left-lateralized coherence and negative values indicate right-lateralized coherence. T-bars represent the standard error of the mean). Tone = 600 Hz tone, NoComp = incomprehensible word string, Comp = word string after comprehension as sentence, T.seq= melody-like sequence of tones, Scale = tone scale. B: Difference-maps and t-maps of the coherence values comparing the two verbal conditions (Comp minus NoComp). Maps are shown in 110° top view and are spherical spline interpolated; nose indicated by triangle. The left graphs show the coherence between the area of the left auditory cortex and all 77 dipole-locations (left cortical input), the right graphs display the coherence between the area of the right auditory cortex and all 77 dipole-locations (right cortical input). For difference-maps, each contour-line represents a step of 0.025, without units. Pink and red colours illustrate higher coherence values after sentence comprehension (Comp>NoComp), grey and black colours illustrate higher coherence values for the incomprehensible word string (Comp<NoComp). For t-maps, significant differences are shown at the 5% level in red (Comp>NoComp) and black (Comp<NoComp).
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| 15500698 | PMC529443 | CC BY | 2021-01-04 16:03:46 | no | BMC Neurosci. 2004 Oct 24; 5:40 | utf-8 | BMC Neurosci | 2,004 | 10.1186/1471-2202-5-40 | oa_comm |
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BMC NeurosciBMC Neuroscience1471-2202BioMed Central London 1471-2202-5-431553325810.1186/1471-2202-5-43Research ArticleMorphological correlates of injury-induced reorganization in primate somatosensory cortex Churchill James D [email protected] Jason A [email protected] Cara L [email protected] Dale R [email protected] Preston E [email protected] Department of Psychology, Saint Louis University, St. Louis, Missouri 63103, USA2 Department of Psychology, Program in Neural Science, Indiana University, Bloomington, Indiana 47405, USA2004 8 11 2004 5 43 43 17 6 2004 8 11 2004 Copyright © 2004 Churchill et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Topographic reorganization of central maps following peripheral nerve injury has been well characterized. Despite extensive documentation of these physiological changes, the underlying anatomical correlates have yet to be fully explored. In this study, we used Golgi impregnation and light microscopy to assess dendritic morphology following denervation of the glabrous hand surface in adult primates.
Results
After survival durations that permit complete physiologically-defined reorganization, we find a systematic change in the dendritic arborization pattern of both layer II/III pyramidal and layer IV spiny stellate cells in the contralateral hand region of area 3b, compared to unaffected cortical areas. In general, our analyses indicate a progressive expansion of distal regions of the dendritic arbor with no appreciable changes proximally. This pattern of distal dendritic elaboration occurs for both basilar and apical dendrites.
Conclusions
These observations are consistent with the notion that latent inputs gain expression in reorganized cortex after nerve injury via their influence through contacts with more distally located termination sites.
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Background
The ability of the nervous system to modify its output in accordance with experiential demands is a central tenet of neuronal plasticity. For many years, the view of critical periods permeated our beliefs; almost dictating that plasticity beyond such epochs was, at best, minimal. The seminal experiments of Merzenich, Kaas and colleagues [1,2] have proved instrumental in moving the field beyond this restrictive mindset by showing that the central representation of the skin surface is subject to dramatic modification following peripheral nerve injury in adult primates. On the foundation of these observations, great strides have been made in understanding the mechanisms [3-9] and extent [10-13] of this phenomenon. These findings have generalized beyond sensory systems and collectively have been interpreted as reflective of fundamental properties of the nervous system.
While physiological techniques are frequently used to characterize topographic (re)organization of central maps, the underlying anatomical correlates have not been thoroughly investigated. Using intracellular injection techniques, thalamic axons have been reported to innervate a much broader sector of cortex than necessary to represent typical receptive field size, suggesting the existence of "latent" inputs [14,15]. Disinhibition is a strong candidate as the primary mechanism during the immediate phase of somatotopic reorganization following nerve injury [16-18]. While unmasking of latent inputs may account for a portion of the overall reorganization [19], modification of central maps is neither complete immediately following nerve injury [1,20-22] nor dependent on a single mechanism [20,22-25]. Moreover, topographic reorganization appears to be permanent in nature [26], while at least some neurochemical changes have shown to be relatively transient [6,18]. Together, these observations suggest that alterations in the underlying anatomical connectivity might provide a stable platform for the maintenance of modified somatotopy.
In this study, we report our examination of neurons in two cortical layers; spiny stellate cells in layer IV, as this is the primary input target of thalamocortical axons; and pyramidal neurons in layer II/III, as supragranular changes have been shown to precede somatotopic modification in the granular cell layer [27]. We predicted that dendritic arborization in the affected areas would be altered following peripheral nerve injury, providing an anatomical correlate of the functional changes. If the anatomical correlates of physiologically-defined changes can be readily observed, our understanding of the mechanisms underlying such changes would be greatly enhanced.
Results
Figure 1 presents typical Golgi-filled layer II/III pyramidal (Fig. 1A) and layer IV spiny stellate cells (Fig. 1B). Figures 1C (pyramidal) and 1D (stellate) are corresponding reconstructions of the same two cell types. Our initial inspection of the data revealed considerable heterogeneity as one moved from proximal to distal regions of the dendritic arbor. With regards to the proximal halves of the arbors, we observed no statistically significant differences between deprived and control groups for either basilar or apical dendrites.
Layer II/III pyramidal neurons
We found a greater number of intersections in the distal halves of dendritic arbors of pyramidal cells in deprived relative to control cortex. For basilar dendrites, there is a 92.0% increase in the number of intersections in the distal arbors of deprived cells relative to control cells (9/9, p < .01; see Fig. 2a). Likewise, we find an 89.5% increase in total basilar dendritic length in the distal sectors of deprived cortical pyramidal cells, compared to control neurons (9/9, p < .01; see Fig. 2b). For apical dendrites, there are 63.1% more intersections in distal portions of the arbors of deprived cells relative to controls (15/16, p < .01; see Fig. 2c). Finally, the average length of the distal apical dendrites of deprived pyramidal cells is 37.4% greater than in controls (14/16, p < .01; see Fig. 2d).
Layer IV spiny stellate cells
A largely comparable set of outcomes were found for spiny stellate cells in layer IV. For the distal sectors of basilar dendrites, there are 66.7% more intersections in arbors of deprived relative to control neurons (9/9, p < .01; see Fig. 3a). Similarly, the overall average length of distal basilar dendrites is 92.4% longer in deprived stellate cells compared to controls (9/9, p < .01; see Fig. 3b). For the distal apical dendritic arbors, deprived stellate cells have, on average, 25.5% more intersections than are found in control neurons (11/12, p < .01; see Fig. 3c). Conversely, while the distal apical dendrites of deprived neurons are 20.5% longer than controls, on average, this difference is not statistically significant (8/12, p > .10; see Fig. 3d).
Differential effects of deprivation on basilar versus apical dendrites
Deprivation of a specific region of somatosensory cortex by nerve transection clearly had a detectable effect on the distal portions of both the basilar and apical dendrites of both layer II/III pyramidal cells and layer IV spiny stellate cells. The data also support the contention that the basilar dendrites of both cell types were more profoundly affected by deprivation than were their apical dendrites. The magnitudes of the deprivation effects are more pronounced in basilar than in apical dendrites (see Fig. 4; Mann-Whitney = 0; p < .01).
Discussion
General observations
In the present experiments, we investigated whether changes in dendritic morphology of neurons in deprived somatosensory cortex are correlated with the well-documented topographic reorganization that follows peripheral nerve injury in adult primates. Our general findings were that in deprived cortical areas, dendritic arbors were expanded distally, while being unaffected proximally. This pattern was found for both the basilar and apical dendrites of layer IV spiny stellate and layer II/III pyramidal neurons, though the effects were more pronounced for the basilar dendritic arbors, a difference that is consistent with previous reports [28]. Measures of dendritic length and the frequency of intersections, both well accepted metrics of dendritic arborization, yielded generally similar patterns of observations. These anatomical changes could well provide the means by which the functional changes in map topography proceed. Alternatively, they could reflect generic neural responses to deprivation per se, and have little or nothing to do with the functional reorganization.
Does nerve injury-induced reorganization reflect the strengthening of normally latent inputs?
Previous research has shown that the spread of thalamocortical arbors is much broader than necessary for the expression of typical receptive field size in primary somatosensory cortex [14,15]. Because of this disparity between the grain of the cortical topographic map [29] and the far more extensive thalamocortical anatomy, we have suggested that all parts of thalamocortical arbors cannot be equally effective in conveying suprathreshold receptive field information to the cortex, and that changes in synaptic efficacy could sustain the topographic plasticity that follows peripheral nerve injury [14,30]. Our observations of subthreshold, latent inputs to the cortex [3], and the emergence of their expression when dominant inputs are attenuated [31] lend support for this idea. Moreover, these presumptive latent inputs are largely prevented from gaining expression in cortex when NMDA glutamatergic receptors are blocked [20,23]. Such a blockade could prevent reorganization by preventing changes in the strength of existing synapses [e.g., [32]], by interfering with neurite outgrowth [33], or both. In any event, such latent inputs become evident only when the normally expressed, dominant inputs are somehow weakened – via pharmacological disinhibition [16,18,34], nerve injury [19,35], or use-dependency [36-38]. The data reported here are consistent with this notion, and suggest that distal sectors of the dendritic arbor may be selectively innervated by these latent inputs.
Our observations that distal regions of apical dendrites, which clearly reside in upper cortical layers, are modified come as no surprise. Previous work has shown that layer IV spiny stellate cells act primarily as intracolumnar signal processors; while pyramidal cells integrate both horizontal and top-down information [39]. Supragranular layers appear to be particularly fertile to altered stimulation patterns as cortical reorganization occurs initially in the outermost layers of cortex, followed later by changes in the granular cell layer [27]. Measures of astrocytic recruitment mirror this outside-to-inside temporal progression of experience-dependent reorganization as well [40]. The selective elaboration of distal regions of the dendritic arbor is also consistent with data that implicate intracortical pathways as playing a major role in cortical reorganization [41,42], though, clearly, the contribution of bottom-up processes cannot be discounted [12].
Is reorganization a secondary consequence of other mechanisms/processes?
While morphological changes may be less likely to account for acute changes in somatotopy after nerve injury, restructuring of the underlying anatomy could well correlate with the longer-term, persistent changes in cortical topographic maps. The modifications of distal dendritic regions reported here may be interpreted from at least three possible, non-exclusive, perspectives. First, expansion of the distal arbor may be a homeostatic response to a reduction in stimulation frequency/pattern following nerve injury. Progressive elaboration of the distal arbor might be an attempt to maintain optimal stimulation levels, and, thus, normal interneuronal trophic relationships. The altered somatotopy could be construed as simply the epiphenomenonal consequence of the activation of a homeostatic response. Second, the elaboration of the distal arbor may be a general property of the nervous system, a mechanism that permits the brain to respond in a dynamic and adaptable manner [43]. This supposes that the functional changes in cortex that follow nerve injury are adaptive, and that has not been convincingly demonstrated. Third, the observation that changes occur distally may simply reflect the fact that areas relatively distant to the soma are more vulnerable/susceptible to changes, regardless of the adaptability of such changes [44]. The reliability of progressively distal changes independent of dendritic location (apical or basilar) is certainly consistent with this idea. While these possibilities are not mutually exclusive, and certainly not all-inclusive, we believe that expansion of the distal arbor reported here is reflective of the altered activation pattern following nerve injury and serves as a long-term trace of this modified stimulation pattern.
Conclusions
Considering the range of survival durations following nerve injury in the current study, the observation of modifications to both layer IV spiny stellate and layer II/III pyramidal neurons was not unexpected. While this broad survival range may have "smeared" our snapshot with respect to the temporal integration of anatomical changes, our intention was simply to determine whether morphological changes were occurring at any point during the reorganization process. Our data clearly indicate that the anatomy in affected cortical areas is subject to modification and that the morphological changes observed may be related to the functional reorganization revealed electrophysiologically. We have begun experiments to better refine the temporal window in which these changes become evident.
In sum, we have shown that just as the functional responsiveness of the mature primate nervous system is susceptible to change, so is the underlying anatomy. Our observations that the anatomical changes appear to be either potentiated in, or possibly restricted to, distal regions of the dendritic arbor provide additional insight into the mechanisms involved in the physiological changes. Further research will be instrumental in determining the exact role that the underlying anatomy plays in this complex reorganization process.
Methods
Adult squirrel monkeys (Saimiri scireus or Saimiri bolivensius) were socially housed with food and water available ad libitum. In six animals, the median and ulnar nerves to one hand were transected following the principles of animal care detailed in NIH publication no. 86–23. The local institutional animal care and use committee approved all procedures prior to initiation of any experiments. Briefly, monkeys were anesthetized with an intramuscular injection of a mixture of ketamine hydrochloride (25–30 mg/kg) and xylazine (0.5–1.0 mg/kg). Their forearms were shaved and prepared for surgery with alternate scrubbings of povidone-iodine and alcohol. Under sterile conditions an incision was made along the midline of the ventral forearm, the median and ulnar nerves were located by blunt dissection and cut about midway between the elbow and wrist. The epineural sheath of the proximal stump was retracted 0.5–1.0 cm, and the exposed nerve avulsed. The empty epineural sheath was re-extended, folded back upon itself and ligated. The nerve stumps were repositioned and the incision closed with sutures. Post-surgically, all subjects received penicillin, dopram hydrochloride, and dexamethasone injections. Subjects were permitted to recover for a period of time previously shown sufficient to permit complete reorganization of the hand representation in cortical area 3b (3–52 months, mean = 15.3). Two additional subjects served as naïve controls.
Following electrophysiological mapping of the affected cortical areas, animals were overdosed and perfused transcardially with 0.9% saline. Brains were extracted and immersed in a modified Golgi–Cox solution for 11 days, thereafter dehydrated and embedded in celloidin. Tissue blocks were sectioned coronally at 150 μm in thickness for morphological assessment and every fourth section in the series was cut at a thickness of 90 μm for Nissl staining to facilitate cytoarchitectonic identification of area 3b borders. Free-floating sections were processed and mounted on glass, according to previously reported procedures [45].
Analysis of dendritic morphology was conducted blind to experimental condition on thoroughly impregnated cells using methods described by Sholl [46]. For each animal, ten layer IV spiny stellate and ten layer II/III pyramidal cells from the hand area of somatosensory area 3b contralateral to the nerve injury were drawn using Neurolucida (MicroBrightfield) at 600 × magnification. In addition, ten area 3b cells of the same two types located outside of the hand representation were drawn to serve as controls.
We have shown previously that dominant and latent inputs from the three nerves innervating the hand have overlapping territories in area 3b [13,32], with the latent inputs gaining expression when the dominant inputs are weakened with peripheral nerve transection. These observations prompted us to treat the proximal and distal portions of the dendritic arbors separately in our statistical comparisons. To accomplish this goal, we divided the arbors into proximal and distal halves using the Sholl ring halfway between the soma and the most distal dendritic process as the dividing point. For dendritic length and intersection comparisons, the deprived and control averages for each Sholl ring were compared. A simple binomial test [47] was then applied to determine whether a systematic, statistically significant difference exists between those sets of means.
Authors' contributions
JDC participated in design of study, conducted the histological processing, drafted the manuscript: JAT conducted some of the histological processing: CLW participated in design of study: DRS participated in design of study: PEG participated in design of study, conducted statistical analyses, conceived and coordinated the study. All authors read and approved the final manuscript.
Acknowledgments
Supported by National Institutes of Health Grant NS37348 (PEG). We thank E.E. Garman and L.L. Arnold for technical assistance.
Figures and Tables
Figure 1 Photomicrographs and reconstructions of Golgi-filled neurons. a: A typical layer II/III pyramidal cell used in the analysis of dendritic arborization. b: A typical layer IV spiny stellate cell. c,d: Reconstructions of a pyramidal and a stellate cell. Scale bar = 50 μm.
Figure 2 Dendritic arborization pattern in the distal sectors of layer II/III pyramidal cells. The figure depicts the extent of arborization as a function of distance from the cell body using a Sholl ring analysis. On average, basilar dendrites in deprived cortex are both more complex (increased number of intersections; Fig 2a) and longer (dendritic length; Fig 2b) than those from controls. Likewise, apical dendrites in deprived cortex are also more complex (intersections; Fig 2c) and longer (length; Fig 2d), compared to controls. All effects were statistically significant.
Figure 3 Dendritic arborization pattern in the distal sectors of layer II/III stellate cells. The figure depicts the extent of arborization as a function of distance from the cell body using a Sholl ring analysis. On average, basilar dendrites in deprived cortex are both more complex (increased number of intersections; Fig 3a) and longer (dendritic length; Fig 3b) than those from controls. Likewise, apical dendrites in deprived cortex are also more complex (intersections; Fig 3c) and longer (length; Fig 3d), compared to controls. All effects were statistically significant.
Figure 4 Comparison of basilar vs. apical effects for all cells analyzed. The figure illustrates that for both cell types (pyramidal and stellate) and metric of dendritic arborization (intersections and length), the magnitude of the effect was reliably larger for basilar dendrites than for apical dendrites.
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| 15533258 | PMC529444 | CC BY | 2021-01-04 16:03:46 | no | BMC Neurosci. 2004 Nov 8; 5:43 | utf-8 | BMC Neurosci | 2,004 | 10.1186/1471-2202-5-43 | oa_comm |
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BMC Cardiovasc DisordBMC Cardiovascular Disorders1471-2261BioMed Central London 1471-2261-4-201551859010.1186/1471-2261-4-20Research ArticleA common genetic factor underlies hypertension and other cardiovascular disorders Williams Frances MK [email protected] Lynn F [email protected] Tim D [email protected] Alex J [email protected] Twin Research and Genetic Epidemiology Unit, St Thomas' Hospital, London SE1 7EH, UK2 Department of Medicine, University of East Anglia, Norwich NR4 7TJ, UK2004 1 11 2004 4 20 20 7 1 2004 1 11 2004 Copyright © 2004 Williams et al; licensee BioMed Central Ltd.2004Williams et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Certain conditions characterised by blood vessel occlusion or vascular spasm have been found to cluster together in epidemiological studies. However the biological causes for these associations remain controversial. This study used a classical twin design to examine whether these conditions are linked through shared environmental exposures or by a common underlying genetic propensity to vasospasm.
Methods
We investigated the association between hypertension, migraine, Raynaud's phenomenon and coronary artery disease in twins from a national register. Phenotype status was determined using a questionnaire and the genetic and environmental association between phenotypes was estimated through variance components analysis.
Results
Responses were obtained from 2,204 individuals comprising 525 monozygotic and 577 dizygotic pairs. There was a significant genetic contribution to all four traits with heritabilities ranging from 0.34 to 0.64. Multivariate model-fitting demonstrated that a single common genetic factor underlies the four conditions.
Conclusions
We have confirmed an association between hypertension, migraine, Raynaud's phenomenon and coronary artery disease, and shown that a single genetic factor underlies them. The demonstration of a shared genetic factor explains the association between them and adds weight to the theory of an inherited predisposition to vasospasm.
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Background
A number of conditions characterised by blood vessel occlusion and/or vascular spasm have been found to be associated in both clinical and epidemiological studies. These include hypertension (HPT), Raynaud's phenomenon (RP), migraine (MIG) and coronary artery disease (CAD) [1-5]. Whether these associations are the consequences of a shared environmental risk factor or represent an underlying propensity to develop the conditions through a common biological mechanism remains controversial. The exploration of the genetic and environmental relationships underlying these conditions is one approach to resolving the biological basis for the association. We have examined the association between these phenotypes in a classical twin study conducted in a large sample. This approach allows us to assess whether the association between HPT, RP, MIG and CAD is explained by a common genetic or environmental aetiology.
Methods
Subjects and methods
Subjects for this study were twins enlisted with the St Thomas' UK Adult Twin Registry [6]. These monozygotic (MZ) and dizygotic (DZ) twin volunteers have been recruited since 1992 using twin registers and successive national media campaigns. For historical reasons most enrolled twins are female. This well studied population is sent regular questionnaires for self-completion concerning a wide range of health issues. Questions relating to HPT, MIG, RP and CAD respectively were contained within large questionnaires sent to the twins in 1998 and 2000. The questions were non-consecutive and respondents were unaware of the hypothesis being tested. The questionnaires also included standard questions relating to zygosity assignment [7]. In addition, fifty-four percent of the respondents had their zygosity assigned with certainty by multiplex DNA fingerprinting using variable tandem repeats on samples taken on previous attendances at the Twin Unit.
Classification of HPT, RP, MIG AND CAD
Classification of the traits HPT, RP, MIG and CAD was based on standard, validated criteria where possible. HPT was classified by asking about a doctor's diagnosis of "high blood pressure" when not pregnant. Questions to determine presence of migraine were based on the UCSD Migraine Questionnaire [8] (including at least five episodes of unilateral, pulsating headache over the previous year, duration four to seventy-two hours, noise and light sensitivity). RP was classified by confirming digital sensitivity to cold and required the respondent to have had recurrent episodes of colour change involving at least two colours [9]. CAD was classified either by respondents having a doctor's diagnosis of heart disease or angina, a previous heart attack or heart operation; or by answering affirmatively to questions taken from the Rose Angina questionnaire [10].
Analysis
Traits were defined categorically as present or absent using the definitions listed above. Phenotypic associations between the four traits were assessed using odds ratios and 95% confidence intervals (CI) corrected to take into account the paired nature of the data. The odds ratios were adjusted for possible confounding by age, smoking and body mass index (BMI). Evidence for a genetic contribution to each trait was examined by estimating casewise concordance [11]. This measures the probability that the co-twin of an affected twin also expresses the trait. Higher casewise concordance in MZ pairs compared to DZ twin pairs indicates a genetic effect.
Where data suggested a genetic influence, a quantitative measure of genetic contribution was estimated using structural equation modelling (Mx software [12]). This standard approach to twin analysis assumes an underlying model in which the observed phenotypic correlation among twins is explained by latent additive genetic influences (A) (which have a correlation of 1 in MZ twins and 0.5 in DZ twins), common environmental influences (C) (having a correlation of 1 in both MZ and DZ twins) and the random environment (E) (uncorrelated among twins). Analysis proceeds on the assumption that the observed categorical phenotype is accounted for by a continuous underlying liability to the trait [13]. Correlation in underlying liability is used as the basis for measuring the association between variables in the modeling. The significance of shared genetic and environmental factors was tested by stepwise deletion in a sequence of models containing the variance components (A, C and E) and assessing the deterioration in chi-squared for the fit of the model. Heritability was estimated from the size of the additive genetic contribution to the final selected model.
Modeling was then extended to consider the genetic and environmental associations between all four variables simultaneously [13]. Three multivariate models were considered:-
(1) the Cholesky decomposition (Figure 1) which includes four independent genetic and environmental factors. The first factor loads on all the traits, the second factor loads on all traits except the first, the third loads on all traits except the first two etc. This provides the fullest potential explanation of the data
Figure 1 The Cholesky AE model. Diagram representing the Cholesky AE model, in which additive genetic (A) effects are shown loading on to the four traits: the unique environment (E) would load similarly
(2) the independent pathway model (Figure 2) is a submodel of the Cholesky model and considers the data to be explained by a single shared genetic and shared environmental factor
Figure 2 The independent AE pathway model. Diagram of the independent AE pathway, in which a single genetic and single unique environment factor loads on to each of the four traits, as well as factors specific to each trait
(3) the common pathway model (Figure 3) which considers a single shared latent phenotype, determined in turn by latent genetic and environment factors.
Figure 3 The common pathway model. Diagram of the common factor pathway in which single genetic and unique environment factors load on to the traits via a phenotypic latent variable
The suitability of the common and independent pathway models may be determined by comparing the model's Akaike information criterion (AIC) with that of the fullest model, the Cholesky. The AIC represents the balance between model fit and the number of parameters (parsimony), with lower values of AIC indicating the most suitable model. Parameter estimates from the most appropriate model were used to calculate the genetic and environmental correlation between variables.
Results
Complete data for analysis were available from 1,102 pairs of female twins, comprising 525 MZ and 577 DZ pairs. The mean (± SD) age of the twins was 48.5 (± 8.2) years. The prevalence of each trait is shown in Table 1.
Table 1 Prevalence of the traits by zygosity and the p value of the difference between zygosities
trait MZ prevalence (%) n = 1050 DZ prevalence (%) n = 1154 p
HPT 131 (12.5) 181 (15.7) 0.052
RP 111 (10.6) 131 (11.4) 0.584
MIG 264 (25.1) 266 (23.1) 0.290
CAD 71 (6.8) 76 (6.6) 0.873
n represents number of twins, MZ monozygotic, DZ dizygotic, HPT hypertension, RP Raynaud's phenomenon, MIG migraine, CAD coronary artery disease
Four of the six combinations of traits showed significant phenotypic association after adjusting for age, smoking and BMI (odds ratios, Table 2). HPT was clearly not associated with RP but there was a suggestion of association of HPT with migraine (significantly so if adjusted for age and smoking only, data not shown). Since smoking status, age and BMI did not influence the size of the odds ratios, these variables were not included in the multivariate modeling. Concordance data and the results of univariate modeling confirmed a significant heritable component to all 4 traits (Table 3). Multivariate model fitting showed that for each of the three models tested, a model containing genetic (A) and unique environmental (E) factors provided the best explanation of the data (Table 4, in bold): incorporation of a shared environmental factor (C) offered no significant improvement in the fit of any model. The independent AE pathway model (Figure 2) offered the best explanation of the data, suggesting that a single common genetic factor loads on the four traits HPT, RP, migraine and CAD. A unique environmental factor loads similarly on the traits. Using this model, genetic and unique environmental correlations between the four traits were calculated (Table 5). Overall, the genetic correlations were greater than the environmental correlations, suggesting a greater role for the genetic factor than the unique environmental factor.
Table 2 Odds ratios (95% confidence interval) of unadjusted and adjusted (for age, smoking and BMI) cross-trait associations
HPT RP MIG CAD
HPT
adj
RP 0.88 (0.59–1.31)
adj 0.99 (0.64–1.54)
MIG 1.31 (0.99–1.73) 1.62 (1.21–2.17)
adj 1.31 (0.95–1.80) 1.68 (1.23–2.3)
CAD 2.85 (1.96–4.16) 2.03 (1.29–3.17) 2.19 (1.53–3.13)
adj 2.41 (1.56–3.73) 2.27 (1.42–3.65) 1.77 (1.19–2.63)
Calculation of odds ratios included a correction factor for familial clustering. Unadjusted odds ratios are shown above and adjusted below, for each combination of phenotypes. BMI represents body mass index, HPT hypertension, RP Raynaud's phenomenon, MIG migraine, CAD coronary artery disease.
Table 3 Casewise concordance rates by zygosity and heritability estimates of the four traits
twin type total pairs discordant pairs (+/-) concordant pairs with trait (+/+) casewise concordance (95% CI) heritability (95% CI)
HPT MZ 525 73 29 0.44 (0.34–0.55) 0.64 (0.49–0.79)
DZ 577 135 23 0.25 (0.17–0.34)
RP MZ 525 75 18 0.32 (0.21–0.44) 0.46 (0.30–0.63)
DZ 577 111 10 0.15 (0.07–0.24)
MIG MZ 525 144 60 0.46 (0.38–0.53) 0.43 (0.30–0.55)
DZ 577 186 40 0.30 (0.23–0.37)
CAD MZ 525 55 8 0.23 (0.10–0.35) 0.34 (0.13–0.55)
DZ 577 72 2 0.05 (0.00–0.12)
Casewise concordance rates (95% confidence intervals, CI) are shown for each trait by zygosity. An estimate of trait heritability (95% CI) is also given.
HPT represents hypertension, RP Raynaud's phenomenon, MIG migraine, CAD coronary artery disease.
Table 4 Results of the model fitting: comparison of the 3 models
model chi2 df p AIC
CHOLESKY ACE 32.95 30 0.33 -27.05
AE 33.35 40 0.76 -46.65
CE 65.82 40 0.01 -14.18
INDEPENDENT ACE 38.98 36 0.34 -33.02
AE 40.82 44 0.61 -47.18
CE 72.03 44 0.01 -15.97
COMMON FACTOR ACE 49.68 42 0.19 -34.32
AE 49.68 47 0.38 -44.32
CE 81.49 47 0.00 -12.51
For each submodel, the chi2 statistic, the degrees of freedom (df) the probability (p) and the Akaike information criterion (AIC) are shown. AIC is used to evaluate the fit, with the best fitting submodel shown in bold in each case.
Table 5 Genetic correlations (bold type, below in table) and unique environmental correlations (above) of the traits
HPT RP MIG CAD
HPT 0.29 0.01 0.17
RP 0.20 0.00 0.05
MIG 0.23 0.36 0.00
CAD 0.35 0.56 0.65
HPT represents hypertension, RP Raynaud's phenomenon, MIG migraine, CAD coronary artery disease
Discussion
This is the first study to examine the role of genetic and environmental factors in explaining the association between HPT, RP, migraine and CAD. The results suggest that all four variables share a heritable basis. These conditions have been shown to be associated with one another and individually each is known to have a genetic basis. The data presented here confirm these previous findings and suggest that shared environmental factors such as diet and lifestyle do not contribute to their expression. In view of the nature of these phenotypes, we speculate that the shared genetic component leads to a predisposition to vasospasm. Indeed, a 'vasospastic phenotype' to account for their co-occurrence has been postulated by others [14]. The demonstration of a single genetic component lends weight to such a theory.
A number of considerations should to be taken into account when interpreting these results. Self administered questionnaires were used for trait ascertainment, introducing the possibility of recall bias. In the present study efforts were taken to minimise recall bias: when surveyed, the twins were unaware of the hypothesis being tested; the questions relating to vascular phenotypes were included non-sequentially and were amongst many other questions in two questionnaires mailed at different times; and the twins completed questionnaires separately with no knowledge of their co-twin's responses. Furthermore there is no reason to suspect differential rates of recall in MZ when compared to DZ twins, hence any effects of recall should not have biased our estimates of genetic influence. Some conditions, such as RP, are notoriously difficult to diagnose regardless of the method employed [15] and it is possible that some subjects have cardiac valve rather than coronary artery disease. No attempt was made to differentiate primary RP and essential HPT from their secondary forms. However, questionnaire diagnoses were based on standard methods [8-10]. Despite these limitations, the traits' prevalences are in keeping with the findings of others for RP (9.6%[3], 5% and 16.8% according to geographical area sampled [16]), migraine (20% [17]) and CAD (8% [18]). In addition, heritability estimates are consistent with published findings for MIG (49–58% [19]), blood pressure (40–70% [20], 57% [21]) and CAD (15–30% [18]). Taken together, these observations suggest that our twins are representative of the adult female population and do not simply reflect a 'healthy volunteer' sample.
The assumptions underpinning twin studies themselves may be questioned. Unequal sharing of the family environment by MZ and DZ twins has been raised as a concern, but this has been shown not to be the case [22]. In addition, twins from this cohort have been shown to be similar to singletons with respect to anti-hypertensive drug use, blood pressure and other phenotypes [23]. The proposed inverse relationship between birth weight and HPT [24] and CAD [25] could potentially bias a study of twins and cardiovascular disease. The explanation for this relationship is still debated, but maternal environment has been suggested to be the main influence on adult blood pressure [26]. As we have demonstrated that the shared environment makes no significant contribution to the vasospastic phenotype, this is not a likely source of error.
It is clear that genetic factors play an important role in all four conditions. The demonstration that they are heritable is consistent with numerous reports of clustering in twin and family studies conducted in a range of settings, including a Dutch kindred with an autosomal dominant condition characterised by vascular retinopathy, migraine and Raynaud's phenomenon [27]. The detection of a common, genetically determined mechanism that contributes to these conditions is important because it points to an intermediate phenotype, vasospasm, and provides a possible focus for future studies. As with all chronic diseases and traits, the difficulty is in establishing which genes are responsible. Vascular tone is controlled at many levels, including local soluble mediators and neurotransmitters. Genes proposed through the study of the individual phenotypes include the beta2-adrenergic receptor gene in hypertension [28]; the muscle acetylcholine- and serotonin receptor genes in RP [29]; and a G protein subunit polymorphism and endothelial nitric oxide gene polymorphism in CAD [30]. A gain of function mutation in a vasoconstrictor or a loss of function mutation in a vasodilator may predispose to vasospasm: many of these genes deserve further consideration with the identification of a common genetic factor underlying HPT, migraine, RP and CAD.
In summary, this twin study has identified phenotypic associations between four vascular conditions and shown that the association is explained by a single common genetic factor. These findings are consistent with the proposed 'vasospastic phenotype' and suggest that studies of genes controlling vascular tone will help to define the genetic basis of these conditions.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
FMKW analysed the data and drafted the manuscript. LFC designed the questionnaires, analysed the data and assisted with the manuscript. TDS collected the twins on the Twin Register and assisted with the manuscript. AJM conceived of the study, collected the twins on the Twin Register, analysed the data and co-drafted the manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
This work was funded by the Arthritis Research Campaign.
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| 15518590 | PMC529445 | CC BY | 2021-01-04 16:30:03 | no | BMC Cardiovasc Disord. 2004 Nov 1; 4:20 | utf-8 | BMC Cardiovasc Disord | 2,004 | 10.1186/1471-2261-4-20 | oa_comm |
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BMC GastroenterolBMC Gastroenterology1471-230XBioMed Central London 1471-230X-4-281550929710.1186/1471-230X-4-28Research ArticleHereditary risk factors for the development of gastric cancer in younger patients Yaghoobi Mohammad [email protected] Naser [email protected] Farhad [email protected] Raheleh [email protected] Yasamin [email protected] Ashraf [email protected] Arezou [email protected] Mohammad Reza [email protected] Mahshid [email protected] Reza [email protected] Digestive Disease Research Center, Tehran University of Medical Sciences, Shariati Hospital, Kargar Shomali St, Tehran 14114, Iran2004 27 10 2004 4 28 28 29 1 2004 27 10 2004 Copyright © 2004 Yaghoobi et al; licensee BioMed Central Ltd.2004Yaghoobi et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
It is believed that the development of gastric cancer (GC) before the age of 50 has a hereditary basis. Blood group A and history of gastric cancer in first-degree relatives have been shown to be risk factors for GC.
Methods
In this case-control study, we enrolled patients with GC who were diagnosed before the age of 50. Patients who were diagnosed as having GC were selected. A total of 534 cases were found; of these, 44 diagnosed before the age of 50 were included in the case group. For the control group, 22 males and 22 females were randomly selected from the remaining subjects, who had diagnoses of GC after the age of 50. All the surviving patients and family members of the dead patients were interviewed about the history of cancer in the family and the age at which other family members developed cancer. The blood group of each subject was also obtained.
Results
forty-four cases under 50 years old (mean age: 36.2 years) and forty-four controls (mean age: 67.1 years) were enrolled in the study. At the time of the study, 59.1% of the study group and 50% of the control group were alive (P value = NS). In the study group, 68.1%, 13.6%, 13.6% and 4.5% had blood groups O, A, B and AB, respectively. In the control group the corresponding figures were 27.7%, 63.6%, 6.8% and 4.5%. First or second-degree relatives with cancer, including gastric (the most frequent), breast, lung, gynecological and hematological malignancies, were noted in 54.5% of the cases and 11.4% of the controls (p < 0.01). Family histories of cancer were accepted as valid provided that they were based on valid medical documents.
Conclusions
It seems that the development of GC before the age of 50 is likely to be accompanied by familial susceptibility. Interestingly, our study showed a significant correlation between blood group O and the development of gastric cancer under the age of 50.
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Background
Gastric cancer is the second most common cause of cancer-related death in the world [1]. Its incidence varies considerably worldwide [2]. In general, it is a larger problem in developing countries than in industrialized nations, and shows a predilection for urban and lower socioeconomic groups [3,4]. The estimated crude rate accounts for approximately 9.9% of cancers worldwide [5]. Gastric cancer rarely occurs before the age of 40. The incidence rises steadily thereafter, peaking in the seventh decade. Men are nearly twice as susceptible as women. This cancer alone is the cause of more than 750,000 deaths per annum in the world [6]. Marked variation within countries has also been observed [3,4], particularly in high-risk countries [7]. In developing countries, the overall incidence of gastric cancer is increasing and projections indicate that the annual number of new cases will increase significantly during the next few decades as a result of adult population growth [6]. A recent cancer survey by the Iranian Ministry of Health and Medical Education revealed that gastric adenocarcinoma is the most common fatal cancer in Iran, with a wide variation of death rate among different provinces [8]. According to recent cancer statistics, deaths due to gastric cancer constitute about 39% of all deaths due to cancer each year in some parts of Iran [9].
The reduced incidence of gastric cancer in western countries reflects a decrease in cancers arising in the distal stomach (body and antrum). In contrast, the incidence of cancer in the proximal stomach and esophagogastric junction has steadily increased, at a rate exceeding that of any other cancer except melanoma and lung cancer [10-13]. In a very recent study, our group showed that cardiac cancer constitutes 49.5% of all sites for gastric cancer in Iran. In contrast, cancers of body and antrum comprise 20.6% and 29.9% respectively [9]. Unlike cancer of the distal stomach, cancers of the proximal stomach and esophagogastric junction are more common among higher socioeconomic classes [6]. Overall, these observations suggest that proximal cancers share a similar pathogenesis, which is distinct from that of distal cancers. Zanghieri and La Vecchia found that about 10% of cases show familial clustering. Epidemiological studies have shown that the risk of gastric cancer in first-degree relatives is increased 2- to 3-fold [14-17]. The relative contributions of inherited susceptibility and environmental effects on familial gastric cancer are poorly understood.
In general, familial genetic mechanisms do not play as important a role in gastric cancer as they do in e.g. colorectal cancer. Nonetheless, in some regions, a family history of gastric cancer may be a risk factor for the disease, although this might reflect environmental factors shared by members of a family [18]. Rate collections of familial aggregates of gastric cancer have been reported, but are distinctly unusual. As yet there is no comprehensive hypothesis for the development of gastric cancer. Gastric cancers are associated with chromosomal aberrations and other genetic defects, but none of these is necessary or sufficient for cancer to occur.
In a review about genetic predisposition to gastric cancer, Bevan and Houlston (1999) concluded that several genes may be associated with increased risk [19]. Gastric cancer is a manifestation of several inherited cancer predisposition syndromes including hereditary nonpolyposis colon cancer, familial adenomatous polyposis, Peutz-Jeghers syndrome and Cowden disease. This suggests the presence of predisposing genes with different effects.
Many studies have addressed the correlation between ABO antigens and the development of gastric cancer, but most of these have indicated a correlation between sporadic cases of gastric cancer and blood group A. This association further supports the role of genetic factors in the development of gastric cancer [21]. Blood type A is more strongly associated with the diffuse histopathological type of gastric cancer than the intestinal type [21,22]. To our knowledge, similar studies on the specific category of gastric cancer in younger patients are scanty. This may be one of the first studies on the role of hereditary factors in the development of the gastric cancer in younger patients.
Methods
The study was designed as a case-control study. We set up an active surveillance to identify patients with gastric cancer. Patients' records in the department of pathology in the main private referral facility in Tehran were scrutinized for gastric cancer cases between 1999 and 2003. Patients are referred here from all regions of the country and from different ethnic backgrounds and they are operated upon in the same hospital, so all the operation and pathology reports were available simultaneously. All the pathology reports were prepared and diagnosed by the same pathologists. The cases were selected from patients who were diagnosed with gastric cancer before the age of fifty. The sex-matched controls were randomly selected and enrolled from patients who were diagnosed over the age of fifty. All the patients and their family were interviewed regarding the history of gastric or other types of cancer over three generations, and the blood groups of affected members were ascertained. Family histories of cancer were accepted as valid provided that they were based on valid medical documents. The transfusion records of the operation were also used to identify the patients' blood groups.
Statistical analysis was performed using the SPSS Statistical Package (version 10.0). The quantitative variables were expressed as means (minimum-maximum) when appropriate. A chi-square test was performed to ascertain the overall effect of blood group on the development of gastric cancer before the age of 50. All statistical tests were two-sided and differences at the 0.01 level were considered statistically significant.
Results
At the beginning of the study, 44 cases (mean age: 36.2, 18–49; m/f = 1) under 50 years old and 44 sex-matched controls (mean age: 67.1, 50–88) were enrolled. Table 1 shows the pathological characteristics of all 88 subjects. At the time of the study, 59.1% of the case group and 50% of the control group were alive; 53.8% of the case group and 38.6% were living in Tehran, but no information on residence background was available. Data regarding first-degree relatives were complete for both groups. These data comprised information on 383 persons in the case group (average 9.1 for each proband) and 498 in the control group (average 11.6 for each proband). Table 2 shows the distribution of blood groups in the two subject groups. Gastric (22 cases) and other types of cancer were reported in 54.5% of the first-degree relatives of the cases and 11.4% of the first-degree relatives of the controls (p < 0.01). The "other types of cancer" among relatives of the case group comprised colorectal (7 cases), breast (3 cases), lung (3 cases), gynecological (2 cases), hematological (1 case) and bladder (1 case) malignancies. For the control group, the corresponding figures were colorectal (2 cases), breast (1 case), lung (1 case) and prostate (1 case) malignancies. Figures 1, 2, 3 show three pedigrees of familial connections.
Table 1 The pathologic characteristics of the tumor in the case and control group (n = 44 in each group).
Pathologic differentiation Pathologic Type Location of the tumor
Case group Well differentiated 1.4% Diffuse 0.5% Cardia 13.6%
Moderately differentiated 5.0% Body 18.2%
Poorly differentiated 1.4% Intestinal 7.3% Distal 68.2%
Control group Well differentiated 7.3% Diffuse 5% Cardia 22.7%
Moderately differentiated 5.0% Body 29.5%
Poorly differentiated 7.7% Intestinal 5% Distal 47.7%
P value NS NS NS
Table 2 Frequency of the different blood groups in the study population (n = 44). The figures in parentheses are the number of the patients.
Blood group O A B AB
Cases (30) 68.1% (6) 13.6% (6) 13.6% (2) 4.5%
Controls (11) 27.3% (28) 63.6% (3) 6.8% (2) 4.5%
P value <0.05 <0.01 NS NS
Figure 1 A family with aggregation with gastric cancer (GC: Gastric Cancer; Ca: History of gastric cancer but not confirmed by a pathologic reports for the histologic type of cancer).
Figure 2 A family with a history of the aggregation with Colorectal cancer (GC: Gastric Cancer; CRC: Colorectal Cancer; PLP: Colorectal Polyps; Numbers in circles: Current age of the persons; CAG: Chronic Active Gastritis).
Figure 3 A family with a history of the aggregation with other cancers as well as gastric cancer (GC: Gastric Cancer; Lung: Lung Cancer; Blad: Bladder Carcinoma).
Discussion
This case control study demonstrates that hereditary factors, especially familial history of cancer and possession of blood group O, are associated with the development of gastric cancer under the age of fifty. To our knowledge, this may be the first study showing a correlation between blood group O and the development of gastric cancer in a specific category of patients.
Risk factors for gastric cancers have been explored in a number of previous studies, including genetic factors such as blood group. Haenszel et al. suggested an association between gastric cancer and blood type A, supporting the view that genetic factors have a role in the development of gastric cancer [21]. Our study emphasizes the role of genetic factors in one subcategory of patients, those who develop gastric cancer under the age of fifty. Blood group A is more strongly associated with the diffuse histopathological type of gastric cancer than the intestinal type [21,22]. In our study, most of the patients had a diffuse rather than intestinal type, so we could not test this association. A larger sample size would be needed. On the other hand, there might be a higher prevalence of Helicobacter pylori in our community, causing a higher incidence of the diffuse type of gastric cancer. However, there were no significant differences in histological type of gastric cancer between the case and control groups. In a study by Su et al. in 2001, a total of 6685 patients with esophageal carcinoma and 2955 patients with cardiac cancer in the Chaoshan district were retrospectively assessed for their association with ABO blood groups. Su et al. showed that the distribution of ABO blood groups in patients with esophageal carcinoma or cardiac cancer was similar to that in the normal local population, but there was an association between blood group B and the development of cancer of cardia in males [23].
In our study, approximately 54% of the case group had a familial history of cancer compared to 11% of the control group. This seems compatible with the findings of a population-based case-control study of stomach cancer in Warsaw, Poland. Here, the investigators interviewed 464 cases and 480 controls to evaluate the role of family history and other risk factors. A greater than threefold increase in risk was associated with a history of gastric cancer in a first degree relative (OR = 3.5), but no excess risk was seen for other forms of cancer. The risk associated with familial occurrence was not significantly modified by gender, age or ABO blood type, and did not vary with Lauren histological classification24. Despite the relatively large sample size in the Polish study, younger patients were not evaluated as a separate category. This may explain the difference between their results and ours. Furthermore, they defined "positive family history" as having a first-degree relative with gastric cancer. In contrast, we considered all types of malignancy in first and second-degree relatives; though the familial incidence of other (non-GI) malignancies in our study may reflect their higher frequency in the community rather than any genetic risk factor. The Polish study did not confirm previous results on the correlation between blood group A and gastric cancer. Moreover, another study by Parsonnet et al on 90 cases and 89 controls showed no association between ABO blood group and malignancy [25]. In a multicentric study in Italy, 1016 patients with gastric cancer and 1623 population controls were interviewed to determine family histories of gastric, esophageal and colorectal cancer. A significant association was found with history of gastric cancer in a sibling or parent (odds ratios 2.6 and 1.7, respectively). Among the adult siblings of controls and cases, the prevalences of gastric cancer reported at interview were 1 and 2.7%, respectively. A further increase was noted in families with at least one affected parent (1.4 and 5.7%). The risk of gastric cancer associated with a positive family history was greater (increased about 2-fold) among residents of low-risk areas. Among the cases, there was no relationship between family history of gastric cancer and blood group A or histological type according to the Lauren classification [26]. In our study, there was no significant relationship between the histological type of the cancer and positive family history or blood group. However, this does not prove that the two variables do not correlate; an association might become apparent with a larger study. Mecklin et al studied the clinical and histopathological characteristics of gastric carcinoma in young patients (under 40 years old) in Finland in 1988. In 94% of the young patients, the carcinoma was of the diffuse type. They showed a poor prognosis, an equal sex ratio, and a strong association with blood group A in their study group. They also found a highly significant over-representation of gastric cancer in the parents of the index cases (p < 0.001) [27]. The difference between Mecklin's study and ours in the blood groups identified as risk factors may reflect ethnic differences; both studies confirm a significant correlation between a specific blood group and the development of gastric cancer. Future studies may use linkage analysis to detect genetic abnormalities in chromosomal regions that are located near the genes encoding the ABO antigens.
Matching the geographical origins of the cases and controls could have improved the power of our study by excluding ethnic factors from the study population. However, this is very difficult to achieve in such studies because there is a high rate of combinations between races in the country. In addition, the sample size was too small for such effects to be excluded. However, there were no significance differences between the two groups in respect of the origins of the subjects.
In conclusion, our results show that familial history of cancer, and hereditary factors including blood group, have a role in the development of gastric cancer in young patients. The role of environmental factors may be more important in older patients and can be considered in future studies.
Competing interests
The authors declare that they have no competing interest.
Authors' contributions
MY designed and assisted in the conduction of the study, analyzing of the data and draft the manuscript.
NR, FS, RB and YJ assisted in the conducting and designing the study and interview with the study cases as well as drafting manuscript.
AM and MA assisted in analyzing of the study and conducting interviews.
AA and MH reviewed and approved the pathology reports of the patients and case collecting.
RM supervised the study scientifically and executively and assisted in drafting the manuscript.
All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The authors wish to thanks Christian Abnet PhD and Sanford Daisy MD, Cancer Prevention Branch, National Cancer Institute, Bethesda, MD, USA for their help in providing the software for pedigree drawing as well as David G. Huntsman, MD, FRCPC, FCCMG, and Pardeep Kaurah, University of British Columbia, Vancouver, Canada for kind help regarding the questionnaire of familial background.
This study has been granted by the Iranian Network of Molecular Medicine and the Digestive Disease Research Center of Tehran University of Medical Sciences.
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| 15509297 | PMC529446 | CC BY | 2021-01-04 16:29:55 | no | BMC Gastroenterol. 2004 Oct 27; 4:28 | utf-8 | BMC Gastroenterol | 2,004 | 10.1186/1471-230X-4-28 | oa_comm |
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BMC GeriatrBMC Geriatrics1471-2318BioMed Central London 1471-2318-4-101551130210.1186/1471-2318-4-10Research ArticleValidity of the Clock Drawing Test in predicting reports of driving problems in the elderly Diegelman Nathan M [email protected] Alan D [email protected] Jeffrey L [email protected] Evangelia [email protected] Michael R [email protected] Department of Psychiatry & Behavioral Sciences, Akron General Medical Center, Akron, OH 44307, USA2 Department of Psychology, Kent State University, Kent, OH 44242, USA3 Department of Psychology, Mount Union College, Alliance, OH 44601, USA2004 29 10 2004 4 10 10 13 7 2004 29 10 2004 Copyright © 2004 Diegelman et al; licensee BioMed Central Ltd.2004Diegelman et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
This study examined the use of the Folstein Mini Mental Status Exam (MMSE) and the Clock Drawing Test (CDT) in predicting retrospective reports of driving problems among the elderly. The utility of existing scoring systems for the CDT was also examined.
Methods
Archival chart records of 325 patients of a geriatric outpatient clinic were reviewed, of which 162 had CDT results (including original clock drawings). T-test, correlation, and regression procedures were used to analyze the data.
Results
Both CDT and MMSE scores were significantly worse among non-drivers than individuals who were currently or recently driving. Among current or recent drivers, scores on both instruments correlated significantly with the total number of reported accidents or near misses, although the magnitude of the respective correlations was small. Only MMSE scores, however, significantly predicted whether or not any accidents or near misses were reported at all. Neither MMSE nor CDT scores predicted unique variance in the regressions.
Conclusions
The overall results suggest that both the MMSE and CDT have limited utility as potential indicators of driving problems in the elderly. The demonstrated predictive power for these instruments appears to be redundant, such that both appear to assess general cognitive function versus more specific abilities. Furthermore, the lack of robust prediction suggests that neither are sufficient to serve as stand-alone instruments on which to solely base decisions of driving capacity. Rather, individuals who evidence impairment should be provided a more thorough and comprehensive assessment than can be obtained through screening tools.
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Background
Assessment of cognitive function pertaining to capacity for safe and independent living among elderly patients is a central responsibility of many geriatric medical clinics and service agencies. Specific concerns pertaining to judgments of driving capacity are also befalling upon the medical profession in primary care settings [1]. To aid in this task, a number of brief assessment screens are often employed to identify cognitive problems that may be indicative of a range of pragmatic concerns, including driving capacity [2]. Specifically, results on assessment instruments purported to assess attention, reaction time, and visuospatial abilities are often used to inform clinical judgment about driving capacity in such settings. Two such screening instruments typically used to gauge general cognitive function, and inform questions pertaining to driving capacity specifically, are the Folstein Mini Mental Status Exam (MMSE) [3] and the Clock Drawing Test (CDT).
The MMSE is a widely used cognitive screening tool, due to its brevity, ease of administration, and relative breadth [4]. Numerous studies over the past 40 years have supported its utility as a valid and reliable indicator of general cognitive function [5]. The MMSE consists of 30 items comprising subscales assessing orientation, word registration, attention (via a serial sevens or spelling task), word recall, and language. Additionally, a figure copy exercise is included to examine visuospatial abilities. The CDT is hypothesized to assess more specific aspects of planning, organization and visuospatial skill. Directions for completing the CDT involve asking a patient to draw the face of a clock, including the numbers, and then to place the hands to designate a certain time, such as "ten minutes after eleven." Although different scoring templates for the CDT exist, most often code for features such relative size, spacing and placement of numbers or hands, disorganization, perseveration, completeness, and other potential errors that are hypothesized to indicate cognitive impairment [6-8].
In addition to the MMSE, results on the CDT are often used in clinical settings to inform clinical impressions pertaining to whether or not patients are impaired to such an extent that they should not be driving [9]. Although empirical reviews note that performance on the CDT should only be examined in conjunction with other assessments in this regard, anecdotal evidence also suggests that the CDT is often used as a stand-alone instrument to inform judgments of driving capacity, in both medical and non-medical settings.
Despite this apparent widespread use, there appears to be a dearth of research addressing the validity of the CDT for detecting driving impairment. Although a small number of studies exist that suggest CDT scores may relate to driving problems, the size of this literature base coupled with methodological concerns indicate a need for further research. For example, one study [10] examined the effectiveness of the CDT and MMSE, in addition to the Trail Making Test, Part A [11] and a visual acuity test, in predicting driving ability as judged by a driving instructor after participants completed a road test. A discriminant function analysis indicated that the set of test scores and participant age correctly identified 80% of drivers judged to be impaired, and 85% of drivers judged not to be impaired, according to driving instructor assessments. The authors reported that the discriminant model did not include the MMSE, however, because it did not add significant discriminatory power. The authors then suggested that the overall battery may be useful as a screening instrument in primary health care settings for detecting potential problems in driving that would warrant further examination. Separate univariate data on the predictive power for each of the separate instruments, however, was not provided. Additionally, the authors incorporated a 4-point scoring system for the CDT that was created for the study and differs from scoring systems used in other studies. Furthermore, given that only the component instruments are typically used in practice as opposed to the more extensive batteries advocated, the unique predictive power of the CDT warrants further investigation.
Additional evidence for the potential utility of the CDT in predicting driving behaviors is provided in an examination of neurophysiologic phenomena related to caregiver reports of driving impairment in 79 individuals with Alzheimer's disease [12]. Single photon emission computerized tomography was incorporated to examine brain function. Additionally, scores on the MMSE, CDT, and caregiver ratings of driving ability were analyzed. CDT scoring was based upon a 5-point system that was constructed for the study. Results indicated that MMSE scores did not significantly differ between individuals based upon driving ability, but that CDT scores were predictive of driving impairment based upon level of impairment and whether participants were instructed to simply copy an existing clock, or construct their own according to specific directions. Furthermore, imaging also indicated that level of driving impairment related positively to changes in cortical function. These authors hypothesized that cognitive tests assessing visuospatial abilities and executive function may thus show greater discriminative power between driving impaired and non-impaired subjects than MMSE scores, which may be impacted to a greater extent by other non-relevant verbal tasks. The validity of the scoring system constructed for the CDT in comparison to other scoring systems, however, was not further explored.
A pilot study examining the comparability of simulated driving tests in predicting actual driving problems also suggested that CDT scores may be significant predictors [3]. A small sample of nine older adults was incorporated, four of whom were classified as cognitively impaired based in part on abnormal CDT and MMSE scores. It was found that simulated driving tasks correlated moderately with actual driving problems across the groups. No data was provided, however, on the extent to which the CDT or MMSE uniquely predicted impairment.
Given the typically low rate of follow-up for patients referred for more formal driving assessments, it would be beneficial to further investigate the relations between scores on the CDT and reports of actual driving problems. Furthermore, the predictive power of the CDT alone and in conjunction with other assessment tools in predicting reported driving problems has yet to be fully assessed. Additionally, previous studies examining CDT scores and driving behaviors have employed markedly small sample sizes, warranting future research with greater numbers of participants. Finally, previous studies differ in terms of what, if any, scoring systems were used to score the clock drawings. Thus, further investigation of the comparability of different scoring systems is needed.
To address these concerns, this study explored the relations of patient scores on the CDT and MMSE to patient or family reports of driving problems. In so doing, the utility and comparability of three scoring systems for the CDT that are commonly used by researchers and practitioners, namely the Shulman et al. [6], Sunderland et al. [7], and Wolf-Klein et al. [8]systems, were also examined. Specifically, the Shulman system incorporates a 1–6 rating scale, where higher scores indicate higher levels of impairment. Conversely, scores on the Wolf-Klein and Sunderland systems range from 1–10, with lower scores indicating greater levels of impairment. Although specific criteria differ, each system codes for elements pertaining to spacing, organization, and comprehension of the task, among other criteria.
Exploratory analyses also were conducted to examine the predictive utility of the CDT and MMSE in predicting whether driving problems, namely accidents or near misses, were reported. Further analyses examined whether linear relationships existed between CDT and MMSE scores and the reported number of such incidents. Finally, regression tests examined whether the CDT and MMSE uniquely predicted the number of reported incidents.
Methods
IRB approval was obtained for the study, and data was collected from archival records of patients seen over a 10-year period at a geriatric assessment center of a general teaching hospital in the Midwest. The center operated as a full-service outpatient clinic, where new patient assessments included a full medical and psychosocial history. This history included patients' and collateral others' reports of driving behaviors within the past year, including whether patients were currently driving or had recently stopped driving within the past year, and number of driving accidents or near misses. The data was often collected during the initial intake assessment, when both the patient and available family members or caregivers were interviewed by a geriatrician, social worker, and/or a nurse specialist. In addition, the MMSE and CDT were typically administered to patients to assess cognitive functioning.
Chart records did not clarify whether the reported driving problems were acknowledged by the patient or caregiver, nor the extent to which any discrepancies existed, but rather only reported the number of incidents. The content of the incidents was also not always documented, but examples that were provided typically included crashes or minor accidents for which the patients were at fault. Nevertheless, despite the subjectivity inherent to such reports, it was the intent of these authors to remain true to the figures documented in the patient charts. Indeed, given that medical professionals typically must rely to some extent on subjective reports of patients or caregivers during intake evaluations to inform initial judgments about patient safety, it was decided that incorporation of such data in the present study would nonetheless be useful.
Data was obtained from charts of 325 patients, including 162 original clock drawings that were scored according to the systems provided by Shulman et al. [6], Sunderland et al. [7], and Wolf-Klein et al. [8]. Two advanced students in psychology were trained in each of the three scoring systems, and subsequently scored the clocks independently of each other and blinded to information about driving. MMSE scores, driving status, and reports of driving problems were also coded for subsequent analyses. The initial sample consisted of 81 men and 242 women (gender data was unavailable for 2 individuals). Of these, 287 (88.3%) were Caucasian, 34 (10.5%) were African American, and one individual was Asian American. Ethnicity data was not available for the other three individuals. The mean patient age was 79.75 (SD = 6.67), and ranging from 58 to 99 years of age. As is typical of many outpatient geriatric populations, there was a range in type and severity of presenting concerns, with some patients reporting relatively few problems and others evidencing diagnoses of vascular dementia, Alzheimer's disease, or depression in addition to other health concerns. Of these, concerns due to cognitive function predominated; approximately 60% of the patient sample was referred to the clinic for evaluation of memory loss, cognitive decline, or dementia. MMSE data was available for 311 patients; of these, 159 also had CDT data sufficient for analysis. Of the 162 charts that had CDT data, only 3 did not also have MMSE data.
Results and discussion
The raters' corresponding CDT scores for each scoring system correlated above 0.70, suggesting adequate inter-rater correspondence. The corresponding scores for each scoring system were then averaged to create three composite scores for each clock drawing, one for each scoring system. Descriptive data pertaining to scores for the overall sample on the MMSE and CDT is provided in Table 1.
Table 1 Descriptive statistics for overall sample scores on cognitive measures
Test N Mean SD Range
MMSE 311 21.51 6.15 0–30
CDT (Shulman Score) 162 3.87 1.25 1–6
CDT (Wolf-Klein Score) 162 6.58 2.10 1–10
CDT (Sunderland Score) 162 6.35 2.49 1–10
Initial exploratory t-tests were conducted to examine whether CDT scores and MMSE scores differed between individuals who had been currently or recently driving, versus those who had not been reported to be driving for a more extensive time period. In each case, current and recent drivers evidenced better scores on all of the cognitive measures than individuals who had not been driving. Results for these analyses are provided in Table 2.
Table 2 Mean differences in CDT and MMSE scores based on driving status
Variable N Mean SD t df
MMSE Score
Currently or Recently Driving 114 24.32 4.87 6.41** 305
Not Currently driving 193 19.98 6.18
Shulman Score
Currently or Recently Driving 61 3.39 1.28 -3.94** 157
Not Currently driving 98 4.16 1.15
Wolf-Klein Score
Currently or Recently Driving 61 7.26 1.83 3.29** 157
Not Currently driving 98 6.18 2.13
Sunderland Score
Currently or Recently Driving 61 7.18 2.28 3.28** 157
Not Currently driving 98 5.89 2.49
Note. **p < .01.
Further analyses examined whether CDT or MMSE scores predicted the presence of reported driving problems among individuals who had been current or recent drivers. Patients who had not been driving for a longer period of time were excluded from the analyses, since no driving problems would have been reported as a function of not driving. Specifically, t-tests were incorporated to examine whether CDT and MMSE scores differed among individuals for whom driving problems had been reported, versus those with none. Drivers with reported problems evidenced significantly lower MMSE scores, but no significant differences were obtained for CDT scores. Nevertheless, the trends for the overall mean differences on CDT scores, although small, were in the same direction as the findings for the MMSE. Specifically, in every case the CDT scores for each scoring system were worse for drivers with reported problems than those with none. Overall, these results suggest that the presence of driving problems may have been reflective of greater levels of cognitive impairment, although the overall differences reflected in CDT scores were nonetheless very small in magnitude. These results are detailed in Table 3.
Table 3 Mean differences in CDT and MMSE scores based on presence of reported driving problems among current or recent crivers
Variable N Mean SD t df
MMSE Score
Did Report Problems 51 23.08 6.03 -2.44* 112
Did Not Report Problems 63 25.32 3.41
Shulman Score
Did Report Problems 27 3.57 1.35 1.03 59
Did Not Report Problems 34 3.23 1.22
Wolf-Klein Score
Did Report Problems 27 7.09 2.25 -.64 59
Did Not Report Problems 34 7.40 1.43
Sunderland Score
Did Report Problems 27 6.72 2.64 -1.41 59
Did Not Report Problems 34 7.54 1.91
Note. *p < .05.
Next, the linear relations for both CDT and MMSE scores in predicted the number of reported problem incidents were examined. Specifically, correlation coefficients were calculated separately for number of reported accidents or near misses, and scores on the CDT and MMSE. Patients who had not been currently or recently driving were excluded from the analysis, since no problems would have been reported if they had not been driving. The number of reported incidents correlated significantly and positively with the level of cognitive impairment as measured by MMSE and CDT scores. Additionally, each of the CDT scoring systems appeared to evidence similar predictive utility, as they correlated highly (above 0.80). Means, standard deviations, and correlations for these variables are provided in Table 4. Given that not all patient charts necessarily contained all of the requisite MMSE and CDT data, cases that were missing data were excluded from some of the cells. Thus, the n of the resultant cases is included for each cell.
Table 4 Means, standard deviations, and correlations for cognitive measures and reported number of problems among current or recent drivers
Variable N M SD 1 2 3 4
1. MMSE Score 114 24.32 4.87
2. Shulman Score 61 3.39 1.28 -.45** (59)
3. Wolf-Klein Score 61 7.26 1.83 .50** (59) -.80** (61)
4. Sunderland Score 61 7.15 2.30 .58** (59) -.82** (61) .83** (61)
5. Reported Number of Driving Problems 116 .62 .90 -.27** (110) .23* (57) -.24* (57) -.27* (57)
Note. ** p < .01, *p < .05. The N for each cell is provided in parentheses. Total reported number of driving problems ranged from 0–4 for each patient.
Finally, hierarchical regression analyses examined whether MMSE or CDT scores uniquely predicted number of reported accidents or near misses. The non-significant R-squared change term in the second step of each regression indicates that neither the MMSE nor set of CDT scores predicted significant incremental variance. Thus, it appears that the variance in reported accidents or near misses predicted by the MMSE and CDT was redundant. Regression results are provided in Table 5.
Table 5 Hierarchical regression analyses predicting number of reported driving problems from CDT and MMSE scores among current or recent drivers (N = 54)
Regression Criterion and Steps R R2 F df R2change Fchange
Reported Number of Accidents or Near Misses
Step 1: CDT Scores .25 .06 1.17 3,51 .06 1.17
Step 2: MMSE Score .31 .10 1.37 1,50 .04 1.92
Step 1: MMSE Score .30 .09 5.19* 1,53 .09 5.19*
Step 2: CDT Scores .31 .10 1.37 3,50 .01 .18
Note. *p < .05
Conclusions
The results of this study suggest that both the MMSE and the CDT appear to have only limited utility in predicting retrospective reports of driving problems among elderly drivers. The finding that MMSE and CDT scores were worse among patients who had not been currently or recently driving may be due to a number of factors, including the possibility that some individuals may have never driven at all before. Nevertheless, it appears likely that many of these individuals probably had been driving in the past, but may have since stopped due to problems related to cognitive impairment. This assertion is supported by the finding that individuals who had been currently or recently driving at the time of the assessment, and who had lower MMSE scores, were more likely to have had reports of accidents or near misses. Although similar mean tests with the CDT were not significant, it is notable that the direction of the obtained differences for each scoring system of the CDT was consistent with the findings of the MMSE. Furthermore, the relatively modest n-sizes within each cell may have limited statistical power.
More robust findings were obtained, however, for the correlations examining number of reported accidents or near misses to CDT and MMSE scores. Among individuals who had been currently or recently driving at the time of assessment, greater levels of cognitive impairment as evidenced by MMSE and CDT scores also predicted greater numbers of reported accidents or near misses. This finding held regardless of which CDT scoring system was incorporated, suggesting that each may have equal utility.
Finally, the results of the regression analyses appear to indicate that the predictive power of the CDT and MMSE are somewhat redundant, since neither added significant incremental variance to prediction. Although it is possible that the regressions may have had limited power to detect significant incremental differences due to the relatively small sample sizes, in each case the increment to R-squared was small nonetheless. Thus, it appears that both the MMSE and CDT served as gross assessments of general cognitive function, versus more specific cognitive capacities, in predicting reported numbers of accidents or near misses.
Although the current results appear to suggest limited predictive utility for the MMSE and CDT in predicting driving problems, an additional cautionary note is in order. The significant predictive power for each instrument as demonstrated by the magnitudes of the correlation coefficients was nevertheless small. Furthermore, significant predictive utility was not obtained for every test in the current research. Additionally, the use of a retrospective design does not necessarily allow for definitive conclusions about predicting instances of future driving problems. Thus, although poor CDT or MMSE scores appear to indicate greater potential for driving problems, the current data do not support the use of the CDT or MMSE alone in making definitive decisions pertaining to driving competence. Rather, the empirical findings of the current research appear to best support the use of the CDT and MMSE solely as their originally intended purpose as screening tools. Thus, scores evidencing impairment on either of these instruments may indicate a need for driver caution, followed by more comprehensive and extensive assessment of driving capacity on which to base decisions regarding safety. As such, the role and utility of these instruments in predicting driving problems may be more fully understood through future research that incorporates a prospective design, along with a more comprehensive assessment of specific and relevant cognitive skills (like psychomotor speed or executive function) and objective assessment of driving abilities (such as can be obtained through simulated or practice driving situations).
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
ND, AG, and JM conceived of the study purpose and design. ND, EB, and MM collected, entered, and analyzed the data. ND conducted the literature review and critique, and drafted the manuscript. AG, JM, EB, and MM provided comments on the manuscript. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgments
This research was sponsored by the Development Foundation of Akron General Medical Center. The authors also wish to thank Chantal Walsh for assisting in this research. An earlier draft of this research was presented at the Eight Annual Research and Scholarly Activities Day sponsored by the NEOUCOM Department of Psychiatry, June 10, 2004 at Akron General Medical Center.
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Folstein MF Folstein SE McHugh PR Mini-Mental State: A practical method for grading the cognitive state of patients for the clinician Journal of Psychiatric Research 1975 12 189 198 1202204
Fleming KC Evans JM Weber DC Chutka DS Practical functional assessment of elderly persons: A primary-care approach Mayo Clinic Proceedings 1995 70 890 910 7643645
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De Raedt R Ponjaert-Kristoffersen I Short cognitive/neuropsychological test battery for first-tier fitness-to-drive assessment of older adults The Clinical Neuropsychologist 2001 15 329 336 11778771
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Ott BR Heindel WC Whelihan WM Caron MD Piatt AL Noto RB A single-photon emission computed tomography imaging study of driving impairment in patients with Alzheimer's disease Dementia and Geriatric Cognitive Disorders 2000 11 153 160 10765046
| 15511302 | PMC529447 | CC BY | 2021-01-04 16:30:12 | no | BMC Geriatr. 2004 Oct 29; 4:10 | utf-8 | BMC Geriatr | 2,004 | 10.1186/1471-2318-4-10 | oa_comm |
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BMC Infect DisBMC Infectious Diseases1471-2334BioMed Central London 1471-2334-4-451551130010.1186/1471-2334-4-45Research ArticleGastro-enteritis outbreak among Nordic patients with psoriasis in a health centre in Gran Canaria, Spain: a cohort study Eriksen Hanne M [email protected] Philippe J [email protected]ård Karin [email protected] Marika 3Marika_Hjertqvist/SMI/SE%[email protected] Jong Birgitta [email protected] Angela MC [email protected] Markku [email protected] Ulrike [email protected] AG [email protected]ør Cato [email protected] Preben [email protected] European Programme for Intervention Epidemiology Training (EPIET), 171 82 Solna, Stocholm, Sweden2 Department of infectious disease control, Norwegian Institute of Public Health, Norway, PO Box 4404, Nydalen, 0403 Oslo, Norway3 Department of infectious disease control, Swedish Institute for Infectious Disease Control, 171 82 Solna, Stocholm, Sweden4 Department of infectious disease control, National Public Health Institute, Finland, Mannerheimintie 166 FIN - 00300 Helsinki, Finland5 Department of infectious disease control, Instituto de Salud Carlos III, Sinesio Delgado, 4 al 12, 28029 Madrid, Spain6 The Gran Canaria Public Health Authority, Las Palmas, Spain7 Department of skin disease, Rikshospitalet, Sognsvannsveien 20, 0027 Oslo, Norway2004 29 10 2004 4 45 45 25 6 2004 29 10 2004 Copyright © 2004 Eriksen et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Between November 2 and 10, 2002 several patients with psoriasis and personnel staying in the health centre in Gran Canaria, Spain fell ill with diarrhoea, vomiting or both. Patient original came from Norway, Sweden and Finland. The patient group was scheduled to stay until 8 November. A new group of patients were due to arrive from 7 November.
Methods
A retrospective cohort study was conducted to assess the extent of the outbreak, to identify the source and mode of transmission and to prevent similar problems in the following group.
Results
Altogether 41% (48/116) of persons staying at the centre fell ill. Norovirus infection was suspected based on clinical presentations and the fact that no bacteria were identified. Kaplan criteria were met. Five persons in this outbreak were hospitalised and the mean duration of diarrhoea was 3 days. The consequences of the illness were more severe compared to many other norovirus outbreaks, possibly because many of the cases suffered from chronic diseases and were treated with drugs reported to affect the immunity (methotrexate or steroids).
During the two first days of the outbreak, the attack rate was higher in residents who had consumed dried fruit (adjusted RR = 3.1; 95% CI: 1.4–7.1) and strawberry jam (adjusted RR = 1.9; 95% CI: 0.9–4.1) than those who did not. In the following days, no association was found. The investigation suggests two modes of transmission: a common source for those who fell ill during the two first days of the outbreak and thereafter mainly person to person transmission. This is supported by a lower risk associated with the two food items at the end of the outbreak.
Conclusions
We believe that the food items were contaminated by foodhandlers who reported sick before the outbreak started. Control measures were successfully implemented; food buffets were banned, strict hygiene measures were implemented and sick personnel stayed at home >48 hours after last symptoms.
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Background
On 5 November 2002, the Department of Infectious Disease Epidemiology at the Norwegian Institute of Public Health (NIPH) was contacted by a dermatologist at Rikshospitalet, a tertiary care university hospital in Oslo, Norway. He reported several cases of gastro-enteritis in a medical care centre in Argúinegúin, Gran Canaria, Spain where Nordic patients with psoriasis undergo climate therapy. The symptoms (diarrhoea and vomiting) were reported to be of short duration (1–2 days). The patient group was scheduled to stay until 8 November. A new group of patients were due to arrive from 7 November. An outbreak management team was created in order to investigate and control the current outbreak, and to prevent similar problems in the following group. The outbreak management team consisted of investigators working in public health institutes in the countries involved (Sweden, Finland, Norway and Spain). The investigation was organised and co-ordinated from the NIPH in Oslo, Norway.
The objectives of this investigation were to assess the extent of the outbreak, identify the mode of transmission, the vehicle and the causative pathogen and recommend appropriate control measures.
Methods
The health centre is a private institution owned by the Norwegian Asthma and Allergy Association. Every three weeks the health centre receives a group of 100–110 Nordic patients suffering from skin diseases (mainly psoriasis). Rikshospitalet, Oslo, Norway administers the project. Patients are residents of Norway, Sweden and Finland. On site, Nordic medical staff (one dermatologist, three nurses and a sport and leisure leader) provides health services for the patients. The centre also hosts private individuals and employees of the Asthma and Allergy Association.
We conducted a retrospective cohort study among all Nordic patients attending dermatological care and employees staying at the health centre between 2 and 10 November. The persons from the Asthma and Allergy Association, and employees eating and staying outside the centre in the same time period were not included since there were no reported cases among this group and they did not eat or use the same facilities as the dermatological patients.
Case definition
A case was defined as a person who 1) attended dermatological care or worked at the Health Centre in Gran Canaria, 2) took meals at the Centre and 3) fell ill between the 2 and 10 November 2002, with symptoms of diarrhoea (3 or more loose stools in 24 hours), vomiting or both.
Recruitment of the cohort
Health personnel at the health centre provided a guest list with name, address, age, sex and bungalow number. For cases they also recorded date of onset of symptoms and stool sampling and results of microbiological analysis.
A standard questionnaire was developed at the Norwegian Institute of Public Health, translated in each national centre and mailed to the entire cohort, with instructions to fill it in and send it back to the public health institute in their country of origin.
The local health authorities in Gran Canaria enquired about gastro-intestinal illness in the community. They contacted health centres, churches and schools in the area.
Exposure
An exposure was defined as a food item eaten or an environmental factor present within the 2 days before onset of illness (the number of cases and the number of persons in the denominator therefore varies for different days of analysis). Possible exposures included consumption of all food items served in the health centre's restaurant, consumption of food served outside the centre, brushing teeth in tap water, drinking tap water, consumption of ice cubes, swimming in one of the two pools, swimming in the ocean, having had contact with an ill person, hand washing habits before meals, and the sharing of bungalow with symptomatic persons. We recorded the number of times food items were eaten to enable the study of potential dose-response relationships.
Analysis
Day by day, we compared food-specific attack rates (AR) for each item for the exposed and the non-exposed. Suspecting the etiological agent to be norovirus and knowing it has a short incubation period (24–48 hours) [1] only food consumption in the two days preceding onset of symptoms were considered as possible vehicle of the infection. Therefore, when considering food eaten on 31 October, only cases that became ill 2 November were in the numerator. Cases ill on or after 3 November were included in the denominator. When considering food eaten on 1 November, cases that became ill on 2 and 3 November were in the numerator. Likewise, persons were removed from the data set after they were reported ill, since they were no longer at risk. Then we pooled the results for the two first days of the outbreak, assuming that person-to person transmission would be low these first days.
We used EpiData software (Epidata Association, Denmark) for data collection and analysed them with SPSS version 10.0 (SPSS Inc. Chicago, Illinois). Attack rates, relative risk and 95% confidence intervals were calculated for each of the food items and other exposures. Only variables with RR greater than 2.0 are presented in this report. A multivariable analysis was run to assess potential confounding, including age, gender and the pooled variables with RR greater than 2.0 in the univariable analysis.
Laboratory and environmental investigations
All cases in the cohort presenting gastro-enteric symptoms were encouraged to deliver a stool sample. The samples were sent to a local private laboratory in Gran Canaria. Both virological and bacterial tests were requested.
Food sampling of some of the served items was performed on 5, 21 and 22 November by the local health authorities in Gran Canaria. The health authorities also took samples on 12 November from water taps and from the pools. Both stool and environmental samples were to be sent to Madrid for virology testing. Due to misunderstandings the samples were never forwarded to Madrid. Virological analyses were therefore not performed.
Results
We mailed 110 questionnaires to the patients who stayed at the Centre between 17 October and 8 November 2002, and employees (n = 6) eating at the centre. Ninety-one questionnaires (response rate = 78%) were returned (2 from employees, 89 from patients at the centre).
Personal characteristics
Among the 91 respondents there were 47 men and 44 women. The median age was 48 years (range 18–80).
Forty-eight persons fulfilled the case definition (attack rate (AR) = 53%). The AR was not significantly different by genders or nationalities. The AR was higher among those above 70 years of age (80%).
Temporal distribution
The outbreak peaked on November 4, with 16 cases (Figure 1). The outbreak extended from 2 to 7 November. In addition, two kitchen workers, without dates of onset, were ill with diarrhoea just before the outbreak started. None of the other staff eating outside the centre were reported ill.
No cases were reported among private individuals and employees of the Asthma and Allergy Association staying in the same compound, but who ate only outside the health centre.
Disease characteristics
Forty-two cases had diarrhoea and 33 vomited (Table 1). The mean duration of symptoms was 3,7 days for diarrhoea (range 1–23 days) and 1 day for vomiting. Twenty-four cases reported having seen a doctor in Gran Canaria. Seven consulted a doctor after returning home. Five persons went to hospital and 11 received intravenous treatment. Six had to stay home from work on average for 1 day. Ten of the respondents reported 24 possible secondary cases among persons they had been in contact with after they returned home.
Cases were distributed among most of the bungalows. The AR for those sharing room with two others were 42% (39/93), AR for those sharing room with one person 38% (3/8) and AR among those 9 living in a single room was 44%. Two cases were employees living private or in a separate section. Six cases fell ill subsequent (more than 10 hours later and within two days) to their roommate.
There were no gastro-enteritis cases reported in the community concurrently to the outbreak in the health centre.
Cohort study, food specific attack rates
From the daily analysis of food consumption, strawberry jam, dried fruit (eaten on both 1 and 2 November) and butter (eaten on 31 October) were associated with risk of illness (RR> 2.0) for those who became ill on 2 and 3 November (Tables 3 and 4). For those ill 4 and 5 November eating pear was associated with risk of illness (RR 2.8, 95% CI: 0.6–14.4) and eating strawberry jam (RR 1.2, 95% CI: 0.4–3.5). None of these items were independent associated with disease onset 4 and 5 November, in the multivariable analysis.
When pooling food consumption 1 and 2 November, eating pear and drinking full milk also increased the risk of developing gastro-enteritis. In the multivariable analysis including sex, age group, and all variables with RR>2.0, consumption of dried fruit (adjusted RR = 3.1, 95% CI: 1.4–7.1) and strawberry jam (adjusted RR = 1.9, 95% CI: 0.9–4.1) were independently associated with disease (Table 3).
AR for those with who became ill on November 2 and 3 increased with the amount of strawberry jam eaten. No dose response was observed for dried fruits (Table 4).
Laboratory results and aetiology
We reviewed the clinical symptoms: 69% vomited, 85% had < 72 h duration of illness and all stool samples were found negative for bacteria. Based on this, norovirus was suspected as the aetiological agent. This hypothesis is supported by Kaplan criteria (Table 2) [2].
Stool samples from 6, non symptomatic food handlers were taken on 8 and 12 November and repeated on 28 November. Two of the food handlers were reported to be symptomatic just prior to the outbreak.
All stool samples taken from employees and patients staying at the centre (altogether 43 stool samples) were reported negative for bacteria at the private laboratory in Gran Canaria. Virological analyses were not performed.
Six food items and water from the two pools were analysed for bacteria: chicken croquettes, frozen chicken breast and leg, sausage, cheese and salmon. Results were negative.
Discussion
We initiated an epidemiological investigation of an outbreak of gastro-enteritis in a health centre in Gran Canaria, Spain. Based on Kaplan criteria, our findings suggest that the aetiological agent was norovirus. Results of the cohort study suggests that the outbreak was initiated by ingestion of dried fruits, strawberry jam or both, followed by person-to-person transmission. There is no supporting microbiological or environmental evidence.
Pathogenesis and mode of transmission
Norovirus outbreaks can often be diagnosed presumptively on clinical grounds from their characteristic epidemiological features [2]. Kaplan has reported four criteria that indicate with a high sensitivity and a relatively high specificity that a gastroenteritis outbreak is caused by norovirus [3]. In this outbreak all four criteria were met. The Kaplan criteria were used since a confirmatory microbiological diagnosis was pending. We collected time of onset of symptoms, but not of recovery. We therfore had to use 0–72 h cut off instead of 12–60 as in the Kaplan criteria. We do not belive this affected our results.
A high proportion of persons in this outbreak reported that they received IV-treatment (n = 11, saw a general practitioner (n = 24), were hospitalised (n = 5) and the mean duration of diarrhoea was 3.7 days. The reported consequences, especially the duration of the illness was severe compared to many other norovirus outbreaks [3-5]. The explanation may be that many of the cases suffered from chronic diseases and were treated with drugs reported to affect the immunity (methotrexate or steroids). This explanation is supported by two recent reports where norovirus gastroenteritis is described not to be so mild in certain groups in the community [6] and in hospital patients' [7].
We believe there were two modes of transmission: a common source for those who fell ill on 2 and 3 November and thereafter mainly person to person transmission. Low RR for all food items at the end of the outbreak and the short incubation period for noroviruses, together with the fact that norovirus outbreaks usually have high rates of person-to-person transmission [8], support this hypothesis.
We did however not find a higher attack rate among those who shared room or bungalow. Only nine persons had a single room.
There were no cases among those persons who stayed at the health centre, but did not eat in the health centre restaurant or in the community during the outbreak. This suggests a foodborne outbreak with its origin at the centre's restaurant.
Vehicles of contamination
Dried fruits, strawberry jam or both were the probable vehicles of contamination. Strawberry jam and dried fruits were handled and kept at the kitchen in the Health Centre and not supplied by the catering service. They were served in buffet style on plates. Both food items are biologically plausible vehicles. Contaminated hands or silverware could explain contamination of these food items, but these modes of transmission were not verified. One hypothesis was that the food items were contaminated by the foodhandlers who reported being ill before the outbreak started. Both foodhandlers were involved in preparing the food for the buffet. Very few organisms of norovirus are needed to transmit the disease [4]. The jam was commercial and cooked. To our knowledge, none of these specific food items has been incriminated as a vehicle in norovirus outbreaks reported in the literature. There are, however, several similar food products that have been involved in norovirus outbreaks [1,8,9].
Dose-response analyses give further support for contamination of the strawberry jam. Those who ate jam twice doubled their risk of developing gastro-enteritis.
Method
Suspecting norovirus, with a short incubation period, as the causal agent [1], we treated each day as a new cohort. We assumed that persons falling ill at the beginning of the outbreak were infected by a common source, while those persons falling ill later could have been infected by person-to-person transmission, by a common source, or both.
When looking at the whole period as a cohort and thus looking at all persons falling ill between 2 and 10 November, then, none of the exposures seemed to increase the risk of disease. The association between food items and disease was probably masked by a high number of cases infected by person-to-person transmission.
Eight cases had not eaten dried fruit during the 2 days before falling ill. The concept of dried fruit is different in Spain from Scandinavia. In Spain, dried fruit means raisins, which were served in the restaurant. In the Nordic countries, dried fruit is in general understood to be a mixture of different types of dried fruits. This difference in concepts may have introduced an information bias.
We asked for food history for 5 days. The food was always served buffet-style. The menus included more than 300 different food items. To reduce potential recall bias, we pooled the food items consumed on either of the 2 days prior to onset. The fact that strawberry jam and dried fruit remained associated for those with onset 2 and 3 November (Table 3) is an argument against the problems related to recall bias.
Intervention
Based on clinical suspicions of norovirus infection, NIPH suggested to the centre medical personnel to apply a guideline on control of norovirus infection in hospital care setting [10]. We recommended implementing these guidelines with a special focus on improved hygiene measures and individually served food instead of buffet meals. The health personnel at the health centre supervised the implementation of the guideline.
We recommended taking new stool samples of the kitchen workers that had been ill with gastro-enteritis symptoms and excluding all symptomatic food handlers from work for 48 hour after their first normal stool. We also recommended looking for structural and operational deficiencies in the health centre kitchen and in the catering company.
Further environmental investigation by the local public health authorities was recommended. The importance of taking stool samples, and analysing them for both virus and bacterial pathogens, was emphasised.
The control measures were successfully implemented. Guidelines to control norovirus for a hospital care setting are more demanding in hygiene measures than guidelines for hotel outbreaks. Taking into account that qualified health personnel were in place, potential cases were more susceptible to the disease because of underlying diseases, so these strict measures were justified.
It was not possible to cancel the planned arrival of the persons due at the centre from 7 November. Stopping the group of patients with scheduled arrival in the end of November was discussed. Patients expecting to travel to Gran Canaria got written information about the situation at the centre. As no new cases were reported after 14 November (see epilogue), we recommended that this group should travel as planned.
Epilogue
Among persons arriving 7 November (n = 100), 18 became ill with gastro-enteritis. There were no cases after 14 November. Four of seven stool samples were positive for Salmonella. This finding did not change the belief that norovirus caused the gastro-enteritis among persons in the outbreak described in this article. Salmonella was not found in any of the 22 stool-samples from our cohort. This, together with the reported symptoms, suggests that there may have been two different outbreaks at the centre during this period.
Conclusion
Between 2 and 7 November, 66 persons fell ill with diarrhoea, vomiting or both in a health centre for Nordic patients with skin diseases at Gran Canaria. Our data suggest that norovirus caused the outbreak.
Our findings suggest that individuals who consumed dried fruit (adjusted RR = 3.1, 95% CI: 1.4–7.1) or strawberry jam (adjusted RR = 1.9, 95% CI: 0.9–4.1) were more likely to contract the disease. One hypothesis is that the food items were contaminated by foodhandlers that had had gastro-enteritis shortly before the outbreak started.
Improved hygiene measures and individually served food were successfully implemented.
Abbreviations
Norwegian Institute of Public Health; NIPH
Attack rate; AR
Relative risk RR
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
HME: In charge of the investigation, data handeling and writing of the article
PJG: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article.
KN: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article.
MH: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article.
BdJ: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article.
AMCR: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article.
MK: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article.
UD: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article.
AGR: Working locally with the outbreak, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article.
CM: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article.
PA: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We acknowledge G. Hernandes, P. Matute Cruz for their contribution to the study.
Figures and Tables
Figure 1 Gastro-enteritis cases (n = 48) by date of onset and exposure to dried fruit and strawberry jam among Nordic patients and employees (n = 91) in a health centre in Gran Canaria, Spain, October-November 2002.
Table 1 Clinical signs and symptoms among the 48 cases and symptoms in non-cases among Nordic patients and employees at a health centre (n = 91) in Gran Canaria, Spain, October – November, 2002.
Symptoms/attribute Number (percent of all cases) Median duration of symptoms in cases Number of non cases with symptoms
Diarrhoea 42 (88) 3 days 2
Blood seen in stool 2 (5*) 0
Vomiting 33 (69) 1 day 0
Nausea 34 (71) 2 days 9
Feeling feverish 12 (25) 3
Abdominal cramps 34 (71) 3 days 4
Headaches 14 (29) 2.5 day 3
General practition visit 24 (50) 1
Stool samples obtained 22 (6 of these after returning home) 0
Intravenous treatment 11 (23) _
Ambulatory visit in hospital 5 (10) _
Home from work 6 (13) 1 day _
• Denominator is only those with diarrhoea (n = 42)
Table 2 Kaplan's criteria (2) for suspecting an outbreak is due to norovirus compared to features of the gastro-enteritis outbreak in Gran Canaria 2002.
Kaplan criteria How our study meet these criteria
1. Vomiting in > 50% of cases 69% vomited
2. Duration of illness 12–60 h 85% had duration of illness -1–72* h
3. Incubation period of 15–36 h Food items eaten within 48 h are possible vehicles
4. Bacterial pathogens are not identified 22 stool samples found negative
* Duration of symptoms was asked for in terms of days. Only time of onset and not hourly information on recovery were collected. We therefore had to use 1–72 h instead of 12–60 h.
Table 3 Food specific attack rates (AR) (uni – and multivariable analysis¤) for pooled food items (food pooled from October 31-November 2) among Nordic patients and workers ill on 2 and 3 November, 2002 at the health centre (n = 91) in Gran Canaria, Spain.
Food item Food eaten Food not eaten Univariable Multivariable
Cases N = 17 Total* AR% Cases Total* AR% RR 95% C.I % cases exposed RR 95% C.I
Strawberry jam 8 24 33 9 63 14 2.3 1.0–5.3 47 1.9 0.9–4.1
Butter 7 28 25 10 59 17 1.5 0.6–3.5 41
Dried fruit 9 21 43 8 66 12 3.5 1.6–8.0 53 3.1 1.4–7.1
Apple 5 25 20 12 62 19 1.0 0.4–2.6 29
Pear 6 17 35 11 70 16 2.3 1.0–5.2 35
Full milk 4 10 40 13 77 17 2.4 1.0–5.7 24
Kiwi 5 21 24 12 66 18 1.3 0.5–3.3 29
4 responders did not remember what they ate
¤ Variables controlled for in the multivariable analysis: age, gender and the pooled variables with RR greater than 2.0 in the univariable analysis.
Table 4 Attack rates of gastroenteritis by amount of food consumed by Nordic patients and workers with onset on 2 and 3 November, 2002 at the health centre (n = 91) in Gran Canaria, Spain.
Food item and dose Ill Not ill. AR RR (95%CI) X2trend
Dried fruit, not eaten 8 58 12 Ref
Dried fruit, eaten once 3 4 43 3.5 (1.2–10.4)
Dried fruit, eaten twice or more 6 8 43 3.5 (1.5–8.6)
Strawberry jam, not eaten 9 57 14 Ref.
Strawberry jam, eaten once 2 6 25 1.8 (0.5–7.0)
Strawberry jam, eaten twice or more 6 8 43 3.1 (1.3–7.4) 6.46 (p = 0.01)
==== Refs
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Turcios RM Widdowson M-A Sulka A Glass RI Reassessment of Kaplan's criteria in identifying foodborn norovirus outbreaks – United States, 1998–2000 Poster presentation, 41st annual meeting of IDSA, San Diego October 9–12, 2003
Evans AS Kaslow RA eds Viral Infections of Humans- Epidemiology and Control 1997 4 New York: Plenum Publishing Corporation 285 324
Lopman BA Adak GK Reacher MH Brown DW Two epidemiologic patterns of norovirus outbreaks: Surveillance in England and Wales, 1992–2000 Emerg Infect Dis 2003 9 71 7 12533284
Billgren M Christenson B Hedlund K-O Vinje J Epidemiology of Norwalk-like human caliciviruses in hospital outbreaks of acute gastroenteritis in the Stocholm area in 1996 J Infect Dis 2002 44 26 32
Rockx B de Wit M Vennema H Natural history of human calicivirus infection: a prospective cohort study Clin Infect Dis 2002 35 246 253 12115089 10.1086/341408
Lopman BA Reacher MH Vipond IB Clinical manifestation of norovirus gastroenteritis in health care settings Clin Infect Dis 2004 39 318 324 15306997 10.1086/421948
Daniels NA Bergmire-Sweat DA Schwab KJ A foodborne outbreak of gastroenteritis associated with norwalk-like viruses: first molecular traceback to deli sandwiches contaminated during preparation J Infect Dis 2000 181 1467 70 10753727 10.1086/315365
Love SS Jiang X Barrett E Farkas T Kelly S A large hotel outbreak of norwalk-like virus gastroenteritis among three groups of guests and hotel employees in Virginia Epidemiol Infect 2002 129 127 132 12211579 10.1017/S0950268802007161
Chadwick PR Management of hospital outbreaks of gastro-enteritis due to small round structured viruses J Hosp Infect 2000 45 1 10 10833336 10.1053/jhin.2000.0662
| 15511300 | PMC529448 | CC BY | 2021-01-04 16:03:31 | no | BMC Infect Dis. 2004 Oct 29; 4:45 | utf-8 | BMC Infect Dis | 2,004 | 10.1186/1471-2334-4-45 | oa_comm |
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BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-4-531553325910.1186/1471-2458-4-53Research ArticleOverweight, obesity, and colorectal cancer screening: Disparity between men and women Heo Moonseong [email protected] David B [email protected] Kevin R [email protected] Department of Psychiatry, Weill Medical College of Cornell University, White Plains, NY, USA2 Department of Biostatistics, Section on Statistical Genetics & Clinical Nutrition Research Center, University of Alabama at Birmingham, Birmingham, AL, USA3 Division of Rheumatology, Johns Hopkins University School of Medicine, Baltimore, MD, USA2004 8 11 2004 4 53 53 20 5 2004 8 11 2004 Copyright © 2004 Heo et al; licensee BioMed Central Ltd.2004Heo et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
To estimate the association between body-mass index (BMI: kg/m2) and colorectal cancer (CRC) screening among US adults aged ≥ 50 years.
Methods
Population-based data from the 2001 Behavioral Risk Factor Surveillance Survey. Adults (N = 84,284) aged ≥ 50 years were classified by BMI as normal weight (18.5–<25), overweight (25–<30), obesity class I (30–<35), obesity class II (35–<40), and obesity class III (≥ 40). Interval since most recent screening fecal occult blood test (FOBT): (0 = >1 year since last screening vs. 1 = screened within the past year), and screening sigmoidoscopy (SIG): (0 = > 5 years since last screening vs. 1 = within the past 5 years) were the outcomes.
Results
Results differed between men and women. After adjusting for age, health insurance, race, and smoking, we found that, compared to normal weight men, men in the overweight (odds ratio [OR] 1.25, 95% CI = 1.05–1.51) and obesity class I (OR = 1.21, 95% CI = 1.03–1.75) categories were more likely to have obtained a screening SIG within the previous 5 years, while women in the obesity class I (OR = 0.86, 95%CI = 0.78–0.94) and II (OR = 0.88, 95%CI = 0.79–0.99) categories were less likely to have obtained a screening SIG compared to normal weight women. BMI was not associated with FOBT.
Conclusion
Weight may be a correlate of CRC screening behavior but in a different way between men and women.
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Background
Colorectal cancer (CRC) is the third most common cancer in the United States with approximately 150,000 cases annually leading to about 57,000 annual deaths [1]. A prospective study of over 900,000 US adults found that, compared to normal weight adults, death rates from CRC were 20% to 84% higher in overweight and severely obese men and 10% to 46% higher in overweight and severely obese women [2]. Although other factors (e.g., age, family history) also contribute to CRC risk, obesity is a significant risk factor [1]. Thus, overweight and obese adults should consider obtaining regular CRC screening because early detection and intervention might reduce mortality [1,3].
However, studies suggest that overweight and obese women are more likely to delay cervical and breast cancer screenings than normal weight women [4,5]. In contrast, data from the 2001 Behavioral Risk Factor Surveillance Survey (BRFSS) indicates that, among men, overweight and obesity associates with obtaining prostate-specific antigen testing (Fontaine, Heo & Allison, under review). Although these cancers are sex-specific, the disparity led us to evaluate whether the obesity CRC screening association differed between men and women.
Methods
The Center for Disease Control and Prevention's BRFSS collects state-based data on preventive health practices and risk behaviors in the non-institutionalized civilian population aged ≥ 18 years [6]. The analyses we report are derived from the 2001 BRFSS. Information on BRFSS design and sampling methods are reported elsewhere [7,8].
Study variables
Body mass index (BMI; kg/m2), calculated from self-reported weight and height, was the predictor.
Outcomes were interval since the most recent use of fecal occult blood test (FOBT), and sigmoidoscopy (SIG) in adults aged ≥ 50 years who reported ever having had the respective screening examination. BRFSS codes FOBT responses as: 'within past year', 'within past 2 years', 'within past 5 years', '5 or more years ago', 'don't know/not sure', or 'refused'. SIG is coded as: 'within past year', 'within past 2 years', 'within past 5 years', 'within past 10 years', '10 or more years ago', 'don't know/not sure', or 'refused'. Consistent with screening recommendations [1], we dichotomized FOBT as 0 = > 1 year since last screening vs. 1 = screened within the past year. For SIG, the American Cancer Society recommends screening every 5 years for adults aged ≥ 501. Thus, SIG was dichotomized as 0 = > 5 years since last screening vs. 1 = screened within the past 5 years.
We included age, education, race, income, self-reported general health status, smoking, employment, and health insurance as covariates.
Statistical analysis
We grouped respondents into 5 BMI-defined categories (18.5–<25 "normal weight", 25–<30 "overweight", 30–<35 "obesity class I", 35–<40 "obesity class II", and ≥ 40 "obesity class III"). Respondents (n = 250; .3%) with BMI's <18.5 ("underweight") were omitted from the analyses.
We used multivariate logistic regression to estimate BMI-screening associations by entering the BMI-defined categories and potential confounders into the model as either continuous (e.g., age [including polynomials up to the third order]) or dichotomous variables (e.g., health insurance). Using the guidelines proposed by Greenland [9], we retained covariates that were statistically significant at the two-sided 0.20 alpha level or caused a ≥ 10% change in any of the BMI-defined categories when deleted. As a result, education, income, self-reported general health, and employment were omitted. Responses coded as 'don't know/not sure', or 'refused' were treated as missing variables and excluded from analyses, as were respondents with missing data on any covariates. To ensure unbiased general population estimates, we used sample weights provided by the BRFSS. BMI categories were investigated as 4 contrasts with the normal weight category serving as the referent.
To evaluate whether sex moderated the BMI-screening association, we ran adjusted logistic models that also included BMI × sex interaction terms. Finally, because we observed a significant BMI × sex interaction, we then analyzed the data for men and women separately. Analyses were performed with SPSS 11.5.
Results
The mean age of the respondents was 65 years (median = 63). The mean BMI was 30.2 (median = 31) and 93% reported having health insurance. Less than half reported ever having either a screening FOBT or SIG (Table 1). Among those who ever had a screening examination 54.1% of men and 52.7% of women (χ2(1) = 6.61, p = .010) reported obtaining a screening FOBT within the previous year, and 84.4% of men and 80.3% of women (χ2(1) = 98.4, p < 0.001) reported obtaining a screening SIG within the previous 5 years.
Table 1 Selected characteristics of respondents aged ≥ 50 years
Characteristic Value* N
Age, yrs 64.6 ± 10.1 84,284
Body mass index (BMI), kg/m2 30.2 ± 6.2 84,284
Sex, %
Men 38.2 32,179
Women 61.8 52,106
Race, %
White 82.3 68,639
Non-white 17.7 14,778
Health insurance, %
Yes 93.0 78,260
No 7.0 5,904
Smoking, %
Current smoker 18.2 15,265
Former smoker 35.4 29,709
Never smoker 46.4 38,959
Ever had fecal occult blood test (FOBT), %
Yes 43.0 37,498
No 53.9 49,123
Ever had screening sigmoidoscopy (SIG), %
Yes 45.3 39,574
No 51.1 46,584
Screening fecal occult blood test (FOBT), %
within past year 53.2 18,449
greater that 1 year 46.8 16,238
Screening sigmoidoscopy (SIG), %
within past 5 years 81.8 30,465
greater than 5 years 18.2 6,771
* Plus-minus values are means ± standard deviation
BMI was not associated with obtaining a FOBT (OR's ranged from 0.90 to 0.98). Compared to normal weight adults, however, those in the overweight (OR = 1.15, 95%CI 1.02–1.31), obesity class I (1.21, 95%CI 1.09–1.35), II (1.17, 95%CI 1.04–1.44) and III (1.27, 95%CI 1.05–1.58) categories were more likely to have obtained a screening SIG within the previous 5 years (p's < 0.05).
The interaction effect between sex and BMI categories on FOBT was not significant (χ2(4) = 8.64, p=.071). However, the interaction effect between sex and BMI categories on SIG screening was significant, (χ2(4) = 114.03, p < .0001). BMI was not associated with obtaining a FOBT for either sex (OR's ranged from 0.87 to 1.05). However, compared to normal weight men, men in the overweight (1.25, 95%CI 1.05–1.51) and obesity class I (1.21 95%CI 1.03–1.75) categories were significantly more likely to have obtained a screening SIG. In contrast, obesity class I (0.86 95%CI 0.78–0.94) and II (0.88 95%CI 0.79–0.99) women were less likely to have obtained a screening SIG compared to normal weight women (see Figure 1).
Figure 1 Adjusted odds ratios (OR) for obtaining a screening sigmoidoscopy according to BMI-defined categories for men and women * Significantly different from normal weight reference group at p < 0.05.
Discussion
These data support an association between BMI and obtaining a screening SIG within the previous 5 years, after smoking, health insurance, race, and age are taken into account. Moreover, the BMI-SIG associations were different between women and men. Women in the obesity class I and II categories were less likely to obtain SIG screening as a function of BMI. This is consistent with associations between BMI and delayed cervical and breast cancer screening [4,5]. On the other hand, men in the overweight and obesity class I categories were more likely to obtain a screening SIG.
The reasons for this disparity are unclear. Perhaps physicians encourage cancer screening more vigorously among their overweight and obese male patients. Differences between men and women on factors such as self-esteem and body image [10] may also contribute to explaining the differential BMI-screening associations. These speculations underscore the importance of identifying barriers that might deter overweight and obese women from obtaining screenings.
This study has limitations including: the BRFSS, a telephone survey, is prone to measurement error; because the BRFSS is an observational study, the BMI-screening associations could be due to residual confounding or confounding from unmeasured variables; the cross-sectional design did not allow testing causal inferences; and people without telephones, approximately 3% of the US population [6], are not surveyed through BRFSS.
Conclusions
These data indicate that weight may be a correlate of CRC screening behavior but in a different way for men and women.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
MH drafted the paper and assisted with the statistical analysis and interpretation. DB assisted with the writing of the manuscript and in the interpretation of the results. KF obtained and analyzed the data and assisted with the preparation of the manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
Supported in part by grants from the Arthritis Foundation, the Blaustein Pain Research Fund, and NIH grant P30DK056336.
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Fontaine KR Heo M Allison DB Body weight and cancer screening among women Journal of Women's Health and Gender-Based Medicine 2001 10 463 470 11445045 10.1089/152460901300233939
Wee CC McCarthy EP Davis RB Phillips RS Screening for cervical and breast cancer: Is obesity an unrecognized barrier to preventive care? Ann Intern Med 2000 132 697 704 10787362
Centers for Disease Control and Prevention Health risks in America: Gaining insight from the Behavioral Risk Factor Surveillance Survey System 1997 Atlanta, GA: US Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention
Centers for Disease Control and Prevention 2001 Behavioral Risk Factor Surveillance Survey- Users Manual 2002 Atlanta, GA: US Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention
Gentry EM Kalsbeek WD Hogelin GC Jones JT Gaines KL Forman MR Marks JS Trowbridge FL The behavioral risk factor surveys: II Design, methods, and estimates from combined state data Am J Prev Med 1985 1 9 14 3870927
Greenland S Modeling and variable selection in epidemiologic analysis Am J Pub Health 1989 79 340 349 2916724
Zayat EN Fontaine KR Cheskin LJ Use of preventive health care services by patients with obesity Obes Res 1999 7 223 10102260
| 15533259 | PMC529449 | CC BY | 2021-01-04 16:28:47 | no | BMC Public Health. 2004 Nov 8; 4:53 | utf-8 | BMC Public Health | 2,004 | 10.1186/1471-2458-4-53 | oa_comm |
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BMC Musculoskelet DisordBMC Musculoskeletal Disorders1471-2474BioMed Central London 1471-2474-5-371549623010.1186/1471-2474-5-37Research ArticleAge-related changes in Serum Growth Hormone, Insulin-like Growth Factor-1 and Somatostatin in System Lupus Erythematosus Denko Charles W [email protected] Charles J [email protected] Department of Medicine/Division of Rheumatic Diseases, Case Western Reserve University School of Medicine, Cleveland, OH 44106-5076 USA2 Department of Medicine/Division of Rheumatic Diseases, and Department of Anatomy, Case Western Reserve University School of Medicine, Cleveland, Ohio, 44106-5076 USA2004 20 10 2004 5 37 37 28 6 2004 20 10 2004 Copyright © 2004 Denko and Malemud; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Systemic lupus erythematosus is an age- and gender-associated autoimmune disorder. Previous studies suggested that defects in the hypothalamic/pituitary axis contributed to systemic lupus erythematosus disease progression which could also involve growth hormone, insulin-like growth factor-1 and somatostatin function. This study was designed to compare basal serum growth hormone, insulin-like growth factor-1 and somatostatin levels in female systemic lupus erythematosus patients to a group of normal female subjects.
Methods
Basal serum growth hormone, insulin-like growth factor-1 and somatostatin levels were measured by standard radioimmunoassay.
Results
Serum growth hormone levels failed to correlate with age (r2 = 3.03) in the entire group of normal subjects (i.e. 20 – 80 years). In contrast, serum insulin-like growth factor-1 levels were inversely correlated with age (adjusted r2 = 0.092). Of note, serum growth hormone was positively correlated with age (adjusted r2 = 0.269) in the 20 – 46 year range which overlapped with the age range of patients in the systemic lupus erythematosus group. In that regard, serum growth hormone levels were not significantly higher compared to either the entire group of normal subjects (20 – 80 yrs) or to normal subjects age-matched to the systemic lupus erythematosus patients. Serum insulin-like growth factor-1 levels were significantly elevated (p < 0.001) in systemic lupus erythematosus patients, but only when compared to the entire group of normal subjects. Serum somatostatin levels differed from normal subjects only in older (i.e. >55 yrs) systemic lupus erythematosus patients.
Conclusions
These results indicated that systemic lupus erythematosus was not characterized by a modulation of the growth hormone/insulin-like growth factor-1 paracrine axis when serum samples from systemic lupus erythematosus patients were compared to age- matched normal female subjects. These results in systemic lupus erythematosus differ from those previously reported in other musculoskeletal disorders such as rheumatoid arthritis, osteoarthritis, fibromyalgia, diffuse idiopathic skeletal hyperostosis and hypermobility syndrome where significantly higher serum growth hormone levels were found. Somatostatin levels in elderly systemic lupus erythematosus patients may provide a clinical marker of disease activity in these patients.
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Background
Systemic lupus erythematosus (SLE) is the protean autoimmune disorder with strong familial penetrance. Immunologically, SLE is characterized by aberrations in T cell and B cell function [1,2], over-production of autoantibodies directed principally against nuclear antigens [3] as well as other tissue antigens, and deficiences in the complement system [4]. SLE is predominantly a disease of young females with peak incidence occuring between 20 and 40 yrs with a female to male ratio of 6–10:1 [5]. Although many of the principal pathophysiological changes associated with SLE indicate organ involvement consistent with vascular inflammation and immune complex deposition [6], several prominent SLE-related pathologic findings suggest systemic disturbances consistent with metabolic abnormalities [7]. However, surrogate blood or serum markers of systemic dysfunction such as erythyrocyte sedimentation rate and C-reactive protein levels, although frequently elevated in SLE compared to normal subjects are often uninformative and unreliable as surrogate markers of SLE disease activity [7].
We have shown that diverse rheumatic and musculoskeletal disorders, including osteoarthritis (OA) [8-12], diffuse idiopathic skeletal hyperostosis (DISH) [12,13] and hypermobility syndrome [14] as well as fibromyalgia [15] were characterized, in part, by elevated serum growth hormone levels. Growth hormone was also found sequestered in erythrocytes in OA and DISH patients at levels that significantly exceeded serum growth hormone levels [16] suggesting a putative mechanism by which "toxic" levels of growth hormone could be confined, or in cases of vascular inflammation, transported to joint synovial fluid or peripheral end-organs [11,16]. Further, medical therapy of OA and DISH principally with non-steroidal anti-inflammatory drugs (NSAIDs) which resulted in pain suppression and reduced stiffness as well as improved range of motion correlated with lower serum growth hormone levels consistent with levels found in normal subjects [10,13]. More recently, we showed that symptomatic rheumatoid arthritis (RA) patients were also characterized by elevated serum growth hormone levels [17], but treatment of RA with prednisone failed to significantly lower serum growth hormone levels.
Insulin-like growth factor-1 (IGF-1) synthesis is coupled to growth hormone via its capacity to stimulate hepatocyte IGF-1 production [11]. In several rheumatic and musculoskeletal disorders, elevated serum growth hormone was correlated with elevated IGF-1 levels [9,11,13,14] with OA [8-10,12] and RA [17] being notable exceptions. In the case of OA, IGF-1 levels are significantly lower compared to normal control subjects [8-10,12]. However, medical therapy of OA principally with NSAIDs resulted in growth hormone and IGF-1 levels approaching normal [10] whereas in DISH patients treated with NSAIDs, reduced serum growth hormone levels failed to result in concomitant changes in IGF-1 [13].
Somatostatin is a 14 amino acid polypeptide whose principal function is to regulate growth hormone release from the pituitary [18]. Elevated serum and synovial fluid somatostatin levels have been associated with inflammatory responses [19] most notably in RA [20]. A recent study showed that patients with symptomatic RA were, in part, characterized by a skewed upward serum growth hormone to somatostatin ratio [17].
The present study was performed to determine the extent to which serum growth hormone, IGF-1 and somatostatin levels were modulated in patients with SLE. A linear regression analysis was performed to determine the relationship between age and serum growth hormone and IGF-1 levels in a group of normal female subjects so that these values could be employed for comparison to a group of predominantly young, female SLE patients.
Methods
All studies were performed at University Hospitals of Cleveland (UHC) and the Wade Park Veterans Administration Medical Center (VAMC), Cleveland, Ohio. The UHC and VAMC Institutional Review Boards approved the study design with the research protocol, which included informed consent, being in keeping with the Declaration of Helsinki. Normal subjects and SLE patients were all volunteers. SLE Patients met the clinical and laboratory criteria for the diagnosis of SLE according to previously published classifications [21]. Patients with co-morbid conditions such as diabetes mellitus or hyperglycemia were excluded from the normal subject group as was any normal individual with evidence for rheumatic disorders in family members. This information was obtained by questioning potential normal subjects.
Blood drawn by venipuncture was clotted at room temperature, centrifuged, serum aliquots separated and stored at -70°C until assayed. Blood samples were generally collected during an identical 3–4 hr morning period to normalize the potential contribution of growth hormone pulses and serum glucose levels to serum growth hormone determinations [8-10]. Serum samples were included for serum growth hormone, IGF-1 or somatostatin determinations only if glucose levels measured by the highly sensitive hexokinase assay [8-10] were between 65 and 135 mg/dl attained either by overnight fasting or a fast of at least 4 hours or more. Insulin levels were measured as previously described [8-10]. An insulin level in the range of 5–27 μU/ml was considered normal.
Basal serum growth hormone and IGF-1 levels were determined by standard radioimmunoassay (RIA) (INCSTAR, Stillwater, MN) as previously described [8-10]. The lower limit of detection for serum growth hormone by the RIA was 0.4 ng/ml [8-10]. Serum growth hormone levels in samples falling at or below the lower limit of detection were excluded from the statistical analysis. Basal somatostatin levels in serum of 112 normal subjects and 55 SLE patients stratified by age (i.e. <45, between 45 and 55 yrs and >55 yrs of age) were separately measured by RIA [17].
The 2-tailed T-test was employed to analyze the differences in means of serum growth hormone, IGF-1 and somatostatin concentrations in groups of unequal size where p < 0.05 was significant. The population sample size was sufficient to detect a 20% difference in serum growth hormone and IGF-1 levels between control subjects and SLE patients and a 15% difference in serum somatostatin levels. The relationship between serum growth hormone and IGF-1 levels as a function of age was analyzed from scatter plots by linear regression analysis employing SPSS 11.1 (SPSS, Inc., Chicago, IL) and SigmaPlot 8.0 (SPSS, Inc.) to calculate the adjusted r2-value and regression line, respectively.
Results and discussion
Glucose and insulin concentration was determined in serum from normal female subjects and from patients with SLE. No normal female subjects were excluded from the study as a result of detecting hyperglycemia or hyperinsulinemia However, 2 SLE patients were excluded from the statistical analysis on this basis (data not shown).
Over the course of this study, SLE patients received medical therapy with NSAIDs, prednisone (10–60 mg/day), hydroxychloroquine sulfate or methotrexate as well as combinations of these drugs. No SLE patients were treated with azathioprine or cyclophosphamide during this study.
Previously it was shown that basal serum growth hormone levels among normal male and female subjects did not significantly differ on the basis of age [16]. As noted, SLE has a high female to male prevalence ratio and is predominant in young females between 20 and 40 yrs of age [5,21]. Thus, it was critical to determine the extent to which serum growth hormone and IGF-1 differed among female normal subjects on the basis of age. Serum growth hormone levels did not correlate with age in normal female subjects between the ages of 20 and 80 (Figure 1A). However, a strong inverse correlation between age and IGF-1 levels (adjusted r2 = 0.269) in this group of normal female subjects was found (Figure 1B).
In contrast to the results obtained from basal serum growth hormone measurements in the entire normal female subject population (Figure 1A), a strong direct correlation (adjusted r2 = 0.092) between age (age, 30.8 ± 7.0, mean ± SD; 95% confidence, 3.74) and basal serum growth hormone levels in the young female normal subjects was found (Figure 2A). However, the correlation between age and basal serum growth hormone levels was weak in the older (age, 60.6 ± 9.4; mean ± SD; 95% confidence, 3.29) normal female subjects (Figure 2B).
Based on the above considerations, basal serum growth hormone and IGF-1 concentration was determined in study groups subdivided by age in normal female subjects and these values compared with basal serum growth hormone and IGF-1 levels in SLE patients. The results showed that SLE was not characterized by elevated serum growth hormone whether or not all normal female subjects or age-matched normal female subjects were employed as the comparison group (Table 1). Serum IGF-1 levels were significantly lower in the normal female subject group compared to SLE patients (Table 1), but there was no significant difference if serum IGF-1 levels in the SLE group were compared to serum IGF-1 levels in the age-matched normal female group (Table 1).
A trend towards elevated somatostatin levels in normal subjects as a function age was previously found [17]. In the present study, there was also a trend towards elevated serum somatostatin levels in the <45 yr old SLE patient group or 45 – 55 yr old group compared to their age-matched normal counterparts (Table 1). However, a significant difference was found only in the older (>55 yrs) SLE patients compared to their age-matched control counterparts (Table 1).
The results of the present study emphasized the critical requirement to control for age and gender when basal serum growth hormone and IGF-1 levels in normal subjects are compared to patients with autoimmune musculoskeletal diseases which, like SLE, are characterized by a strong age and gender association.
Several studies from our laboratory have consistently shown basal serum growth hormone to be higher in females than in males [8,9,14]. One study, in particular, examined the correlation between age, gender and race with basal serum growth hormone and concluded that, in general, older Causcasian women had slightly higher growth hormone levels compared to older African-American women [8]. However, in that study (8) no statistical differences were shown when serum growth hormone levels in young Caucasian women (age, 28 ± 6; mean ± SD) were compared to serum growth hormone levels in African-American women (age, 34 ± 10). This finding is particularly noteworthy to studies of SLE because, in most cases, SLE onset is prominent among young females during their reproductive years, and African-American women are over-represented in the SLE patient population [21].
The present analysis also extends the results of previous studies [8,9,14] and partially supports the conclusions of Ghigo et al. [22] who showed that basal growth hormone levels were similar in young and older individuals. Ghigo et al. [22] further suggested that the somatotroph response in young versus older individuals to the combined administration of arginine and growth hormone-releasing substance also did not vary with age.
In contrast, the present results do not support the conclusions that growth hormone decreases as a function of age as reported by Kelijman [23]. In fact, the results of the present study showed a strong correlation between age and serum growth hormone only in the circumscribed young normal female (age 20 – 46 yrs) group (Figure 1A).
The decrease in basal serum IGF-1 levels with age (Figure 1B) confirmed previous studies by Hochberg et al. [24] who studied patients with osteoarthritis of the knee as well as earlier studies by Ghigo et al. [22] who reported a significant difference in IGF-1 levels between young and older individuals. Thus, it was not unexpected that basal serum IGF-1 levels in SLE was significantly elevated when compared to basal serum IGF-1 levels in the general population of normal subjects, but not so, when basal serum IGF-1 levels from SLE patients were compared to their age-matched counterparts (Table 1). In this regard, Bennett et al. [25] also failed to find differences in serum IGF-1 levels when normal subjects (age, 45.1 ± 8.6) were compared to 15 age-matched SLE patients (age, 42.5 ± 7.0).
The relationship between putative abnormalities in the hypothalamic-pituitary axis, systemic disturbances and SLE pathogenesis and progression remains conjectural. In this regard, Rovensky et al. [26] found no correlation between plasma prolactin, growth hormone, interleukin-6, cortisol or C-reactive protein in adult SLE patients. However, studies by Chikanza et al. [27] reached a different conclusion. They suggested that a "pro-inflammatory hormonal bias" existed in juvenile SLE which was identical to adult SLE. They also concluded that the role of the neuroendocrine-immune system in adult SLE was, at the present time, limited to deficiencies in prolactin. Of note, two recent case reports suggested a link between growth hormone and exacerbation of lupus nephritis in a male teenager with SLE [28] as well as in juvenile SLE [29] where when growth retardation treated with growth hormone was terminated, clinical improvement in lupus symptoms was observed. These findings suggested that exogenously-administered growth hormone may result in "toxic" levels of growth hormone accompanied by lupus "flares" with progressive autoimmune dysfunction.
A recent study from this laboratory showed that the growth hormone to somatostatin ratio was skewed upward in patients with RA [17]. In the present study, somatostatin levels in the age groups encompassing the average age of the SLE patients were not different from than of normal subjects (Table 1). Although previous studies have suggested that somatostatinergic activity increased with age [20], the present analysis (Table 1) does not support that view (at least from measurements of basal somatostatin levels) as lower somatostatin levels in the older SLE patients reached statistical significance when compared to age-matched controls with the caveat that the present study did not relate changes in somatostatin to SLE disease activity.
Although somatostatin may alter growth hormone effects and immune responses in chronic autoimmune diseases, the relationship between somatostatin and "specific" somatostatin receptor (sSR) in SLE remains to be elucidated. In this regard, van Hagen [30] showed that 97% of patients with sarcoidosis, 100% of patients with tuberculosis or Wegener's granulomatosis, 75% of patients with Sjogren's syndrome but only 50% of SLE patients exhibited sSRs on mitogen-activated human peripheral lymphocytes compared to 97% in normal individuals. Of note, somatostatin receptor levels appeared to be unrelated to disease progression or remission. In the present study, a trend towards reduced serum somatostatin levels was seen only in the older SLE patients (Table 1). As functional somatostatin may change in autoimmunity and result in altered growth hormone release, reduced somatostatin levels could also influence basal levels of growth hormone in elderly SLE patients. Thus, changes in somatostatin could be one of several environmental stress factors resulting in the progression of clinically active disease in older SLE patients [31].
The therapeutic implications and diagnostic utility of serum growth hormone, IGF-1 and somatostatin measurements in SLE as well as in other musculoskeletal disorders appears central to assigning a role for these factors in disease progression. Serum growth hormone remained elevated in some DISH and OA patients where clinical symptoms were significant [9,11,12]. Thus, single serum growth hormone determinations appear to accurately reflect a pattern of serum growth hormone levels associated with these clinical disorders. Further, improvement in the clinical symptoms in OA and DISH patients with medical therapy [10,13] resulted in a sustained reduction in serum growth hormone levels reaching levels comparable to those found in normal subjects. Although longitudinal measurements of serum somatostatin in SLE and other rheumatic diseases have not yet been performed, reduced somatostatin levels appear to be most strongly associated with joint inflammation (as was seen in RA) [17] as well as in older patients (>55 yrs) with the inflammatory complications of knee OA (Denko and Malemud, submitted). Thus, it could be informative if elevated somatostatin levels correlated with clinical improvement in SLE patients.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
CWD participated in the design of the study and performed the clinical analysis. CJM participated in the design of the study and performed the statistical analysis. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
This study was supported, in part, by NIH P60 AR-20618 (Northeast Ohio Multipurpose Arthritis Center). The authors' thank Ms. Betty Boja for technical assistance and the personnel in the clinics at University Hospitals of Cleveland and Veterans Administration Medical Center for providing their support for this study.
Figures and Tables
Figure 1 Linear regression analysis. Serum growth hormone levels (Panel A) or serum IGF-1 levels (Panel B) were plotted as a function of age in a normal female subject group. The results indicated that while serum growth hormone levels were not correlated with age (adjusted r2 = 3.03), IGF-1 levels were inversely correlated to age (r2 = 0.269).
Figure 2 Linear regression analysis. Serum growth hormone levels were plotted as a function of age in young (age range, 20 to 46 yrs) normal female subjects (Panel A) or older (age range, 47 to 80 yrs) normal female subjects (Panel B). The results indicated that serum growth hormone levels correlated with age (r2 = 0.092) only within the circumscribed group of younger normal female subjects.
Table 1 Serum Growth Hormone (GH), Insulin-like Growth Factor-1 (IGF-1) and Somatostatin (SOM) levels in normal female subjects and Systemic Lupus Erythematosus (SLE) patients
Group Age (yrs)* GH ng/ml)* IGF-1 (nM/L)* SOM (pg/ml)* p-value
All Normals (35) 57.1 ± 13.1 1.17 ± 0.40 14.9 ± 3.6
(77) <45 25.2 ± 11
(17) 45–55 32.6 ± 12
(18) >55 36.2 ± 10
Age- Matched Normals (18) 30.8 ± 7.1** 1.58 ± 1.16** 25.0 ± 7.3†
SLE (17) 35.9 ± 8.6† 1.45 ± 0.88† 22.8 ± 7.1**
(22) < 45 29.8 ± 12 P > 0.05
(12) 45–55 35.4 ± 14 P > 0.05
(21) >55 29.9 ± 9 P < 0.05
AMNv.SLE **P > 0.05 **P > 0.05 **P > 0.05
ANv.SLE †P < 0.001 †P > 0.05 †P < 0.001
• Mean ± SD
• N: number of samples
AMN: age-matched normal subjects
AN: all normal subjects
SLE: systemic lupus erythematosus patients
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| 15496230 | PMC529450 | CC BY | 2021-01-04 16:03:43 | no | BMC Musculoskelet Disord. 2004 Oct 20; 5:37 | utf-8 | BMC Musculoskelet Disord | 2,004 | 10.1186/1471-2474-5-37 | oa_comm |
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BMC SurgBMC Surgery1471-2482BioMed Central London 1471-2482-4-151551626810.1186/1471-2482-4-15Research ArticlePudendal nerve decompression in perineology : a case series Beco Jacques [email protected] Daniela [email protected] Michèle [email protected] Gynaecology, CHU Sart-Tilman, University of Liège, B-4000 Liège, Belgium2 Perineology, CHC-Clinique Sainte-Elisabeth, 17 rue du Naimeux, B-4802 Heusy, Belgium3 Research, Institut d'Enseignement Supérieur Parnasse-Deux Alice, Avenue Mounier 84, B-1200 Brussels, Belgium4 Physiotherapy, CHR La Citadelle, Boulevard du 12ème de Ligne, B-4000 Liège, Belgium2004 30 10 2004 4 15 15 20 3 2004 30 10 2004 Copyright © 2004 Beco et al; licensee BioMed Central Ltd.2004Beco et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Perineodynia (vulvodynia, perineal pain, proctalgia), anal and urinary incontinence are the main symptoms of the pudendal canal syndrome (PCS) or entrapment of the pudendal nerve. The first aim of this study was to evaluate the effect of bilateral pudendal nerve decompression (PND) on the symptoms of the PCS, on three clinical signs (abnormal sensibility, painful Alcock's canal, painful "skin rolling test") and on two neurophysiological tests: electromyography (EMG) and pudendal nerve terminal motor latencies (PNTML). The second aim was to study the clinical value of the aforementioned clinical signs in the diagnosis of PCS.
Methods
In this retrospective analysis, the studied sample comprised 74 female patients who underwent a bilateral PND between 1995 and 2002. To accomplish the first aim, the patients sample was compared before and at least one year after surgery by means of descriptive statistics and hypothesis testing. The second aim was achieved by means of a statistical comparison between the patient's group before the operation and a control group of 82 women without any of the following signs: prolapse, anal incontinence, perineodynia, dyschesia and history of pelvi-perineal surgery.
Results
When bilateral PND was the only procedure done to treat the symptoms, the cure rates of perineodynia, anal incontinence and urinary incontinence were 8/14, 4/5 and 3/5, respectively. The frequency of the three clinical signs was significantly reduced. There was a significant reduction of anal and perineal PNTML and a significant increase of anal richness on EMG. The Odd Ratio of the three clinical signs in the diagnosis of PCS was 16,97 (95% CI = 4,68 – 61,51).
Conclusion
This study suggests that bilateral PND can treat perineodynia, anal and urinary incontinence. The three clinical signs of PCS seem to be efficient to suspect this diagnosis. There is a need for further studies to confirm these preliminary results.
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Background
The objective of perineology is to treat each defect of the perineum with the right procedure [1-3]. Pudendal nerve decompression (PND) is theoretically a basic procedure in perineology thanks to its ability to treat the defect "pudendal neuropathy".
Before going into details of this procedure, it is necessary to remember the anatomy of the pudendal nerve. This anatomy is still controversial.
While summarizing the data of the literature and the results of our dissections, the likeliest anatomy of the pudendal nerve presents itself as follows. The pudendal nerve is a mixed nerve carrying motor and sensory fibers. Its fibers are derived from the sacral roots S2, S3 and S4 [4,5]. Once the roots traverse the sacral foramen, they divide into autonomic branches forming the pelvic plexus (parasympathetic supply of the pelvic organs) and somatic branches merging to form the pudendal nerve travelling under the piriformis muscle. Near its formation point, it gives a levator branch running on the inner (upper) surface of the levator plate and providing the innervation of this muscle [4]. For Barber et al [6], this levator nerve originates directly from the S3, S4 or S5 roots. Some somatic fibers coming from S2 and S3 run close to the pelvic plexus to innervate the levator ani and the urethral sphincter [4]. Caudally, the pudendal nerve enters a small space ("clamp") between the sacro-spinal and sacro-tuberous ligaments very near the ischial spine. Just inferior to the ischial spine, the nerve gives its first branch, the dorsal nerve of the penis [4] or the clitoridal nerve. These nerves are separated from the main trunk by the pudendal vein and artery. Then, it enters the Alcock's canal formed by a division of the obturator muscle aponeurosis. In the canal the nerve crosses the sharp edge of the sacro-tuberous ligament (falciform process) [7,8]. Caudally, at the level of the anus, the nerve gives medially the inferior rectal nerves (usually two branches) which innervate the anal sphincter (and probably the pubo-rectalis) and the skin of the posterior perineum and anterolaterally the transversus perinei branch (for this muscle, for the ischiocavernosus muscle and maybe for the urethral sphincter) [4]. The remaining part of the nerve is usually called the perineal nerve. This nerve gives a bulbocavernosus branch and finally divides into a sphincteric branch (innervation of the urethra) and a branch which innervates the skin of the anterior perineum [9].
The pudendal canal syndrome (PCS) and its surgical treatment have been described by Shafik in 1991 [5]. This syndrome is induced by the compression or the stretching of the pudendal nerve in the Alcock's canal. The complete syndrome presents with anal incontinence, pain, hypo or hyperesthesia and urinary incontinence (and impotence in males). Some important studies were done earlier by Amarenco [10] and Robert [7] but these authors focused mainly on pain which is only a part of the syndrome.
The cause of the PCS is not always clear but it is often possible to find a compression (biking, long time sitting, haematoma...) or a stretching (descending perineum, surgery, delivery....) of the pudendal nerve in the Alcock's canal [10-19] in the history of the patient. A change in the shape or orientation of the ischial spine induced by some athletic activities during the youth could also explain some cases [20].
The clinical signs and investigation results proposed by Shafik [5] to confirm the diagnosis of PCS before surgery are: tenderness over the pudendal canal in the ischio-rectal fossa, diminished perineal sensation, weak or absent anal reflex, reduced EMG activity of the external anal sphincter and increased PNTML. The surgical procedure described by this author (trans-perineal approach) consists in opening the Alcock's canal to give the pudendal nerve a sufficient length to be unstretched and/or to suppress compression.
The trans-gluteal approach proposed by Robert to treat pudendal neuralgia aimed to open also the "clamp" between the sacro-spinal and the sacro-tuberous ligament by cutting one or two of them [8].
Since Shafik's study in 1991, some questions about the effect of PND on the PCS remained open. No peer reviewed publications confirming the efficacy of this surgery on anal incontinence or on urinary incontinence could be found. Even the existence of a genuine PCS has never been validated.
The aim of this study was to answer the following questions:
Is there any effect of the bilateral PND according to Shafik on:
- three main symptoms of PCS ?
- perineodynia (vulvodynia, perineal pain, proctalgia) [21]
- anal incontinence
- urinary incontinence
- three clinical signs of PCS ?
- abnormal sensibility
- painful Alcock's canal
- painful "skin rolling test" [22]
- two neurophysiological tests ?
- electromyography (EMG) of the anal sphincter and of the bulbocavernosus muscles.
- pudendal nerve terminal motor latencies (PNTML) of the anal and perineal branches.
What is the clinical value of the aforementioned three clinical signs in the diagnosis of PCS ?
Methods
Studied sample
A retrospective analysis to study the effects of a bilateral PND. The studied sample comprised 74 female patients who underwent a bilateral PND between 1995 and 2002 done by the same surgeon. The average age was 56.1 years (range: 28 – 77). All these patients underwent a complete history and clinical examination following the three perineal axis (gynaecological, urological and colo-proctological) according to the concept of perineology [1-3].
The frequency of the 3 main symptoms of the PCS (anal incontinence, perineodynia, urinary incontinence) in the 74 patients before surgery is presented in Figure 1.
Figure 1 Frequency of the 3 main symptoms of the pudendal canal syndrome (perineodynia, anal incontinence, urinary incontinence) before surgery.
Associated surgical procedures were performed at the same time as the PND to treat all the defects revealed by the clinical examination, and are presented in Table 1.
Table 1 Procedures associated with the bilateral pudendal nerve decompression
Associated procedures Operated (n = 74) Reviewed after one year or more (n = 56)
None 17 10
MVT according to Mouchel [48-50] 46 38
Correction of rectocele 49 42
Correction of cystocele 20 17
Vaginal hysterectomy 16 13
Levatorplasty according to Shafik [26] 14 13
Urethral meatotomy 4 3
Prepubien section [45] 2 1
Anal sphincteroplasty 2 2
Urethrolysis 1 1
Diagnostic tools for PCS
The following variables were used:
Three main symptoms of the PCS
Perineodynia
For perineodynia, four situations were encountered: no pain, proctalgia, unilateral pain, bilateral pain. The effect of surgery was estimated by the patient using one of the following proposals: cured, improved, unchanged or worsened.
Anal incontinence
For anal incontinence, a four levels ordinal scale was used: no incontinence, gas incontinence, liquid incontinence, solid incontinence. "Cured" was defined as "no incontinence". The patient was considered "improved" if there was a change of at least one level in the scale going from "solid" to "gas" incontinence. The patient was defined as "worsened" if there was a change of at least one level in the opposite direction.
Urinary incontinence
For urinary incontinence, a four levels ordinal scale was used: no incontinence, mild incontinence, moderate incontinence and severe incontinence. The two types of urinary incontinence, stress and urge incontinence, were evaluated separately even if both were present in the same patient (mixed incontinence). "Cured" was defined as "no incontinence". The patient was considered "improved" if there was a change of at least one level in the scale going from "severe" to "mild" incontinence. If the change observed was in the opposite direction the patient was considered "worsened".
Three clinical signs of the PCS (the examinations were done in gynaecological position)
Abnormal anal or vulvar sensibility
Sensibility was tested with a needle comparing the left and the right sides of the vulva and of the skin 2 cm lateral to the anus. The interpretations of the results were done using a four levels ordinal scale: 0 = total anaesthesia, 1 = reduced sensibility, 2 = normal sensibility, 3 = hypersensibility. 0, 1 and 3 were considered as "abnormal sensibility".
Painful Alcock's canal on rectal examination
The pain induced by the palpation of the pudendal canal by rectal examination was evaluated using a seven levels ordinal scale : 0 = no pain, 1 = mild pain, 2 = mild pain with Tinel sign (irradiation of the pain), 3 = moderate pain, 4 = moderate pain with Tinel sign, 5 = severe pain, 6 = severe pain with Tinel sign. The Alcock's canal was considered "painful" if the pain was 4 or more.
Painful "skin rolling test"
Beginning from 5 cm behind the level of anus the skin was pinched and then rolled to the front until the skin fold was at the level of the clitoris. The skin rolling test was considered "painful" if it induced a severe pain at least at one level (Figure 2).
Figure 2 Skin rolling test : the skin of the perineum is pinched just beneath the level of the anus and then rolled to the front searching for a sharp pain at one level. This sign is well known in the diagnosis of neuralgia.
Two neurophysiological tests
Concentric needle EMG
Concentric needle EMG was done at rest and during voluntary contraction on both sides of the external anal sphincter and on each bulbocavernosus muscle. The richness of the EMG was grossly evaluated using a six levels scale: 1 = simple, 2 = poor, 3 = intermediate poor, 4 = intermediate, 5 = intermediate rich and 6 = rich.
Anal and perineal PNTML
Anal and perineal PNTML were measured before and after the operation using the St Marks electrode to stimulate the pudendal nerve by the rectal route just under the ischial spine. For anal PNTML the electrical potentials induced in the striated anal sphincter were collected using the ring of this electrode. For the perineal PNTML the method described by Kiff [23] and Snooks [24] was modified according to Amarenco [25]. The electrical potentials were collected with a concentric needle in the two bulbocavernosus muscles. In our laboratory the normal values were: less than 2.5 msec for the anal PNTML and less than 5 msec for the perineal PNTML. EMG and PNTML were done by the same physician before and at least one year after the operation.
Minimal criteria for surgery
At least one of the 3 following symptoms resistant to conservative treatments (physiotherapy, drugs, infiltrations, modification of diet or behaviour):
a. Anal incontinence
b. Perineodynia
c. Urinary incontinence
Associated with at least two of the five following criteria:
a. increased anal or perineal PNTML
b. pathological EMG of the anal sphincter or bulbocavernosus muscles (neurogenic trace, reduced activity: richness "poor" or "simple").
c. painful Alcock's canal on rectal examination (at least on one side)
d. abnormal perineal sensibility (at least at one level)
e. painful "skin rolling test" (at least on one side).
Surgical procedure
Surgical procedure as described by Shafik in 1991 [5]. The operations were done under spinal or general anaesthesia. The patients were installed in the gynaecological position.
The different steps of the procedure were:
- Vertical incision of the skin between the anus and the ischial tuberosity.
- Opening of the ischio-rectal fossa with scissors.
- The inferior rectal nerve is hooked under the finger and followed to the entrance of the Alcock's canal (Figures 3 and 4).
Figure 3 Left Alcock's canal (showed by the tip of the forceps) viewed from the mid side on a female cadaver: on the left the pudendal nerve, on the right the inferior rectal nerve on the finger.
Figure 4 Alcock's canal viewed from below like in the operating room (right side of a female cadaver): inferior rectal nerve (horizontal) showing the entrance of the canal.
- Opening of this canal (without opening the clamp between the sacro-spinal and sacro-tuberous ligament).
- Control of the haemostasis.
- Self draining closing of the skin with nylon.
Evaluation of PND
The efficacy on the symptoms, on the clinical signs and on the neurophysiological tests was evaluated during a follow up consultation one year or more after the surgical procedure because the nerve healing can be very slow [5].
Statistical methods
Firstly, the efficiency of the PND on the symptoms and clinical signs was studied by means of descriptive statistics. Tests of hypothesis were done to compare the mean values of the neurophysiological tests before versus after PND.
Secondly, the diagnostic value of the clinical signs was evaluated in a "case control" setting. A subject belongs to the "controls group" when PCS is considered to be absent, namely if the patient does not present any of the following symptoms, signs or risk factors for PCS: perineodynia, anal incontinence, prolapse, previous surgery in the area, dyschesia. The clinical signs are not used to decide if a subject belongs to the controls or cases group. The statistical comparison was done between the patient's group before ("cases group") and after the operation, and the "controls group" of 82 women (average age: 48.8 years, extremes 27–76).
Statistical analysis of differences was performed using chi-square testing for categorical variables and t-tests for continuous variables.
Results
Effects of surgery
Effect on the symptoms of the PCS
The effect of PND on the symptoms of PCS is presented in Table 2.
Table 2 Effect of PND on the 3 main symptoms of the PCS
Parameters Perineodynia (pain) Perineodynia (pain) Anal incontinence Anal incontinence Stress urinary incontinence Stress urinary incontinence Urge incontinence Urge
incontinence
All Without: levat All Without: sphincteroplasty, levat, recto All Without: levat, mvt, cysto, prepubien, meato, urethrolysis All Without: levat, mvt, cysto, prepubien, meato, urethrolysis
Number of cases studied 74 59 74 22 74 22 74 22
Number of pathological results 26 22 46 9 47 4 33 4
Follow up less than 1 year or lost 8 8 10 4 10 3 6 0
Follow up 1 year or more 18 14 36 5 37 1 27 4
Mean follow up in months (range) 22,2 (12–48) 24,5 (12–48) 26,4(12–70) 17,2 (12–26) 32 (12–96) 12 26,7(12–72) 18,5 (12–26)
Cured (%) 11 (61,1%) 8 (57,1%) 23 (63,9%) 4 (80%) 26 (70%) 0 (0%) 17 (62,9%) 3 (75%)
Improved (%) 3 (16,6%) 2 (14,3 %) 7 (19,4%) 1 (20%) 7 (18,9%) 1 (100 %) 6 (22,2%) 0 (0%)
No change (%) 4 (22,2%) 4 (28,6%) 4 (11,1%) 0 (0%) 4 (10,8 %) 0 (0 %) 3 (11,1%) 0 (0%)
Worse (%) 0 (0 %) 0 (0 %) 2 (5,5%) 0 (0%) 0 (0%) 0 (0 %) 1 (3,7%) 1 (25%)
levat = levatorplasty, recto = cure of rectocele, cysto = cure of cystocele, prepubien = prepubien section, meato = meatotomy.
In order to treat completely the patient, PND was frequently associated with other procedures which might have an effect on the symptom studied. Therefore, the results for each symptom were presented in two columns: the first corresponds to the entire sample ("all") and the second to the small group of patients in which the symptom was treated by PND only ("without").
Effect on perineodynia
On the 26 patients with pain before the operation, 18 were reviewed 12 months or more after the operation. The pain had disappeared in 11 and was reduced or had another origin (painful puborectalis) in 3. The cure rate with a mean follow-up of 22,2 months was 61,1 % (77,7% cured or improved).
As none of the surgical associated procedures used in this study were known to improve or cure perineodynia, the only procedure removed was levatorplasty. Theoretically this operation can reduce the stretching on the pudendal nerves by reducing the sagging of the levator plate. The results were similar in the group without levatorplasty.
Effect on anal incontinence
On the 46 patients with anal incontinence before the operation, 36 were reviewed 12 months or more after the operation. 23 of them were cured, 7 improved, 2 were worse and 4 reported no change. The cure rate with a mean follow-up of 26.4 months was 63.9 % (83,3% cured or improved).
The results according to the severity of incontinence are presented in Table 3.
Table 3 Effect of PND on anal incontinence according to the incontinence level.
Cured Improved Unchanged Worsened
Solid (n = 5) 3 2 0 0
Liquid (n = 20) 12 5 3 0
Gas (n = 11) 8 0 1 2
To study the real impact of PND on anal incontinence, we removed the following cases in which PND was associated with anal sphincteroplasty, levatorplasty or cure of rectocele by fascia and perineal body restoration:
- 2 patients had anal sphincteroplasty together with the PND: one was cured and the other improved.
- Levatorplasty according to Shafik [26] was used in 8 patients who had a severe levator plate sagging. This procedure could have a "post-anal repair effect" [27] and therefore improve anal incontinence.
- 30 cases had a cure of rectocele by fascia and perineal body restoration (Ayabaca and coll. [28] found an improvement of anal incontinence in 25 of their 34 patients. Nevertheless, none of them gained full continence post-operatively).
In the small group of patients with PND only, 5 were reviewed one year or more after the operation: 4 were cured (3 incontinences for liquid and 1 for gas) and 1 improved (liquid incontinence).
Anal ultrasound was done before the operation in 13 of the 36 patients reviewed. Only 4 were normal, 3 showed a rupture of the internal and external sphincters, and 6 presented a disruption of the external sphincter alone.
In the 7 patients who had a rupture of the anal sphincter (5 external only, 2 internal and external) without anal sphincteroplasty and a follow up of more than 12 months (mean 18.5 months), 4 were cured and 3 were a failure.
5 patients who were still incontinent after 1 year follow up (2 incontinences for flatus and 3 for liquid) became continent 2 years after the operation.
Effect on urinary incontinence
Five patients presenting urinary incontinence (4 urge and 1 stress incontinence) had PND without any other procedure around the urethra. The mean follow up was 18,5 months for the 4 patients with urge incontinence. In this small group, 3 patients were cured and one was a failure. The patient with stress urinary incontinence was improved (the number of pads used per day was reduced from 9 to 4) one year after surgery.
Effect on the clinical signs
The effect of PND on the three clinical signs is described in Table 4.
Table 4 Effect of PND on the three clinical signs
Parameters Abnormal sensibility (at least at one level) Abnormal sensibility (at least at one level) Painful Alcock's canal (at least on one side) Painful Alcock's canal (at least on one side) Painful skin rolling test (at least on one side) Painful skin rolling test (at least on one side)
All Without: levat All Without: levat All Without: levat
Number of tests before surgery 42 27 46 32 39 26
Follow up less than 1 year or lost 11 9 19 16 22 16
Follow up 1 year or more 31 18 27 16 17 10
Normal test before (reviewed 1 year or more after) 15 8 9 6 8 2
Abnormal test before (reviewed 1 year or more after) 16 10 18 10 9 8
Mean follow up (range) 27,7 (12–68) 32,2 (12–68) 28,1 (12–68) 32,8 (12–68) 28,7 (12–60) 33,7 (12–60)
Normal before => Normal after 14 8 9 6 7 2
Normal before => Abnormal after (%) 1 0 0 0 1 0
Abnormal before => Normal after (%) 11 (68%) 6 (60%) 11 (61%) 7 (70%) 6 (66,6%) 5 (62,5)
Abnormal before => Abnormal after 5 4 7 3 3 3
Because levatorplasty can theoretically reduce the stretching of the pudendal nerve, the evaluation of the effect on the clinical signs has been done for the entire sample ("all") and for the same group without the patients who had a levatorplasty ("without: levat").
The cure rate of the 3 clinical signs was between 60 and 70 % depending of the sign and of the type of sample studied.
Effect on EMG and PNTML
38 patients underwent a complete EMG evaluation before and after surgery. A relevant comparison was possible in 35 patients. 3 patients were excluded because one of the two EMG explorations was insufficient for technical reasons. The average follow up was 16,9 months (range: 12 – 35,4 months).
Left and right values for one parameter correspond to two different nerves. Therefore, these values were considered independent (maximum number of analysed cases: 35 × 2 = 70).
The effect of PND on EMG and PNTML is presented in Table 5.
Table 5 Effect of PND on EMG and PNTML
Anal Richness (range 1 to 6) BC Richness (range 1 to 6) Anal PNTML (msec) Perineal PNTML (msec)
All subjects 74 74 74 74
Follow up less than 12 months or lost 39 39 39 39
Analysed cases(left and right) 70 61 70 51
Mean Before 2,70 2,23 3,38 5,63
Mean After 3,11 2,44 2,63 5,21
t-test p-value(one-tail) 0,00007 0,06989 0,00004 0,00816
Left and right values for each level (anal and perineal) were included in the same group. P-values at the bottom line correspond to one-tailed significance tests of the mean differences "before" versus "after".
The "Anal Richness" on EMG after surgery was significantly higher than before. The mean "Bulbocavernosus Richness" after surgery was slightly higher than before but this difference was not significant. Both anal and perineal PNTML after PND were significantly reduced compared to values before.
The box-plots of the 4 studied parameters are presented in Figures 5 and 6.
Figure 5 Effect of PND on anal and bulbocavernosus (BC) richness on EMG. The box is defined by the sample mean plus or minus one standard error of the sample mean. The probability to obtain a value in the box is 67 %. The whiskers represent the 95% confidence intervals of the population means.
Figure 6 Effect of PND on anal and perineal PNTML. The box-plots definitions are the same as in Figure 5.
Evaluation of the clinical signs
We present here the results concerning the evaluation of the three clinical tests: abnormal sensibility, painful Alcock's canal and painful "skin rollling test" as diagnostic tests for PCS.
The statistical analysis is based on the following contingency tables presented in Tables 6 to 9.
Table 6 "Abnormal sensibility" in the diagnosis of pudendal canal syndrome
Cases Controls
Abnormal sensibility Before Surgery After Surgery
Abnormal 24 7 19
Normal 18 36 63
Total 42 43 82
Chi-square versus Controls 12,691 0,449
P-value < 0,001 0,503
At the bottom lines, p-values and chi-square test statistics correspond to the homogeneity tests comparing the "Cases Before Surgery" to "Controls". The results obtained for the homogeneity test comparing "Cases After Surgery" versus "Controls" are also reported in the tables, although this does not contribute to the sensibility/specificity analysis.
Table 7 "Painful Alcock's canal" in the diagnosis of pudendal canal syndrome
Cases Controls
Painful Alcock's canal Before Surgery After Surgery
Abnormal 32 10 24
Normal 14 30 58
Total 46 40 82
Chi-square versus Controls 17,842 0,0776
P-value < 0,001 0,781
Table 8 "Painful skin rolling test" in the diagnosis of pudendal canal syndrome.
Cases Controls
Painful skin rolling test Before Surgery After Surgery
Abnormal 21 6 13
Normal 17 28 69
Total 38 34 82
Chi-square versus Controls 17,968 0,001
P-value < 0,001 0,97
Table 9 The three clinical signs in the diagnosis of pudendal canal syndrome
Cases Controls
The three clinical signs Before Surgery After Surgery
Abnormal – All positive 13 3 6
Abnormal – Two positive 8 3 9
Abnormal – One positive 5 3 20
Normal – All negative 6 24 47
Total 32 33 82
Chi-square versus Controls 26,528 3,834
P-value < 0,001 0,280
The proportions of observations in the "Cases Before Surgery" and "Controls" columns of the contingency table vary significantly from row to row (p-values <0,001), whereas no significant difference is observed between the proportions of observations between "Cases After Surgery" and "Controls" (p-values >0,05).
Estimated sensibility, specificity, predictive values (positive and negative) and odd ratio (estimated values and 95% confidence intervals) corresponding to each of the three clinical tests and combinations of all of them are presented in Tables 10 and 11, respectively. The predictive values were calculated for a PCS prevalence of 20 %. All the indicators in Tables 10 and 11 are estimated from data in Tables 6 to 9, columns "Cases Before Surgery" and "Controls".
Table 10 Evaluation of each of the three clinical signs of pudendal canal syndrome
Tests SE SP PPV NPV OR 95%IC(OR) Nb of Cases Before Nb of Controls
Abnormal sensibility 0,57 0,77 0,38 0,88 4,42 1,99 – 9,82 42 82
Painful Alcock's canal 0,70 0,71 0,37 0,90 5,52 2,51 – 12,15 46 82
Painful skin rolling test 0,55 0,84 0,47 0,89 6,56 2,74 – 15,68 38 82
SE = sensibility, SP = specificity, PPV = positive predictive value, NPV = negative predictive value, OR = odd ratio, 95%IC (OR) = 95 % confidence interval of the odd ratio. PPV and NPV for a prevalence of 20 %.
Table 11 Evaluation of different combinations of the three clinical signs of pudendal canal syndrome
Test: All Three Clinical Signs SE SP PPV NPV OR 95%IC(OR) Nb of Cases Before Nb of Controls
All positive vs. All negative 0,68 0,89 0,60 0,92 16,97 4,68 – 61,51 19 53
At least 2 positive vs. At least 2 negative 0,66 0,82 0,47 0,90 8,53 3,40 – 21,39 32 82
At least 1 positive vs. All negative 0,81 0,57 0,32 0,92 5,82 2,16 – 15,66 32 82
SE = sensibility, SP = specificity, PPV = positive predictive value, NPV = negative predictive value, OR = odd ratio, 95%IC (OR) = 95 % confidence interval of the odd ratio. PPV and NPV for a prevalence of 20 %.
The most sensible test is the "Painful Alcock's canal" and the most specific is the "Skin rolling test".
Using the three signs altogether, the most sensible combination is "At least 1 positive versus All negative" and the most specific combination is the "All positive versus All negative".
Side effects
During one operation a heavy bleeding coming from the pudendal artery just near the pudendal nerve was very difficult to treat (selective ligature). This patient had a blood transfusion but no long term side effect. Since the operation, one patient has presented sometimes a short lasting clitoridal pain. This patient had also an increase in her anal incontinence (gas incontinence became a liquid incontinence). Three patients had wound healing problems which resolved with simple disinfection.
Prevalence
In the literature there is no data available about the prevalence of the PCS. Therefore, it seems to be a rare event. In this study, we evaluated the prevalence of the PCS in an outpatient perineology clinic. By using three different methods during the last 24 months (percentage of pudendal nerve decompressions in the treatment of prolapse or incontinence : 13/78; percentage of anal incontinence and/or perineodynia in our outpatient consultation : 78/316 ; percentage of positive skin rolling tests : 9/55) the estimated prevalence should be around 20%.
Discussion
Effects of surgery
Before discussing the results of surgery, the first important issue is about the interest of a bilateral decompression. The benefit – risk ratio must be studied. The results of Shafik were obtained after bilateral decompression [5,29,30]. For urinary and anal incontinence, it seems logical to treat both nerves because the sphincters have a bilateral innervation and if one nerve is suffering maybe there is a problem on the other. The EMG exploration is not very sensitive and doesn't study the sensory pathway, which could be very important for continence. For the same reason, bilateral decompression seems logical in the treatment of proctalgia. For unilateral pain, the dilemma is more important. The risk is to induce pain at the "normal" side. Until now we have been performing bilateral operations without such a side effect but a controlled randomised study would be necessary to conclude.
The treatment of pain starts with a holistic approach of the woman (drugs, psychotherapy, relaxation...) with exclusion of other causes of pain: piriformis syndrome, coccygodynia, interstitial cystitis, endometriosis... The other neurological causes must be excluded by a complete electrophysiological study of the perineum (sacral latencies, PNTML, detection EMG and sensory evoked potentials) and imaging of the spinal cord [31]. If the diagnosis is confirmed an infiltration of the Alcock's canal under scanner control can be tried. This infiltration is successful in 57% in the short term but only in 15 % of the cases after one year [32]. It can be repeated maximum 3 times to avoid a nerve irritation. In the treatment of pain the results of this study are similar or better than those obtained in previous studies [32-34].
Even with the transgluteal approach where the "clamp" between the sacro-spinal and the sacro-tuberous ligament is opened by sectioning the sacro-spinal ligament, the cure rate remains around 50%. In the 4 cases of proctalgia fugax the results were better (3 cured and 1 improved). Shafik [5] had also very good outcomes with this type of pain (100% cured). In this study, the results were worse if the pain was bilateral.
For Robert early diagnosis appears to be the determining factor in improving results [35]. He used the infiltration of the Alcock's canal with lidocaïne-corticoïds as a test before operating. According to him a sufficient pain relieve, lasting during a short period, is a good indication for surgery. Mauillon et al also thinks that complete disappearance of pain for at least two weeks after a nerve block repeated twice before surgery may be the best criterion to predict success [34]. In this study, the patients presenting with perineodynia only (n = 10; Figure 1) had an infiltration before surgery but the number of cases was not sufficient to give a relevant impression about the infiltration test.
For anal incontinence, our results were in the same range as Shafik [29]. In a previous study we also had similar results [36,37]. The exclusion of the patients who had sphincteroplasty, levatorplasty (possible post-anal repair effect [27,28]) and/or a cure of rectocele did not change the cure rate.
More interesting was the cure rate in the group of patients with a clear rupture of one or the two anal sphincters. The traumatic rupture of the anal sphincter (delivery, sphincterotomy...) usually induces an immediate anal incontinence. In the patients who remained continent the power of the broken muscles remain sufficient to avoid flatus or faeces leakage. In the long term the continence is probably maintained with the help of the fibrous tissue located between the two edges of the ruptured muscle which acts like a bridge and therefore enables the sphincter to be efficient during many years. The aging process of the muscle and the pudendal neuropathy reduce the power of the muscle (and probably the sensibility in the anal canal) with time and explain the appearance of an anal incontinence. Therefore it is logical to restore continence by improving the conduction in the pudendal nerve. This fact can also explain why the results of sphincteroplasty decrease with time especially in the non diagnosed or treated pudendal neuropathies [38].
The fact that 5 patients were cured from their anal incontinence only 2 years after surgery emphasized the importance of a long follow up period to obtain relevant cure rates. Surprisingly, the cure rate seems to be not dependant of the degree of anal incontinence but the number of solid incontinences (5 cases; 3 cured and 2 improved) was too small to validate this impression.
The results of the pudendal nerve decompression seem to be equivalent to these of neuromodulation [39] and the procedure is far less expensive because there is no need for a special material. If this study is confirmed by others, the treatment of the neuropathy should be done before any trial of neuromodulation. In fact it is logical to repair the electric cable before enabling the current to pass.
For urinary incontinence the number of cases is too small to give a relevant cure rate but there were enough cases to suggest that this surgery can treat some patients with stress or urge incontinence.
In a previous study, 3 of the 7 patients presenting a stress urinary incontinence were cured by bilateral pudendal nerve decompression alone [36,37].
In Shafik's study 6 patients were cured from their stress urinary incontinence, 5 improved and one was a failure [30]. For this author, the efficacy of the pudendal nerve decompression on stress urinary incontinence is due to an increase of the external urethral sphincter EMG activity and to a decrease in the straining urethral reflex latency (time between the expiration involved with the cough and the first deflection of the reflex muscle action potential complex) and PNTML. For Shafik [40] the increase of urethral pressure during abdominal hyperpressure is not only passive but is induced by an active contraction of the urethral sphincter. After an injection of lidocaïne in the sphincter the urethral hyperpressure was suppressed. Thind & al. clearly demonstrated the role of the pudendal nerve in urinary continence. These authors showed a clear reduction of the maximum urethral pressure and a decrease in the adjunctive urethral closure forces during stress after bilateral pudendal blockade [41,42]. This is also in agreement with the study of Constantinou which demonstrated that a fast-acting contraction occurs in the distal third of the urethra 240 plus or minus 30 msec before the bladder hyperpressure [43]. Furthermore, Ko and Kim demonstrated that pudendal nerve block with a 7% phenol solution is very effective in the treatment of external urethral sphincter hypertonicity in patients with spinal cord injury [44].
This study is the first one dealing with a possible effect of the pudendal nerve decompression on urge incontinence. It is probably due to a better control of the urethral sphincter which can reduce urethral instability [45] and improve the inhibition of the detrusor activity.
One weakness of this study is the rough evaluation of the symptoms. We did not use any scoring system, pad test, quality of life questionnaires or "visual analog pain scale". Furthermore the number of anal and urethro-vesical manometries done before and after PND was too small to give relevant results.
The objective evaluation of PND was done using two neurophysiological tests and the clinical examination. Like Shafik [29,30] we found a significant increase in anal richness on EMG, a significant reduction of anal and perineal PNTML after surgery and a significant reduction in the frequency of the clinical signs. The skin rolling test was improved as much as the perineal sensibility and the Alcock's canal pain, thus showing its relevant link with the PCS.
Evaluation of the clinical signs and minimal criteria needed for the diagnosis
Shafik described many clinical signs of the PCS [5,29,30]. In this study only three signs were studied carefully. Shafik described two of them: abnormal perineal sensibility and pain during the palpation of the Alcock's canal by a rectal examination. The third one is the "skin rolling test" which is well known in the diagnosis of neuralgia in other parts of the body [22]. This study is the first one in which this test was utilized as a clinical sign of PCS.
Compared to patients with negative clinical signs, those having positive clinical signs have a 4,42 ; 5,52 and 6,56 higher likelihood of PCS for "Abnormal sensibility", "Painful Alcock's canal" and "Painful skin rolling test", respectively (Table 10). When patients with all three signs positive are compared to patients with all three signs negative, the odd ratio is 16,97 (Table 11). All the estimated 95% confidence intervals for the odds ratios are significantly higher than 1, indicating that the clinical signs can be considered as valuable signs in the diagnosis of PCS.
The most specific sign was the "Painful skin rolling test" and the most sensitive was the "Painful Alcock's canal". The association of the three positive tests had a very high specificity in the diagnosis of a PCS (89 %). This high specificity was confirmed by the low frequency of this association after the operation (return to the same level as the control group).
Therefore, in some cases the clinical examination should be sufficient to prove the existence of a PCS. For example, a patient presenting anal incontinence, an intact sphincter proven by ultrasound and the three clinical signs positive has almost certainly a PCS. Of course, from a scientific point of view it is still interesting to perform a complete electrophysiological study and a precise neurological examination to exclude a central problem (multiple sclerosis, tumor...) or a polyneuropathy.
However, making the diagnosis of PCS is not usually an easy task. Many times, there is a high degree of suspicion but, as in many illness, all the symptoms or signs are not present. In this study, we decided to operate when at least two of the five clinical and neurophysiological signs described in the methods section were associated with one or more of the 3 classical symptoms (perineodynia, anal incontinence and urinary incontinence). At the beginning of this study, it was usually "increased PNTML" and "painful Alcock's canal". With the introduction of the "skin rolling test" and of the "sensibility test", clinical examination became more important in the decision. The more symptoms (especially anal incontinence and perineodynia) and signs were present, the more confident we were in the diagnosis of PCS. Further studies are necessary to validate this and to define more precisely the minimal criteria needed for the PCS diagnosis.
Side effects
The pudendal nerve decompression by the perineal route is a blind procedure. The search for the inferior rectal nerve and the opening of the Alcock's canal are done under finger control. In our experience it is not easier with retractors. Therefore it is necessary to have a clear anatomical vision of this area before performing the operation.
Maybe the use of a laparoscope would help [46] but the procedure will become more expensive and time consuming. To suppress the blind character of the procedure the transgluteal approach proposed by Robert [8] or the more recent transvaginal approach from Bautrant [47] could be other ways to treat the PCS. Until now the results on pain are the same as those obtained by the Shafik's approach but with the concurrent sections of one or two ligaments of the pelvis (sacro-spinal and/or sacro-tuberous ligaments). However, we should be aware that the long term effects of these sections on the stability of the pelvic region are until yet unknown. Therefore, if the "clamp" must be open efforts should be done to open it without cutting a ligament. Up to now no data are available about a potential effect of the transgluteal or transvaginal procedures on urinary or anal incontinence.
Despite the blind character of the procedure we only had one heavy bleeding probably coming from the pudendal artery. One patient still presents with a mild intermittent clitoridal pain and a worsening of anal incontinence. Because the nerve of the clitoris leaves the pudendal nerve just before the entrance into the Alcock's canal this problem is probably the result of a too large dissection in the upper part of this canal. The two cases of anal incontinence worsening (gas incontinence becoming liquid incontinence), including the aforementioned patient, are difficult to explain. Maybe the neuropathy increased with the stretching involved in the procedure, the scarring process or a too large dissection. It could also be the result of the changes in the posterior level anatomy induced by concomitant procedures (easier expulsion of gas or faeces). For the 2 patients the EMG data and the clinical examination after the operation did not improve therefore showing that the neuropathy was not healing.
Prevalence
Because the roughly estimate prevalence of PCS is around 20%, this "defect" seems to be a very frequent problem in Perineology. Therefore it should be logical to search for it in each clinical examination of a patient presenting with prolapse, perineodynia, urinary or anal incontinence.
Conclusions
Pudendal neuropathy is probably a frequent "defect" in perineology. Pudendal nerve decompression seems to be the defect specific procedure indicated in such a problem. In fact it can treat perineodynia, anal and probably urinary incontinence. Anal incontinence can be cured by pudendal nerve decompression alone even in the presence of a clear disruption of the anal sphincter on anal ultrasound. Anal richness on EMG increases and PNTML decrease significantly after surgery proving an objective effect on the nerve. The frequency of abnormal puncture sensibility, painful Alcock's canal and painful "skin rolling test" are significantly reduced by the operation. This study suggests that the three clinical tests could be used in practice to confirm or suspect the diagnosis of pudendal neuropathy in case of pain, urinary and/or anal incontinence. However, further studies are necessary to confirm these preliminary results.
Abbreviations
PCS = pudendal canal syndrome
PND = pudendal nerve decompression
EMG = electromyography
PNTML = pudendal nerve terminal motor latency
Competing interests
The authors declare that they have no competing interests.
Authors' contribution
JB conceived the study, carried out surgery and clinical follow up and drafted the manuscript.
DC participated in the design of the study and performed the statistical analysis.
MB carried out the neurophysiological examinations.
All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
Thanks to Professor Alain Carlier who allowed us to realize dissections of fresh cadavers in the department of anatomy of Liège University and to Georges Nélissen D.O. and Jack Mouchel M.D. for their relevant help in these dissections. The valuable assistance of Els Bakker, PT, PhD, in revising the manuscript is greatly appreciated. Figure 2 can be published thanks to the patient's consent.
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| 15516268 | PMC529451 | CC BY | 2021-01-04 16:28:03 | no | BMC Surg. 2004 Oct 30; 4:15 | utf-8 | BMC Surg | 2,004 | 10.1186/1471-2482-4-15 | oa_comm |
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BMC DermatolBMC Dermatology1471-5945BioMed Central London 1471-5945-4-171553324810.1186/1471-5945-4-17Case ReportFibromatosis of the hand associated with EMO syndrome: A Case report Appelhans Carolin [email protected] Frank [email protected] Andreas [email protected] Peter [email protected] Alexander [email protected] Dept. of Dermatology, Ruhr-University Bochum, Gudrunstr. 56, D-44791 Bochum, Germany2 Dept. of Internal Medicine, St. Josef Hospital Bochum, Gudrunstr. 56, D-44791 Bochum, Germany2004 8 11 2004 4 17 17 6 4 2004 8 11 2004 Copyright © 2004 Appelhans et al; licensee BioMed Central Ltd.2004Appelhans et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
EMO syndrome, defined as a triad including exophthalmus, pretibial myxedema and osteoarthropathia, is a rare condition in patients suffering from hyperthyreosis.
Case presentation
We here describe an interesting case of EMO syndrome associated with unilateral fibromatosis of the hand and an initial stage of generalized myxedema of the skin. To our knowledge a similar case has not yet been described in literature though reports about associated fibromatosis, e.g. located retroperitoneally, already exist. Familiar explanations include its initiation by autoimmune processes or aberrant T-cell cytokine stimulation leading to an overwhelming production of glycosaminoglycans.
Conclusion
Interpreting our case in context with previous reports we conclude that associated fibromatosis induced by autoimmune processes may affect a variety of different localizations and therefore requires careful monitoring. A therapeutical attempt by using UVA1 irridation for pretibial myxedema remained without a satisfying regression.
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Background
EMO syndrome is a rare condition seen in patients suffering from hyperthyreosis. It is defined as a triad of exophthalmus, pretibial myxedema and osteoarthropathia occurring in less than 1% of patients suffering from Graves' disease [1]. We here describe an unusual case of EMO syndrome associated with unilateral fibromatosis and an initial stage of generalized myxedema of the skin.
Case presentation
In October 2003, a 64-year-old male Caucasian patient was admitted with aggravating pretibial myxedema. Five years ago, he was diagnosed with hyperthyreosis caused by Plummer's disease. Laboratory findings revealed a repressed TSH of 0.01 mU/L (0.4–4.0 mU/L). The patient was treated with radioiodine therapy. Today he suffers from hypothyreosis and a daily substitution of 100 μg L-Thyroxin is performed. Within the months following the radioiodine therapy, an erythema progressing into a manifest pretibial myxedema developed, followed by exophthalmus and fibromatosis of the right hand within the next three years. Surgical orbita decompression due to massive exophthalmus was performed, followed by a subsequent correction of the ocular muscles. Additionally, the fibromatosis of the right hand was excised. Retrospective histological findings revealed a regressive sclerotic fibrosis of firm kollagenous fibers embedded in soft and fat tissue were consistent with a generalized fibrosing process as known in myxedema. Within the last months, decent mucous plaques of the upper limps decreased. The patient now reported progressing congestion of lymph and decreasing flexibility of the lower legs caused by the pretibial myxedema. Clinical examination confirmed massive pretibial myxedema, lymphatic congestion of the lower legs, generalized myxedema accentuating the upper limps, a residual postsurgical nodular tumor of the ulnar aspect of the right hand, exophthalmus, and severe hypertrophic osteopathy of the distal phalanges with clubbed fingers and hippocratic nails (Fig. 1). A Complete check-up including autoantibodies remained unremarkable. Under substitution of 100 μg L-Throxin daily we found the following thyroidal values: T3 0.94 ng/ml (0.59–1.74), T4 10.17 μg/dl (4.50–12.00), TSH 0.47 μIE/ml (0.38–4.70), fT3 2.64 pg/ml (1.45–3.48), fT4 1.44 ng/dl (0.71–1.85). There were no hints for additional fibromatosis. The classical combination of exophthalmus, pretibial myxedema and acropachy led to the diagnosis of EMO syndrome associated with fibromatosis of the right hand and a beginning generalized myxedema of the skin.
Figure 1 Pretherapeutic clinical appearance of a 64-year-old patient consistent with the diagnosis of EMO syndrome, exophthalmus (a), combined lymphatic congestion and pretibial myxedema (b) and palmoulnar fibromatosis (intrasurgical, c).
Conclusions
Grave's disease is known to eventually develop after radioiodine therapy [2] and this may be followed by a EMO syndrome. To our knowledge this is the first report about a EMO syndrome combined with coexistent palmoulnar fibromatosis. The patient was treated with lymphdrainage and physiological compressive therapy. As Farr et al. [3] reported the successful use of PUVA in a case of scleromyxedema, we initiated a therapeutic attempt with conventional UVA1 irradiation five times a week for one week followed by three times per week for three consecutive weeks (20 J/cm2 single dose, 280 J/cm2 cumulative dose). While the congestion of lymph clearly improved, pretibial myxedema remained without any signs of regression.
A massive recurrence after excision of thyroid dermopathy, as described in literature, could not be observed in our patient during a follow-up of 2.5 years.
Generalized fibrosing processes are thought to be based on an accumulation of glycosaminogylcans. Common explanations comprise its initiation by autoimmune processes, e.g. thyreotropin receptors on fibroblasts, or aberrant T-cell cytokine stimulation, leading to overwhelming glycosaminoglycan production [4] Fibrosing processes such as retroperitoneal or sellar associated with EMO syndrome or multifocal fibrosclerosis, respectively, have been previously reported [5,6]. Nevertheless, coexistent palmar fibromatosis so far has not been described.
Therefore, we conclude that concomitant fibromatosis might appear in various localizations requiring elaborated diagnostic procedures and monitoring in all patients affected. We started low-dose UVA1 irradiation of the pretibial myxedema known to be able to degrade pathologic collagenous architecture by inducing dermal matrix-metalloproteinases as well as by decreasing abnormal cytokine liberation following T-cell apoptosis [7]. Nevertheless, our patient discontinued UVA1 phototherapy due to non response after a cumulative dosage of 280 J/cm2 UVA1 (seven treatment sessions). However, we suggest that phototherapy might also be considered as an adjunct therapeutic alternative in persistent EMO syndrome.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
C.A. attended to the patient, participated in the design of the case report, and drafted the manuscript. A.K. conceived the study. F.B., A.B. and P.A. participated in its design and coordination.
All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
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| 15533248 | PMC529452 | CC BY | 2021-01-04 16:29:15 | no | BMC Dermatol. 2004 Nov 8; 4:17 | utf-8 | BMC Dermatol | 2,004 | 10.1186/1471-5945-4-17 | oa_comm |
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BMC BiotechnolBMC Biotechnology1472-6750BioMed Central London 1472-6750-4-251550068210.1186/1472-6750-4-25Methodology ArticleEnhanced cell-permeant Cre protein for site-specific recombination in cultured cells Lin Qing [email protected] Daewoong [email protected] Kassatihun D [email protected] H Earl [email protected] Department of Microbiology and Immunology, Room AA5206, MCN, Vanderbilt University, School of Medicine, 1161 21st Ave South, AA4210, Nashville, TN 37232-2363, USA2004 22 10 2004 4 25 25 29 5 2004 22 10 2004 Copyright © 2004 Lin et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Cell-permeant Cre DNA site-specific recombinases provide an easily controlled means to regulate gene structure and function in living cells. Since recombination provides a stable and unambiguous record of protein uptake, the enzyme may also be used for quantitative studies of cis- and trans-acting factors that influence the delivery of proteins into cells.
Results
In the present study, 11 recombinant fusion proteins were analyzed to characterize sequences and conditions that affect protein uptake and/or activity and to develop more active cell-permeant enzymes. We report that the native enzyme has a low, but intrinsic ability to enter cells. The most active Cre proteins tested contained either an N-terminal 6xHis tag and a nuclear localization sequence from SV40 large T antigen (HNC) or the HIV Tat transduction sequence and a C-terminal 6xHis tag (TCH6). The NLS and 6xHis elements separately enhanced the delivery of the HNC protein into cells; moreover, transduction sequences from fibroblast growth factor 4, HIV Tat or consisting of the (KFF)3K sequence were not required for efficient protein transduction and adversely affected enzyme solubility. Transduction of the HNC protein required 10 to 15 min for half-maximum uptake, was greatly decreased at 4°C and was inhibited by serum. Efficient recombination was observed in all cell types tested (a T-cell line, NIH3T3, Cos7, murine ES cells, and primary splenocytes), and did not require localization of the enzyme to the nucleus.
Conclusions
The effects of different sequences on the delivery and/or activity of Cre in cultured cells could not be predicted in advance. Consequently, the process of developing more active cell-permeant recombinases was largely empirical. The HNC protein, with an excellent combination of activity, solubility and yield, will enhance the use of cell-permeant Cre proteins to regulate gene structure and function in living cells.
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Background
The Cre recombinase from bacteriophage P1 has been widely used to induce DNA sequence-specific recombination in mammalian cells [1]. The enzyme, which catalyzes recombination between 34 nucleotide LoxP sequences during P1 genome replication, has been used in a variety of genetic applications to regulate gene structure and function. These include conditional mutagenesis, gene replacement, chromosome engineering, and regulated gene expression [2-4]. However, the use of site-specific recombination in genetic studies is frequently hampered by difficulties expressing the recombinase in cells at the desired time and place [5]. Moreover, the use of Cre expression vectors is constrained by the fact that prolonged exposure to the enzyme can be lethal to cells [4,6,7].
To address these problems, we [8] and others [9-12] have developed membrane-permeable Cre recombinase proteins that are capable of entering cells by a process of protein transduction. Protein transduction exploits properties of specific protein sequences [termed protein transduction domains (PTDs)] that enhance the delivery of macromolecules – including peptides, proteins, and DNA fragments – into living cells [13-15]. Cell-permeant Cre proteins provide an effective means to regulate gene structure and function in living cells, and Cre-mediated recombination provides a potentially useful reporter system with which to study the process of protein transduction itself. In particular, recombination provides a stable and quantitative record of protein uptake that circumvents problems of distinguishing between internalized and cell-associated proteins [16].
In our previous report, recombinant fusion proteins bearing the 12 amino acid membrane translocation sequence (MTS) from fibroblast growth factor 4 (FGF-4) were used to deliver enzymatically active Cre proteins directly into mammalian cells. Of the four recombinant proteins tested, an enzyme containing an N-terminal 6xHis affinity tag, a nuclear localization sequence (NLS) from SV40 large T antigen, and the FGF-4 MTS (HNCM), displayed the best combination of yield, solubility, nuclear localization and enzymatic activity within cells. Recombination was observed in greater than 70% of cells treated with 10 μM HNCM for 2 hours. Widespread recombination was also observed in mice following intraperitoneal administration of HNCM, indicating that a wide variety of terminally differentiated cell types can internalize cell-permeant Cre and are competent to undergo site-specific recombination.
In the present study, eleven recombinant Cre proteins were prepared in order to evaluate sequences affecting the uptake and/or activity of the enzyme in cultured cells and if possible to develop more active recombinases. Several constructs were designed to compare the activities of different PTDs, including the FGF-4 MTS [17], sequences from HIV TAT [18] and a (KFF)3K sequence that was previously used to deliver peptide nucleic acid (PNA) conjugates into cells [19]. Only the Tat sequence promoted the delivery of active Cre into cells, while all three PTD sequences adversely affected the solubility of recombinant proteins containing polyhistidine tags. The contribution of the SV40 large T antigen NLS [20] was also examined to understand apparent differences in the behaviour of cell-permeant Cre and proteins expressed following gene transfer. Thus, the activity of cell-permeant Cre was enhanced by the SV40 large T antigen NLS [8,10], whereas, the native Cre protein appears to possesses a functional NLS, whose activity was not augmented by the T antigen NLS [21]. We report that polyhistidine tags (6xHis) frequently used for protein affinity purification [22] and the large T antigen NLS each separately enhance cellular uptake of enzymatically active Cre, and we describe the development of a cell-permeant Cre recombinase with an excellent combination of activity, solubility and ease of purification.
Results
Recombinant Cre fusion proteins
Eleven recombinant Cre proteins were prepared in order to evaluate sequences affecting the uptake and/or activity of cell-permeant enzymes (Figure 1A). Native Cre (Cre) corresponds to the protein encoded by the P1 phage genome [23]. HC and H6C have amino-terminal hexahistidine tags consisting of MGSSHHHHHHSSLVPRGSH and MHHHHHH, respectively, while CH6 is similar to H6C except the His tag is on the C-terminus. His-NLS-Cre (HNC) is similar to HC except a nuclear localization sequence (PKKKRKV) from SV40 large T antigen [20] is positioned between His and Cre. HT7N'C is similar to HNC except the His tag contains 11 additional amino acids (MASMTGGQQMG) from the pET28a(+) polylinker and the arginine of the NLS sequence was converted to a lysine. This change, which resulted from an altered PCR primer, is unlikely to affect nuclear localization activity. HNCM, described previously [8], contains a membrane translocation sequence (MTS) from the leader sequence of FGF-4 positioned at the C-terminus of HNC. Finally, four different sequences, each reported to have protein transduction activity [17,19,24], were placed on the amino terminus of CH6. These consisted of the FGF-4 MTS (MCH6), an SV40 large T antigen nuclear localization sequence (NCH6), the HIV Tat sequence (TCH6), and a (KFF)3K sequence (KCH6).
Cre proteins were expressed from pET28a(+) plasmids in E. coli and, except for native Cre, were purified by Ni2+ affinity chromatography under non-denaturing conditions [8]. The native enzyme was purified by a combination of hydroxyapatite column chromatography and Sephacryl S-100 HR FPLC. All proteins were expressed at high levels, with yields of purified proteins ranging from 5 to 41 mg/L of E. coli culture (Figure 1). All of the enzymes except TCH6 and KCH6 could be dialyzed against DMEM or RPMI media and stored at -20°C until use. However, TCH6 precipitated under these conditions and was dialyzed instead against PBS supplemented with 0.3 M NaCl (0.45 M total NaCl) and 8% glycerol. The pH of the buffer (ranging from 7.5–8.5) had no obvious effect on protein solubility. The KCH6 protein was insoluble over a range of pH values and salt concentrations (up to 0.8 M), and the protein was not evaluated further. The remaining proteins could be prepared at final concentrations above 1 mg/ml except HNCM, which precipitated out of solution at protein concentrations above 500 μg/ml. The specific activities of the tagged fusion proteins were similar, ranging from 4.3–8.5 × 104 U/mg protein, corresponding to 47–92% of the activity of the native enzyme (Figure 1).
Native Cre recombinase has protein transduction activity that is enhanced by polyhistidine and NLS sequences
The uptake and enzymatic activity of Cre proteins was monitored in Tex.loxP.EG cells [8]. These cells contain a single integrated retrovirus (Fig 2A) in which the expression of an enhanced green fluorescent protein (EGFP) gene is prevented by a "stop cassette" consisting of multiple polyadenylation sites positioned between two loxP sites. Deletion of the stop cassette by Cre mediated-recombination activates the expression of the EGFP reporter gene. Cells were exposed to Cre, HC, HNC, HT7N'C, HNCM, H6C, CH6, NCH6, and TCH6 for 2 hrs at concentrations ranging from 0 to 8 μM. The cells were then washed extensively with PBS, were cultured for 24 hrs to provide time for EGFP expression, and the percentage of EGFP-expressing cells was determined by flow cytometry. Recombination was also monitored by Southern blot hybridization (Fig 2C), thus confirming that expression of the EGFP reporter accurately reflected the extent of template recombination.
As shown in Figure 2B, all of the proteins tested induced recombination in a concentration-dependent manner. The native enzyme had the lowest activity, inducing recombination in up to 17% of the cells. Uptake and/or activity was increased by polyhistidine tags positioned on either the amino- (HC and H6C) or carboxy-terminal (CH6) ends of the enzyme. HC, which contains the 6xHis tag from pET28a(+), and H6C and CH6, which contain simple 6xHis sequences, induced similar levels of recombination, ranging between 45 and 60% of cells. Activity was further increased by the addition of an SV40 large T antigen nuclear localization signal (HNC, HT7N'C and NCH). At low concentrations, TCH6 was the most active protein tested, but higher levels of the protein were toxic to cells, presumably because of the concentration of NaCl required to maintain protein solubility during preparation of the enzyme. Finally, presence of the FGF-4 MTS sequence proved inhibitory for recombination in cultured cells. HNC, which lacks the MTS sequence, was approximately 8 times more active than HNCM as determined by the concentration of enzyme required to induce recombination in 50% of the cells. Thus, while the FGF-4 MTS has been used to transduce a variety of proteins and peptides into mammalian cells, the sequence suppressed the activity of cell-permeant Cre. This confirms the results of a earlier study, reported while this work was in progress [10].
Temperature-dependent protein transduction
Transduction of cargoes containing the FGF-4 MTS [17], including the HNCM protein used in this study [25], is greatly decreased at 4°C as compared to 37°C. By contrast, there have been conflicting reports with regard to the transduction of proteins containing the Tat and Antennapedia PTDs [26-33]. Cre fusion proteins with (HNCM) and without (HNC, HT7N'C and HC) the FGF-4 MTS were therefore tested for their ability to enter cells at 4°C (Fig 3A). Tex.loxP.EG cells were incubated with varying concentrations of the proteins for one hour at either 37°C or 4°C; were washed extensively and were cultured at 37°C to allow time for EGFP expression. In each case, the uptake of the enzyme was inhibited at 4°C, indicating that the inhibitory effects of low temperature are not limited to cargoes containing the FGF-4 MTS. Low levels of recombination observed in cells treated with higher concentrations probably results from cell-associated enzyme that the washing steps fail to remove and that gains entry when cells are later cultured at 37°C.
Serum and cell density effects on protein transduction
Serum has been reported to inhibit the transduction of cargoes containing the FGF-4 MTS; whereas, transduction of proteins containing the Tat and Antennapedia PTDs appears to be unaffected by serum. The effects of serum on protein transduction were assessed by treating TexloxP.EG cells with either HC (5 μM) or HNC (2 μM) for 1 hour in the presence of varying concentrations of either fetal bovine serum (Figure 3B, FBS) or normal mouse serum (Figure 3B, MS). Recombination induced by both proteins was inhibited by up to 60% and 80% in media containing 10% FBS and MS, respectively. Serum appeared to inhibit protein transduction specifically, since it had no discernable effect on either the stability or activity of the proteins in vitro (data not shown). These results indicate that the inhibition of protein transduction by serum is not limited to cargoes containing the FGF-4 MTS.
To assess the effects of cell density on protein transduction, TexloxP.EG cells were seeded at different concentrations in 2 cm2 culture dishes and were treated with HNC (2 μM) for 1 hour (Figure 3C). Recombination efficiencies increased by about 40% as the number of number of cells was increased from 104 to 6 × 104 cells/cm2, and then declined sharply at concentrations above 2 × 105 cells/cm2. Tex.loxP.EG is a T-cell line and is non-adherent; however, the cells settle to the bottom of the culture dish. The optimum density for recombination was similar to the number of cells (.75 × 104 cells/cm2) required to cover the culture dish.
Uptake and localization of cell-permeant Cre proteins
The kinetics of cell-permeant Cre uptake in cultured cells was monitored by examining cells for recombination after exposure to cell-permeant Cre for different lengths of time (Figure 4). Cells were treated with HC (5 μM), HNC (3 μM) and HNCM (10 μM) for 5 to 120 mins, were washed extensively and the extent of recombination was monitored 24 hours later by flow cytometry. Thus, the assay measures the time required for extracellular enzyme to become committed to cell entry. The uptake of HC, HNC and HNCM increased with time, reaching half-maximum levels after 15–20 min. Recombination was induced in cells following exposure to HNC and HNCM proteins for less time than to HC, suggesting a direct role for the NLS in promoting cell binding and/or entry. Note that the observed differences (apparent within minutes after exposing cells to Cre) cannot reflect potential differences in nuclear trafficking since a delay in nuclear import of a few minutes would not affect the extent of recombination measured 24 hours later.
The uptake and localization of cell-permeant Cre in cultured cells was also monitored in living cells by using proteins labeled with the fluorescent dye, Alexa 488 (Figures 5 and 6). The uptake of tagged HC, HNC and HNCM increased with the time as measured by flow cytometry (Figure 5). Again, HNC and HNCM appeared to enter cells more rapidly than HC, which lacks an NLS. Note that the magnitude of the fluorescence is less important than the rate of increase (slope) since the proteins were not labelled to the same extent with Alexa 488.
The localization of Alexa 488-tagged HNC and HC proteins was monitored in living cells by fluorescence microscopy. The HNC protein was localized to the nuclei of Cos7 cells, but was predominately cytoplasmic in NIH3T3 cells (Fig 6B). Cre protein was also localized to the nuclei of Tex.loxP.EGcells (data not shown).
Recombination in different cell types
Several mammalian cell types were analyzed for their ability to undergo Cre-mediated recombination. Clones of Cos7 and NIH3T3 cells containing a single pBABE.lox.stp.EGFP provirus were treated with HNC for two hour and analyzed by Southern blot hybridization (Figure 7). Although the concentration of enzyme necessary to achieve nearly complete recombination was approximately 10 times higher in Cos7 cells than in NIH3T3 cells (10 versus 1 μM, Figure 7), the recombination efficiency did not correlate with localization of the enzyme to the nucleus (Figure 6). Extensive recombination was also observed in murine embryonic stem cells containing a floxed IKK γ gene (Figure 7) and in primary splenocytes explanted from ROSA26R mice (Figure 8). Efficient recombination was therefore observed in all mammalian cell types examined.
Discussion
Cell permeant Cre proteins have generated considerable interest as genetic tools to regulate gene structure and function in mammalian cells [5]. In addition, Cre mediated recombination provides a quantitative reporter for studies on the protein transduction process itself. In the present study, 11 recombinant fusion proteins were analyzed to characterize sequences and conditions that affect protein uptake and/or activity and to develop more active cell-permeant enzymes. We report that the native enzyme has a low, but intrinsic ability to enter cells. Uptake and was enhanced by the addition of a 6xhistidine-tag on either the amino or carboxyl terminal ends of the protein and was enhanced further by the addition of a nuclear localization sequence from SV40 large T antigen or the transduction sequence from the HIV Tat protein. Finally, the hydrophobic membrane translocation sequence (MTS) from fibroblast growth factor 4 (FGF-4) had a net deleterious effect on Cre-mediated recombination in cultured cells.
We had hoped that the native or 6xHis-tagged enzymes would lack transduction activity so that the effects of additional sequences on cell entry, nuclear transport and recombination could be compared. However, we found that the native Cre protein has an intrinsic ability to enter cells, thus confirming observations by Will, et al. [11]. The mechanism by which Cre gains entry into cells remains to be determined. The enzyme may possess a protein transduction domain analogous to those described for HIV Tat, Antennapedia and a growing list of proteins that can enter cells directly, without requiring specific receptor and transporter systems [34]. The fact that Cre is a basic protein [23] is potentially significant considering that many basic peptides are capable of entering cells [24,35-38].
The transduction activity of the native enzyme hinders quantitative studies of sequences incorporated into recombinant Cre proteins, since structural changes associated with each modification may have varying effects on the intrinsic ability of Cre to enter cells. Even so, the 6xHis sequence appeared to facilitate cell entry, since two different 6xHis sequences enhanced Cre-mediated recombination while having little effect on the activity of the enzyme in vitro. Moreover, 6xHis sequences were active when positioned on either the amino- or carboxyl-terminal ends of the enzyme. L-histidine heptamers have been shown to enter cells, although much less efficiently than arginine homopolymers [37,38]. Positively charged histidine sequences also bind cell surface heparin sulfate proteoglycans [39,40], and thus may enhance uptake as has been suggested for the HIV Tat transduction sequence [41]. The transduction activity of the 6xHis sequences is potentially significant given their widespread use as affinity tags to purify recombinant fusion proteins [22].
We [8] and others [10] have shown that the activity of cell-permeant Cre fusion proteins in cultured cells can be enhanced by the addition of an SV40 large T antigen nuclear localization sequence (NLS). The NLS has been shown to enhance the activity of Cre expression vectors [42], presumably by targeting the protein to the nucleus. However, in the present study the NLS enhanced the delivery of Cre fusion proteins into cultured cells as assessed either by cell-based recombination or by uptake of fluorescent Cre proteins. Moreover, nuclear localization did not appear to contribute to cell type-specific differences in the activity of the HNC protein. These results are consistent with the observation that the T antigen NLS, like a number of other basic sequences, has protein transduction activity [24].
HNC, which consisted of a 6xHis tag and T antigen NLS attached to the amino-terminus of the enzyme, was highly active despite the absence of a canonical protein transduction sequence. The 6xHis tag and NLS sequences separately contributed to the transduction of the enzyme, which was 5–8 times more active in cultured cells than the HisNLSCreMTS (HNCM) protein described in our earlier study [8]. By virtue of their positive charge, these elements share similarities with basic protein transduction domains such as HIV Tat and Antennapedia. Early studies suggested that the basic PTDs entered cells through an energy-independent process that was relatively unaffected by low (4°C) temperature [26-30]. However, later studies suggest that the positively charged PTDs promote cell uptake by endocytosis possibly by binding negatively charged proteins on the cell surface [12,31-33,43,44]. Similarly, the uptake of Cre fusion proteins is consistent with an endocytic mechanism. In particular, uptake (time required for commitment to entry) was relatively rapid (10 to 15 min for half maximum uptake), was greatly decreased at 4°C, and was inhibited by up to 80% by serum. The inhibition by serum appeared to involve cell binding and/or entry specifically, since serum had no discernable effect on the stability or activity of Cre fusion proteins in vitro (data not shown). Recombination was markedly suppressed at higher cell densities, possibility because binding sites in or on cells sequester the enzyme, thus lowering the effective protein concentration. Alternatively, cell density may suppress protein transduction by reducing cell size and/or available surface area [25]. In either event, similar observations have been reported for a TatCre protein that lacks a 6xHis tag [9], illustrating the need to control for cell density when comparing the effects or cell-permeant proteins in different cell populations.
Conclusions
The effects of different sequences on Cre uptake or activity were difficult to predict in advance, and consequently, the process of developing a more active cell-permeant recombinase was largely empirical. Proteins containing amino-terminal Tat and (KFF)3K sequences (TCH6 and KCH6) were poorly soluble when dialyzed against isotonic media, and the FGF-4 MTS interfered with the activity of the enzyme in cultured cells. Since Cre is probably not unique in this regard, investigators seeking to develop other cell-permeant proteins would be advised to test a variety of sequences positioned at different places on the protein. In the present case, the HNC protein possesses an outstanding combination of activity, solubility and yield that will enhance the use of cell-permeant Cre to regulate gene structure and function in living cells.
Methods
Cre expression vectors
Native Cre and Cre fusion proteins were expressed in E. coli from pET28a (+)-based plasmids (Novagen). A plasmid expressing native Cre (Cre) was constructed by using the Cre #1 and Cre-stop-HindIII primers to amplify Cre coding sequences from MBP-NLS-Cre-MTS [8] which were cloned between the NcoI and HindIII sites of pET28a (+). HC, His6C, HNC, and HT7C were similarly constructed by PCR amplification, using primers Cre #2, Cre #3, Cre #4 and Cre #5, respectively, together with Cre-stop-HindIII primer. The amplified DNA fragments were cloned into NheI and HindIII sites of pET28a (+). CHis6, NCHis6, MCHis6, TATCHis6, and (KFF)3KCHis6 were constructed by using primers Cre #6, Cre #7, Cre #8, Cre #9 and Cre #10, respectively, together with the Cre-XhoI primer, and then cloned into the NcoI and XhoI sites of pET28b(+).
Cre #1: AGAGAGCCATGGGCTCCAATTTACTGACCGTACACCAA
Cre #2: GTACATGCTAGCTCCAATTTACTGACCGTACACCAA
Cre#3: AGAGAGCCATGGGCCATCATCATCATCATCACAGCTCCAATTTACTGACCGTACACCAA
Cre#4: GTACATGCTAGCCCAAGAAGAAGAGGAAGGTGCTCCAATTTACTGACCGTACACCAA
Cre#5: GTACATGAATTCTCCAATTTACTGACCGTACACCAA
Cre#6: AGAGAGCCATGGGCTCCAATTTACTGACCGTACACCAA
Cre#7: AGAGAGCCATGGGCCCCAAGAAGAAGAGGAAGGTGTCCAATTTACTGACCGTACACCAA
Cre#8: AGAGAGCCATGGGCGCAGCCGTTCTTCTCCCTGTTCTTCTTGCCGCACCCTCCAATTTACTGACCGTACACCAA
Cre#9: AGAGAGCCATGGGCGGTCGCAAGAAACGTCGCCAACGTCGCCGTTCCAATTTACTGACCGTACACCAA
Cre #10: AGAGAGCCATGGGCAAATTCTTTAAGTTCTTTAAGTTCTTTAAGTCCAATTTACTGACCGTACACCAA
Cre-stop-HindIII: GATACGAAGCTTCTACTAATCGCCATCTTCCAGCAGGCGC
Cre – XhoI: AGAGAGCTCGAGATCGCCATCTTCCAGCAGGCGCACCATTGCCCCTGT
Protein purification
Polyhistidine-tagged Cre fusion proteins were expressed in E. coli strain BL21 (DE3) and purified by Ni2+ affinity chromatography as described previously [8]. Bacterial cultures (2L) were grown to an A600 of 0.6–1.0, were induced with 0.4 mM IPTG, and after harvesting the cells were lysed in 40 ml 50 mM Tris (pH 8.0), 50 mM sodium phosphate and 300 mM NaCl. After affinity chromatography, recombinant proteins were dialyzed against DMEM or RPMI media, except TCH6 and KCH6, which precipitated under these conditions. TCH6 was dialyzed instead against PBS supplemented with 0.3 M NaCl (0.45 M NaCl final) and 8% glycerol. The native recombinase was similarly expressed; however, lysates were applied to a 50 ml hydroxyapatite column and step-eluted with sodium phosphate buffers (pH 8.0) of increasing concentration (50, 100, 200, 300, and 500 μM). The peak fraction (200 μM) was dialyzed against PBS, was concentrated by centrifugal ultra filtration to about 20 mg/ml and was fractionated by Sephacryl S-100 HR gel filtration FPLC. Peak fractions identified by SDS PAGE, were concentrated and dialyzed against PRMI-1640 medium containing 1% streptomycin/penicillin and 2% glycerol.
In vitro assays of Cre enzyme activity measured the release of a circular plasmid inserted into a λ phage (Novagen, Madison, WI) by transformation of E. coli [8]. One unit (U) of enzyme produces 104 colonies (equivalent to 2 × 106 circular molecules) in a 30 minute reaction containing 200 ng DNA substrate in 50 mM Tris HCl, pH 7.5, 33 mM NaCl and 10 mM MgCl2 in a total volume of 15 μl. All Cre proteins were stable for at least 6 months at -80°C without significant loss of enzymatic activity.
Cell culture and protein transduction
Tex.loxp.EG, 3T3.loxp.EG and Cos7.loxp.EG cells were derived from Tex (a murine thymoma line derived from p53-deficient mice), NIH3T3 and Cos7 cells, respectively, following infection with the pBABE.lox.stp.EGFP retrovirus [8]. Cells were incubated with serum-free RPMI 1640 (Tex.loxp.EG) or DMEM (3T3.loxp.EG and Cos7.loxp.EG) containing Cre fusion proteins for 2 hours, then washed with PBS twice and cultured in normal growth medium at 37°C incubator for 24 hours. Cre mediated recombination, which induces the expression of an enhanced green fluorescence protein (EGFP) in Tex.loxp.EG cells, was measured by flow cytometry using a FACSort instrument (Becton Dickinson). Alternatively, recombination was monitored by Southern blot hybridization [8]. For experiments involving low temperature protein transduction, the cells and all solutions were maintained at 4°C until after washing with cold PBS, after which the cells were returned to normal medium at 37°C.
Preparation of fluorescent HNC, HC, and HNCM proteins
Fluorescent HNC, HC, and HNCM proteins were prepared by using the Alexa 488 protein labeling kit (Molecular Probes, catalog number A-10235), according to the manufacturer's instructions. The proteins were dialyzed against PBS, were labeled at a concentration of 2 mg/ml and purified by gel filtration through a Sephadex G50 column. Cells were treated with the fluorescent proteins at a final concentration of 1 μM.
The localization of fluorescent Cre proteins was monitored in living NIH3T3 and Cos7 cells by fluorescence microscopy. The cells were cultured to 50–80% confluence in a slide chamber (NUNC), washed with PBS and then incubated with fluorescent Cre proteins at a final concentration of 1 μM for 1.5 hours. The cells were washed three times with PBS, counterstained with 1 μg/ml 4',6-diamidino-2-phenylindole (DAPI) in PBS for 20 minutes, washed three times with PBS and mounted in anti-fade fluorescence mounting medium. The stained cells were photographed with an Olympus BX60 fluorescence microscope using green and blue filters.
Ex vivo recombination by HNC recombinase in primary cells
Splenocytes from ROSA-26R mice were cultured for one day in RPMI 1640 medium containing 10% FBS and were treated with HNC recombinase in serum free media for 2 hours. The cells were washed, cultured for 24 hours, and stained with a fluorescent β-galactosidase substrate (ImaGene green™, C12FDG) according to instructions provided by the manufacturer (Molecular Probes, Inc.) Recombination, which activates the expression of a floxed lacZ gene, was assessed by flow cytometry.
Authors' contributions
QL developed and characterized most of the recombinant Cre proteins described in this study. DJ provided the HNCM protein and assisted with in vitro assays for Cre activity. KDGA. investigated inhibition of Cre activity by serum. ER supervised the project and drafted the final manuscript. All authors approved the final manuscript.
Acknowledgements
We thank Gene Oltz for providing ES cells with a floxed IKK γ allele and Derya Unutmaz for assistance with flow cytometry. This work was supported by Public Health Service Grants (R01NS43952 and R01RR13166 and to H.E.R.) and by a grant from the Kleberg Foundation. Additional support was provided by a Cancer Center Support grant (P30CA42014) to the Vanderbilt-Ingram Cancer Center.
Figures and Tables
Figure 1 Recombinant Cre fusion proteins. (A) Structures of recombinant Cre fusion proteins. Cre sequences from nucleotide 484 to 1513 (GenBank X03453) were expressed unaltered or incorporated into fusion proteins containing one or more of the following elements: His (MGSSHHHHHHSSLVPRGSH); His6 (MHHHHHH or HHHHHH for N- and C-terminal sequences, respectively); NLS (PKKKRKV); NLS' (PKKKKKV); T7 (MASMTGGQQMG); MTS (AAVLLPVLLAP); TAT (YGRKKRRQRRR) and (KFF)3K (KFFKFFKFFK). The table on the right lists the name, size (number of amino acids), yield following purification from E. coli (mg/L), relative solubility, and in vitro specific activity (units per milligram of enzyme) of each fusion protein. (B). Analysis of purified Cre fusion proteins. Purified Cre fusion proteins were fractionated by SDS-PAGE and stained with Coomassie blue.
Figure 2 Cre-mediated recombination in cultured cells. (A). Structure of the recombination substrate in Tex.loxP.EG cells. The pBABE.lox.stp.EGFP retrovirus contains multiple polyadenylation sequences (stop cassette) flanked by loxP sites such that Cre-mediated recombination activates the expression of an enhanced green fluorescent protein (EGFP) reporter gene. (B). Dose-response of Cre-mediated recombination. Tex.loxp.EG cells were treated for two hours with increasing concentrations of each soluble Cre recombinase; were washed twice with PBS; and after 24 hours in normal growth medium, the percentage of GFP expressing cells was determined by FACS analysis. For clarity, the results obtained using different fusion proteins are plotted in two panels. The Upper Panel shows results for HNC (solid squares), HT7N'C (solid circles), HNCM (open squares), HC (open circles), and Cre (solid triangles). The Lower Panel plots the results for NCH6 (solid squares), H6C (solid circles), and CH6 (solid triangles). (C). Southern blot analysis of Cre-mediated recombination. Tex.loxp.EG cells were treated with 0.5, 1.0, 2.0, and 4.0 μM of HNC, HNCM and Cre proteins as described above, except after 24 hours DNA was extracted, digested with EcoR1 and analyzed by Southern blot analysis. Cre-mediated recombination results in the conversion of the loxP-containing fragment (upper band, U) to the recombination product (lower band, R).
Figure 3 Temperature, serum and cell density effects on Cre-mediated recombination. (A) Transduction of cell-permeant Cre is inhibited at 4°C. Tex.loxp.EG cells were treated for 2 hours with increasing concentrations of the indicated enzymes at 4 or 37°C and the cells were processed and analyzed for Cre-mediated recombination as described in Figure 1. (B) Effect of serum on Cre-mediated recombination. Tex.loxp.EGFP cells were treated for 2 hours with 2 μM HNC or with 5 μM HC in the presence of increasing concentrations of fetal bovine serum (FBS) or normal mouse serum (MS) and analyzed for Cre-mediated recombination as described in Figure 1. The extent of recombination (percent GFP positive cells) was normalized to cells treated in the absence of serum. (B) Effect of cell density on Cre-mediated recombination. Increasing concentrations of Tex.loxp.EGFP cells were treated for 2 hours with 2 μM HNC and were analyzed for Cre-mediated recombination as described in Figure 1.
Figure 4 Kinetics of Cre binding and/or uptake. Tex.loxp.EGFP cells were treated with HNC (3 μM), HNCM (10 μM) and HC (5 μM) for different lengths of time; were washed three times with PBS and were analyzed for Cre-mediated recombination as described in Figure 1.
Figure 5 Uptake of fluorescent Cre fusion proteins. Tex.loxp.EG cells were treated for different lengths of time with the indicated Alexa 488-labeled Cre fusion proteins at 4 or 37°C and protein uptake was monitored by flow cytometry. (A) Kinetics of protein uptake. The mean fluorescence of cells treated with Alexa 488-labeled Cre fusion proteins, as calculated by the CellQuest software, increased with time and was inhibited at 4°C. (B) Representative FACS profiles of cells treated with the fluorescent HC protein. Cells were treated with Alexa 488-labeled HNCM at the indicated temperature for 0 (light gray, solid), 15 (medium gray) and 120 min (black) and analyzed by flow cytometry. Profiles of cells treated for 30 and 60 min. have been omitted for clarity.
Figure 6 Intracellular localization of fluorescent Cre fusion proteins. Alexa 488-labeled HNC or HC proteins (1 μM) were incubated with Cos7 or NIH3T3 cells for 1.5 hr, and the living cells were directly visualized by fluorescence microscopy (green fluorescence, left panels). Cell nuclei counter stained with DAPI (blue fluorescence) are shown in the right panels and the merged images are shown in the middle panels.
Figure 7 Cre-mediated recombination in different cell types. Cos7 and NIH3T3 cells containing a single integrated copy of the pBABE.lox.stp.EGFP retrovirus and murine embryonic stem (ES) cells containing a floxed IKK γ gene were treated with 0, 0.5, 1.0, 2.0, 5.0, and 10 μM HNC (Cos7 and NIH3T3) or with 0, 0.25, 0.5, 1.0, 2.0, and 5.0 μM HNC (ES cells) for two hours. Cellular DNA was extracted, digested with EcoRI, and analyzed by Southern blot analysis. Cre-mediated recombination results in the conversion of the loxP-containing fragment (upper band, U) to the recombination product (lower band, R).
Figure 8 Cre-mediated recombination in primary splenocytes. Splenocytes from ROSA-26R mice were treated with 0 (thin black line), 1.0 (light grey line), 2.0 (dark grey line) and 5.0 (thick black line) μM HNC recombinase in serum free media for 2 hours. The cells were washed, cultured for 24 hours, stained with a fluorescent β-galactosidase substrate (ImaGene green™, C12FDG) and analyzed by flow cytometry. Cre-mediated recombination, which activates the expression of a floxed lacZ gene, results in increased fluorescence.
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| 15500682 | PMC529453 | CC BY | 2021-01-04 16:02:56 | no | BMC Biotechnol. 2004 Oct 22; 4:25 | utf-8 | BMC Biotechnol | 2,004 | 10.1186/1472-6750-4-25 | oa_comm |
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BMC Womens HealthBMC Women's Health1472-6874BioMed Central London 1472-6874-4-81550714310.1186/1472-6874-4-8Research ArticleScreening for known mutations in EIF2B genes in a large panel of patients with premature ovarian failure Fogli Anne [email protected] Fernande [email protected] Raphael [email protected] Vien H [email protected] Vladimir K [email protected] Lawrence M [email protected] Odile [email protected] INSERM UMR 384, Faculté de Médecine, 28 place Henri Dunant BP 38, 63001 Clermont-Ferrand cedex, France2 Developmental and Metabolic Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA3 National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA2004 26 10 2004 4 8 8 8 7 2004 26 10 2004 Copyright © 2004 Fogli et al; licensee BioMed Central Ltd.2004Fogli et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Premature Ovarian Failure (POF), defined as the development of hypergonadotropic amenorrhea before the age of 40 years, occurs in about 1% of all women. Other than karyotype abnormalities, very few genes are known to be associated with this ovarian dysfunction. Recently, in seven patients who presented with POF and white matter abnormalities on MRI (ovarioleukodystrophy) eight mutationswere found in EIF2B2, 4 and 5.
Methods
To further test the involvement of known mutations of EIF2B genes in POF, we screened 93 patients with POF who did not have identified leukodystrophy or neurological symptoms. We evaluated these eight mutations and two additional mutations that had been found in patients with milder forms of eIF2B-related disorders. We used restriction enzymes and direct sequencing.
Results
None of the known mutations in EIF2B genes, either homozygous or heterozygous, were identified in our 93 patients with pure 46,XX POF. The upper 95 % confidence limit of the proportion 0/93 is 3.2%.
Conclusions
We conclude that eIF2B mutations, already described in cases of POF associated with white matter abnormalities, are an uncommon cause of pure spontaneous premature ovarian failure.
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Background
Premature Ovarian Failure (POF) can present as a primary or secondary amenorrhea and is associated with elevated gonadotropins before 40 years of age. POF affects 1% of all women and occurs in 0.1% before the age of 30 years [1]. POF has been associated with karyotype abnormalities, including various X chromosome aberrations such as Turner syndrome, which causes depletion of ovarian follicles during development [2]. While conditions such as autoimmune diseases are also associated with POF, the cause is unknown in about 90 % of cases. However, since many affected women have a family history of the condition, predisposition to POF may be inherited [3]. To date, mutations associated with POF have been identified in a small number of genes [4], including those encoding the inhibin alpha [5], the FSH receptor [6], the LH/choriogonadotrophin receptor [7], and the forkhead transcription factor 2 [8]. No more than 10% of women with ovarian failure have mutations in these different genes [8].
Recently three of the five EIF2B genes (EIF2B2, 4 and 5) were reportedly involved in seven patients who presented with POF and white matter abnormalities on MRI (ovarioleukodystrophy) [9]. These genes encode the five subunits of the eucaryotic initiation factor 2B (eIF2B alpha to epsilon), which is involved in the first step of protein synthesis. eIF2B-related disorders include a large group of phenotypes with a recognizable MRI pattern but different clinical severities. The clinical spectrum can range from a rapid course leading to death in severe congenital forms to asymptomatic MRI findings in adult patients [10,11]. Ovarioleukodystrophy might present in a phase without neurological symptoms and an apparently isolated form of POF [9]. Therefore, we screened a series of 93 patients with apparently pure, karyotypically normal POF for mutations in EIF2B genes.
Methods
Selection of patients with premature ovarian failure
In the current study, we evaluated the presence of EIF2B mutations in 93 unrelated and well-characterized women with POF. An institutional review board approved the study and all participants gave a written informed consent. Referring physicians made the diagnosis of premature ovarian failure based on the following criteria: development of at least 4 months of amenorrhea before age 40 associated with two serum FSH levels in the menopausal range. Women with premature ovarian failure as a result of surgery, radiation, chemotherapy, or known karyotype abnormalities were not included in the study. There were 6 Asians, 12 Blacks, 4 Hispanics and 71 Caucasians. The median age at the onset of menstrual irregularity was 24.5 years (range 13 to 39). Eighteen women had a family history of POF. All women underwent a history and physical examination and laboratory screening to confirm the diagnosis of POF and all had a normal karyotype. None of the women had evidence of a neurological disorder.
EIF2B mutations screening
Genomic DNA was extracted from peripheral blood using standard procedures.
The exons of the genes EIF2B2, 4 and 5 which contain mutations found in POF patients or in milder forms of eIF2B-related disorders were amplified by the polymerase chain reaction (PCR) as previously described (Table 1)[11].
Table 1 Sequences of PCR primers used and their PCR conditions.
Nucleotide change tested (gene) Primers sequences (5'-3') PCR conditions
C512T (EIF2B2), C547T (EIF2B2) F: GCAAAACCGTTCTTAC
R: CCTACCCATCTCTCGTTTAT PCR preparation: 1.5 mM MgCl2, 0.225 mM dNTP, 0.8 μM primers, 100 ng primers, 1 unit AmpliTaq Gold™(Applied Biosystems), 1X Taq buffer.
607–612del/insTG (EIF2B2), A638G (EIF2B2) F: GGAAATTATGTGCTGGATATG
R: ACTTTATTCTCTCACCGTGGAT
P243L (EIF2B4) F: ATGCTCAAGCTCCCTTTCAA
R: CTTCACAACTTACAAAGCCT
R374C (EIF2B4) F: ATTCAAGCACCTGGCATGAT
R: CGCTGCACTCCATCCTTATC PCR reaction: 95°C 12 min, 35 cycles (94°C 30 s, 55°C 30 s, 72°C 45 s), 72°C 10 min, 4°C.
T1393C (EIF2B4), T1465C (EIF2B4) F: TGTCCTGTAAGTAGGGGACCTT
R: AAGGGGTTGTGAAGTCTGGA
G338A (EIF2B5), C583T (EIF2B5) F: GAGAAGGACTGTGAGTGCTGA
R: GCCTTCTAAGGGGACAATAAC
F: forward primer, R: reverse primer
Nine mutations were tested by restriction enzymes directly on PCR products (Table 2): 500 ng of PCR products were incubated with 1 unit of specific restriction enzyme from Biolabs® Inc. for 90 minutes, according to the supplier's instructions. Restriction fragments were analyzed by standard acrylamide gel electrophoresis.
Table 2 EIF2B2, 4 and 5 mutations tested with restriction enzymes.
Mutation tested: nucleotides changes (amino acid changes) Mutated gene Restriction enzyme used PCR product size (base pairs) Restriction profile (number of restriction fragments: their size in base pairs)
No mut* Het mut* Hom mut*
C512T (S171F) EIF2B2 Hpy188III 251 4 fragments: 51, 29, 16 and 155 bp. 5 fragments: 51, 29, 16, 155 and 171 bp. 3 fragments: 51, 29 and 171 bp.
607–612del/insTG (M203fs) EIF2B2 HphI 313 2 fragments: 310 and 3 bp. 4 fragments: 310, 142, 164 and 3 bp. 3 fragments: 142, 164 and 3 bp.
C547T (R183stop) EIF2B2 EcoNI 253 2 fragments: 120 and 133 bp. 4 fragments: 120, 133, 14 and 119 bp. 3 fragments: 120, 14 and 119 bp.
A638G (E213G) EIF2B2 BsmAI 313 2 fragments: 153 and 160 bp. 3 fragments: 313, 160 and 153 bp. 1 fragment: 313 bp
P243L (C728T) EIF2B4 AciI 612 3 fragments: 353, 223 and 36 bp. 4 fragments: 576, 353, 223 and 36 bp. 2 fragments: 576 and 36 bp.
R374C (C1120T) EIF2B4 HpyCH4IV 640 2 fragments: 447 and 193 bp. 3 fragments: 640, 447 and 193 bp. 1 fragment: 640 bp.
T1393C (C465R) EIF2B4 BsrDI 694 3 fragments: 368, 32 and 294 bp. 4 fragments: 368, 32, 294 and 326 bp. 2 fragments: 368 and 326 bp.
T1465C (Y489H) EIF2B4 NlaIII 707 3 fragments: 109, 310 and 288 bp. 5 fragments: 109, 310, 288, 57 and 231 bp. 4 fragments: 109, 310, 57 and 231 bp.
G338A (R113H) EIF2B5 Fnu4HI 800 3 fragments: 115, 633 and 52 bp. 4 fragments: 115, 633, 52 and 748 bp. 2 fragments: 748 and 52 bp.
* No mut: no mutation; Het mut: heterozygous mutation; Hom mut: homozygous mutation.
The C583T (R195C) mutation in the EIF2B5 gene was tested by direct sequencing of exon 4 as previously described [11].
Results
None of the eight mutations already described in ovarioleukodystrophy were detected in our 93 patients with pure 46,XX POF, neither in a homozygous nor in a heterozygous state. In addition, the mutations C728T and C1120T (EIF2B4) described in milder forms of eIF2B-related disorders were not found in this series of 93 patients with POF. The upper 95 % confidence limit of the proportion 0/93 is 3.2%.
Discussion
eIF2B-related disorders include a large group of phenotypes with different clinical severities. Individuals can be classified into three clinical groups according to their age at disease onset: <2 years (group 1), 2 to 5 years (group 2) and > 5 years (group 3) [11]. Group 3 corresponds to individuals with the milder form of the disease, including the six families (seven patients) already described presenting with ovarioleukodystrophy [9].
In these six eIF2B-mutated families, neurological symptoms with abnormalities of the cerebral white matter on MRI were associated with primary or secondary amenorrhea due to POF [9]. A correlation was observed between the age of onset of the neurological deterioration and the severity of the ovarian failure, suggesting a common pathophysiological pathway [9].
The mutated eIF2B may be responsible for both increased apoptosis of ovarian follicles leading to POF, and a defect in glial cell development causing abnormal formation of white matter structures. In ovarioleukodystrophy, a phase of amenorrhea without neurological symptoms can be observed, suggesting that an apparently isolated case of POF might be due to EIF2B mutations. In the present study, we tested for EIF2B mutations a series of 93 patients with pure, karyotypically normal POF without identified signs of cerebral dysfunction.
In eIF2B-related disorders, a correlation exists between genotype and disease onset [11]. The mutations G338A (EIF2B5 gene) and A638G (EIF2B2 gene) are found in 71% of families with late onset forms of eIF2B-related disorders (group 3) [11]. In ovarioleukodystrophy, 4/6 families have a G338A or A638G mutation in a heterozygote or a homozygote state. Thus, to further evaluate involvement of eIF2B mutations in apparently isolated cases of POF, we restricted our screening to the 10 mutations associated with the late onset form (group 3) of eIF2B-related disorders. In the present series of 93 patients with pure, karyotypically normal POF, no mutations were detected, suggesting a low frequency of EIF2B mutations in women with POF who have no apparent neurological signs.
Conclusions
For patients presenting with POF without neurological signs or MRI abnormalities, the routine screening of the EIF2B mutations is not clinically indicated.
Competing interests
The author(s) declare that they have no competing interests.
Authors'contributions
AF and FGB carried out the molecular genetics studies, including enzyme restrictions (AF) and sequencing (AF and FGB). AF drafted and conceived of the study. RS participated in the coordination of the study. VHV, VKB, LMN recruited and evaluated the patients, collected DNA samples, participated in the design and coordination of the study, and helped in drafting the manuscript. OBT conceived of the study, and participated in its design and coordination. All authors participated in the writing of the manuscript and have read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgments
We gratefully acknowledge the participation of the patients. This work was supported by a grant from the European Leukodystrophy Association (ELA), the Fondation pour la Recherche Médicale (FRM, ARS 2000) and the Jean Pierre and Nancy Boespflug myopathic research foundation.
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| 15507143 | PMC529454 | CC BY | 2021-01-04 16:30:33 | no | BMC Womens Health. 2004 Oct 26; 4:8 | utf-8 | BMC Womens Health | 2,004 | 10.1186/1472-6874-4-8 | oa_comm |
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BMC Med EthicsBMC Medical Ethics1472-6939BioMed Central London 1472-6939-5-610.1186/1472-6939-5-6Research ArticleClinical education of ethicists: the role of a clinical ethics fellowship Chidwick Paula [email protected] Karen [email protected] Dianne [email protected] Laurie [email protected] University of Toronto Joint Centre for Bioethics, Ontario, Canada2 Trillium Health Centre, Mississauga, Ontario, Canada3 William Osler Health Centre, Brampton, Ontario, Canada4 Sunnybrook and Women's College Health Sciences, Toronto, Ontario, Canada5 Centre for Clinical Ethics (a shared service of Providence Healthcare, St. Joseph's Health Centre and St. Michael's Hospital), Toronto, Ontario, Canada6 St. Joseph's Healthcare, London, Ontario, Canada7 Department of Physical Medicine and Rehabilitation, Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario2004 8 11 2004 5 6 6 2 6 2004 8 11 2004 Copyright © 2004 Chidwick et al; licensee BioMed Central Ltd.2004Chidwick et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Although clinical ethicists are becoming more prevalent in healthcare settings, their required training and education have not been clearly delineated. Most agree that training and education are important, but their nature and delivery remain topics of debate. One option is through completion of a clinical ethics fellowship.
Method
In this paper, the first four fellows to complete a newly developed fellowship program discuss their experiences. They describe the goals, structure, participants and activities of the fellowship. They identify key elements for succeeding as a clinical ethicist and sustaining a clinical ethics program. They critically reflect upon the challenges faced in the program.
Results
The one-year fellowship provided real-time clinical opportunities that helped them to develop the necessary knowledge and skills, gain insight into the role and scope of practice of clinical ethicists and hone valuable character traits.
Conclusion
The fellowship enabled each of the fellows to assume confidently and competently a position as a clinical ethicist upon completion.
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Background
Bioethics is being integrated into healthcare settings more widely and systematically than ever before. In Canada, clinical ethicists are employed in many teaching hospitals and their presence is increasing in community hospitals and long-term care facilities. Although individuals who work in the field come from diverse backgrounds with a variety of skills and training, the roles that clinical ethicists fill have some commonalities. Most clinical ethicists serve as resource persons and engage in consultation services, research, education and policy development within a healthcare setting, as well as engage in organizational ethics activities [1].
The American Society for Bioethics and Humanities has developed a set of core competencies for health care ethics consultation [2,3]. The Society suggests that ethics consultants should have skills in three general areas (assessment skills, process skills, and interpersonal skills) and knowledge in nine areas. The Society suggests that competencies can be acquired through a variety of different approaches. Although there is general agreement that education and training for clinical ethicists are important, the most effective methods of delivering that training have not yet been clearly identified [1,4-9]. The fit between the education and training students receive and the ability to assume a position in bioethics upon completion has been questioned [9,10]. There is also debate as to whether education and training programs should become more uniform and homogenous or remain heterogeneous [9,10].
A clinical ethics fellowship is perceived by some to be one of the ways in which necessary core competencies can be acquired [2]. Currently, however, clinical ethics fellowship opportunities for individuals wishing to pursue a career as a clinical ethicist are relatively limited. In a fellowship, individuals are provided with real-time clinical opportunities to help them develop necessary knowledge and skills, gain insight into the role and scope of practice of clinical ethicists and hone their character over a period of time. Specifically, clinical ethics experience may assist individuals in the development of their abilities to identify and analyze ethical problems, use reasonable clinical judgment, communicate effectively, negotiate and facilitate when there is conflict, and act as a resource for healthcare professionals who are faced with the daily challenges of delivering ethical care.
The University of Toronto Joint Centre for Bioethics (hereafter referred to as the JCB) developed and implemented a clinical ethics fellowship program to assist in meeting the identified need for clinical knowledge and skills [2,3,6,11]. In this article, a description of the fellowship is provided, including its goals, structure, participants and activities. By reflecting on their experiences, the authors, who were the program's first four participants, discuss how the clinical ethics fellowship helped prepare them to work as clinical ethicists. They identify key elements they perceive as necessary for success as a clinical ethicist and for developing an effective clinical ethics service. As well, they critically reflect upon the challenges faced as they progressed through the program.
Method
University of Toronto Joint Centre for Bioethics Clinical Ethics Fellowship
The JCB is a collaborating centre of the World Health Organization. It was formed in 1995 and is a partnership among the University of Toronto and its affiliated hospitals. With a membership of over 160, approximately 20 of whom work full-time in bioethics, it represents the largest multi-disciplinary group of in-hospital ethicists in Canada. Its members are widely published and actively engaged in a number of locally and nationally funded ethics research projects. In addition to the clinical ethics fellowship, the JCB offers two bioethics graduate programs.
The first two participants in the JCB's clinical ethics fellowship entered the program in July 2001. The second cohort of two fellows began the program in August 2002. (In September 2003, the program expanded to include three fellows, and in September 2004 grew to five fellows.) The primary purpose of the one-year fellowship program is to provide the necessary preparation individuals require for a smooth transition from academic and clinical education, training and experience to the position of clinical ethicist. The fellowship provides multi-site clinical ethics opportunities at both specialty and general hospitals, exposes fellows to a variety of multi-disciplinary approaches to clinical ethics, supports the work of the ethicists at the JCB's affiliated hospitals, and, lastly, expands and strengthens the network among clinical ethicists, both within the JCB and across Canada. To be eligible for the fellowship candidates must have a graduate degree in bioethics or a professional degree with significant bioethics training. Preference is given to candidates with previous exposure to clinical bioethics including consultation and teaching experiences.
Structure of the fellowship
In the first two years the program of the program, each fellow rotated through four of the JCB's eight affiliated teaching hospitals, both specialty and general. As the number of affiliated hospitals has continued to grow, so too has the number of fellows. The fellowship was structured so that each fellow was concurrently assigned to two hospitals for a six-month period of time, averaging about two days per week on site at each hospital. A minimum of one day per week was spent working at the JCB where the fellows shared well-equipped office space. This functional arrangement promoted opportunities for collaboration, reflection and mutual support among the fellows. The fellows received a monthly stipend that was sufficient for covering basic costs of living.
Results
Activities in the fellowship
Throughout the year, fellows attended and actively participated in the weekly Wednesday meetings and case conferences of the JCB's Clinical Ethics Group, as well as the weekly seminars hosted by the JCB that were open to the university community and the public. The Clinical Ethics Group is comprised of the ethicists who work at the JCB's affiliated hospitals. The focus of these weekly meetings is to develop exemplary models of clinical ethics practice in diverse healthcare settings. Activities include research and practice collaborations, sharing of ideas and resources, strategic planning and policy discussions. Fellows actively participated as full members of the group in these meetings. For example, one of the projects that fellows worked on was the conception, development, and implementation of the Project for Examining Effectiveness of Clinical Ethics (PEECE). PEECE was an ongoing research initiative. Its purpose was twofold: to describe the current state of affairs of clinical ethics across sites and through interviews with key stakeholders to identify benchmarks of effectiveness. Fellows participated in all aspects of the project from reviewing literature, developing a proposal, collecting and analyzing data to preparing papers for publication. Policy discussions revolved around such varied topics as sexuality in long-term care, pharmaceutical sponsorship, gift-giving in the context of professional/provider relationships and end-of-life care.
During the weekly case conferences, individual ethicists bring complex and challenging cases forward for broader consultation and review. For example, in the second year of the fellowship, a pressing clinical situation arose, with an accompanying set of complex ethical questions. This was the emergence of Severe Acute Respiratory Syndrome (SARS). The weekly case conference discussions during this time period focused on ethical issues such as the professional duty to care for and treat patients, limits of confidentiality and visitor restrictions. Among the many other cases that came to the case conference were situations of conflict around end-of-life treatment and defining futility, moral distress of staff providing care in the context of serious resource limitations, elder abuse in the community, and pregnancy termination for genetic anomalies. Fellows who were involved in the cases collaborated and co-presented with the hospital ethicist. Fellows also provided background literature, developed presentation materials and other resources for the Clinical Ethics Group on specific ethical issues as requested. In addition to providing a mechanism for acquiring broader consultation on a particularly challenging and complex case, the case conferences served as a quality assurance mechanism for the affiliated hospitals.
The weekly meetings and case conferences were a resource for the clinical ethicists and clinical ethics fellows to receive collegial support and networking opportunities. The weekly seminars featured local, national and international speakers on a wide range of topics.
Fellows were encouraged and mentored to participate in a wide range of activities at each of the affiliated hospitals. The fellows were warmly welcomed into the various institutions by the clinical ethicists, staff and patients. Participation in the preparation and delivery of formal and informal educational activities comprised the largest element of the fellowship, and occurred on at least a weekly basis and frequently more often. Educational activities included presenting at Grand Rounds on ethics topics such as clinical ethics decision-making, moral distress, and advance care planning; leading unit-based rounds on topics such as artificial hydration and nutrition at the end-of-life; facilitating brown bag lunches on topical ethics issues; teaching segments of undergraduate and graduate programs; and developing and implementing innovative curriculum for ethics committee members.
Second, case consultations were another activity in which the fellows routinely engaged. Initially, fellows participated in the preparation for case consultations and then observed the consultation process as it unfolded. They provided support for the hospital ethicists by gathering background information about the case, reviewing the relevant literature and documenting the consultation in the health record. As the Fellows progressed through the program and their skills and confidence increased, they assumed more responsibility in consultations by chairing or facilitating meetings. In addition, fellows had the opportunity mentor graduate bioethics students by including them in consultations. Throughout the fellowship, fellows received immediate feedback on the progress and outcomes of the consultations from the hospital ethicist. This debriefing opportunity was invaluable for fellows, enabling them to gain insights into the context of the case, the nature of the conflict or difficulty and the unique and recurring themes that were encountered within and across consultations. Teachable moments, individual strengths and areas for further skill and knowledge development were also identified. The number of consultations varied from one site to another, but over the course of the fellowship, each fellow was exposed to a wide variety of consultation experiences. Case consultations differed in terms of their length, from a very short 10-minute conversation to up to 6 hours in a single day with continuing follow-up over subsequent days, weeks and sometimes months.
Third, fellows participated in policy and guideline development, although these activities consumed less time than educational and consultation duties. For example, one fellow developed guidelines for the administration of blood and blood products to pediatric Jehovah's Witness patients. She then took the draft to focus groups consisting of various stakeholders both internal and external to the hospital and redrafted the guidelines based on this input.
Fourth, fellows participated in clinical ethics research and research ethics board activities. An example of such research was a chart audit conducted by a fellow to examine how consent and capacity issues were being addressed in a particular facility. Several practice concerns were identified and subsequently a facility-wide educational program was implemented. In addition, the fellows engaged in a variety of other scholarly activities including writing, presenting and publishing on ethics-related topics in a variety of forums, which allowed the fellows to develop a comprehensive understanding of a wide variety of strategies for building a sustainable, integrated and accountable ethics program. These experiences, which built professional knowledge, skill and confidence, laid the foundation for the fellows in developing their professional identity as clinical ethicists. Observing the hospital ethicists in action, the fellows realized that these clinical ethics roles were developed over time and with effort. This helped to shape realistic goals and expectations for the early phase of a clinical ethics career.
The first fellows
In July 2001, Paula Chidwick and Laurie Hardingham were the first fellows accepted into the JCB's clinical ethics fellowship program. Dr. Chidwick holds a PhD in Philosophy and prior to entering the fellowship program completed an ethics internship at Sunnybrook and Women's College Health Sciences JCB. She has taught bioethics at the University of Toronto. Laurie Hardingham is a registered nurse who has worked in a variety of healthcare settings. She has comprehensive academic education in philosophy, completing a Masters in Philosophy and doctoral course work in philosophy. She has taught philosophy and ethics at the University of Calgary and Mt. Royal College, as well as planned and coordinated the Provincial Health Ethics Network in Alberta.
Karen Faith and Dianne Godkin were selected for the 2002/2003 clinical ethics fellowship program. Karen Faith completed a Masters in Science majoring in bioethics through the Collaborative Program at the Joint Centre for Bioethics and the Institute of Medical Sciences, University of Toronto. After completing her degree in bioethics, Ms. Faith was a part-time ethics consultant to several healthcare organizations. Previously, she was a social worker who worked in the area of mental health. Ms. Faith has taught at York University, Seneca College and Centennial College in Toronto. Just prior to beginning the clinical ethics fellowship, Dianne Godkin completed a PhD in Nursing. During her doctoral studies she focused on ethics and gerontology, particularly in the areas of end-of-life decision-making and advance care planning. While studying at the University of Alberta, she taught an interdisciplinary graduate course in health ethics and was an observer on a healthcare ethics committee. The objectives that the fellows set out to accomplish during the fellowship included gaining expertise in the clinical consultation process, further developing their teaching and researching skills, increasing their confidence in working through difficult ethical situations as they unfold and expanding their multi-disciplinary network of contacts.
Discussion
Preparing fellows to work as clinical ethicists
The fellowship helped prepare the fellows to make the transition to clinical ethicists by providing real-time clinical opportunities. Although there were opportunities to attend lectures, seminars and conferences and to participate in research projects and the activities of research ethics boards, the focus of this fellowship was clinical practice.
"Real-time" clinical opportunities
Generally, bioethics education is largely theoretical, focusing on academic course work in philosophy and ethics, as well as other disciplines, at the graduate level. Practical clinical experiences for individuals wishing to pursue a career as a clinical ethicist have been very limited historically and offered only sporadically. In this fellowship, ethical challenges unfold and are addressed within the day-to-day experiences of hospital life. Although hypothetical or retrospective cases studied in the classroom are useful in applying theory to clinical cases, the value of experiential knowledge gained when cases are encountered in the here and now involving real people with tangible consequences cannot be overstated. One fellow recalls a case involving a family having a very difficult time coming to terms with the imminent death of a loved one. The family was adamant that "everything be done", a phrase that often is bandied about in these sorts of discussions and requires considerable exploration. In this case, "everything" was defined by the family to include CPR and admission to intensive care. The fellow attended a meeting with the family and the healthcare team to discuss the plan of care, but the patient, although capable, was too ill to attend. The fellow had not met the patient. The description of the patient by the healthcare team was completely different from that given by the family. The fellow was uncomfortable with the decisions made without directly hearing the patient's voice. It was not until the fellow met with the patient and the physician alone, that she began to understand the situation. Seeing the physical frailty, but clear thinking and comprehension of the patient fuelled her wish to see that the patient received the care that she desired. It mattered what decisions were made, the situation was no longer hypothetical, but was real and the stakes were high.
Through their daily work and interactions with staff in the various hospitals, the fellows became familiar with the fast-paced clinical environment and culture, the healthcare providers' values and practices and the complexity and diversity of ethical issues. Given the unpredictability of when consultation requests would surface, fellows found themselves needing to be flexible and accommodating, often leaving writing or research activities to respond to requests for consultation. Fellows could be called to the intensive care unit, coronary care unit, emergency department, or hospital boardroom at any time and some of these consultations required an immediate response. Consultations of a less emergent nature were scheduled for a later time and often included meetings with the healthcare team, families and patients. Fellows carried a pager so that they could be reached immediately.
Other learning opportunities included the following: developing and implementing an ethics program through participation in strategic planning activities; raising the profile of ethics in a hospital using a variety of networking, public relations and communication strategies; reaching out to those who questioned the value of ethics programs by establishing an ongoing presence on units that were struggling with a particular ethics issue; building trust and establishing credibility with healthcare professionals by recognizing, understanding and responding first to their most urgent needs; identifying opinion leaders in the organization and integrating them into the ethics program; building bridges with senior management; and supporting the work of ethics committees as well as other hospital committees
Skill development
Throughout the year, the fellows each worked with a number of clinical ethicists with varied approaches, backgrounds, training and expertise. As a result there were numerous and ongoing opportunities to develop a multiplicity of skills. Through the observation and mentoring of the clinical ethicists, fellows honed their mediation, communication and negotiating skills. They developed political, practical and conflict resolution skills in both observing and responding to conflicts pertaining to patient care decisions They learned to use wisdom or judgment, particularly in establishing credibility, gaining trust and responding to challenges regarding their role and duties. For example, when a fellow witnessed a clinical ethicist's role being challenged by a senior hospital staff member, the clinical ethicist modeled a respectful but assertive approach, demonstrating both good judgment and clarity of purpose. They acquired skills in the recognition, prevention and management of moral distress and moral residue. Many of the clinical ethicists shared personal experiences of morally distressing situations and modeled the need for broad consultation through the JCB consultation group and debriefing with colleagues as a way to cope with stress. The development of this last skill has proven invaluable as the role that moral distress and residue play in the clinical setting becomes increasingly acknowledged and better understood [13,16].
The skills that were nurtured and developed during the fellowship mirror the ethical assessment skills, process skills and interpersonal skills that have been identified as core competencies for ethics consultation [2,3] As the fellows moved through the program, they received ongoing critique of their skills. They participated in educational and practice activities to support their skill development (for example, conflict negotiation workshops).
Insights into the role and scope of practice of clinical ethicists
The fellows observed that the scope and practice of the clinical ethicists included four primary areas of focus: building capacity, acting as a resource, organizational ethics and scholarly work. The goals of capacity building within the organization included promoting ethical sensitivity and discernment, increasing ethics knowledge and skills and enhancing ethical behavior in the delivery of healthcare. This was accomplished through formal and informal educational activities, committee work, consultations and daily interactions with staff. As a resource, clinical ethicists were called upon to do ethics consultations, provide information and share expertise in various areas of ethical concern. Clinical ethicists' organizational ethics activities were diverse and included the development of policy, guidelines and procedures, collaborative initiatives with other departments and professionals and strategic planning. As well, all of the clinical ethicists were engaged in scholarly activities such as research, writing and publishing, presenting at conferences and teaching at universities and colleges.
As a result of working with clinical ethicists in a variety of healthcare settings with different educational backgrounds, the fellowship experience offered a broad perspective on the role and scope of clinical ethics practice. Because clinical ethics is a relatively young field that continues to evolve and define itself [6,7,17,18], seeing and working with clinical ethicists in action, demonstrating their skills and knowledge, was instructive and assisted the fellows in developing their own professional identity and understanding of what an ethicist's role and responsibilities were and were not in the healthcare setting. The fellows learned that common misperceptions of the clinical ethicist's role included that of moral expert, judge of right and wrong, legal expert, risk manager, ethics police, ombudsperson, locus of ethics for the institution and final decision-maker [19,20].
Character development
By observing and participating with the clinical ethicists in their daily activities the fellows identified certain important character traits for this role, such as humility, respect for others, self-knowledge, self-awareness and courage. Although other character traits were also observed, the fellows agreed that these particular traits were both necessary and desirable and thus worthy of emulation in their own practice.
The fellows observed that clinical ethicists who modeled humility recognized that their role was neither that of judge nor moral expert, but as a member of the team who was able to engage in a collegial process of deliberation and ethical decision-making. As well, with humility came the recognition that one ethicist cannot be knowledgeable in all areas and that it was essential to build up a network of colleagues from different educational backgrounds with whom to consult. Similar traits such as self-knowledge and self-awareness involved the ability of the ethicist to recognize his or her strengths and limitations. The extent to which the ethicist demonstrated self-knowledge and self-awareness influenced their own self-care practices and ability to manage work demands and work related-stress and thus avoid burnout. Fellows observed ethicists maintaining an attitude of respect toward the opinions of all concerned parties; they ensured that each individual's voice was heard and his or her perspective considered. When clinical ethicists upheld an ethical position in the face of considerable opposition the fellows concluded that ethicists modeled courage. The traits deemed important by the fellows reflect many of the character traits that are considered to be prerequisites to successful healthcare ethics consultation [2,11]. Further contemplation on these traits by the fellows raised their own level of self-awareness and their desire and ability to integrate and exhibit these traits in their daily practice.
Key elements for success
Through their fellowship experiences in a variety of ethics programs at differing stages of development, the fellows recognized certain elements that appeared to contribute to an effective clinical ethics program. First, a clinical ethics program needs to be integrated throughout the organization. Integration was key in building capacity from bedside to boardroom and dispelling myths about the role of ethics and ethics programs. Embedding ethical considerations into all aspects of decision-making is achieved through an understanding of how ethics can be a resource for the staff when they face ethical dilemmas. Indicators of a well-integrated ethics program included a clear understanding of the program by staff, visibility within the organizational structure and accessibility of the ethicist to staff, patients and families.
Second, a sustainable ethics program requires organizational support and a commitment through the provision of a dedicated budget for ethics including administrative support, adequate physical space and resources, as well as support for continued education. Organizational commitment can be demonstrated through a clearly defined and stable reporting structure and the clinical ethicist's participation in decision-making at the management level. Such organizational commitment allows the ethicist the resources and time to provide the services that support excellence in patient care and to help staff when faced with ethical issues. The clinical ethicist needs to have clear goals and parameters for the work and establish reasonable expectations in order to provide an effective service, reducing ethicists' moral distress and burnout.
Third, clinical ethicists cannot work in isolation and need the support of a network of colleagues both within and outside of the field of ethics, especially when confronted with complex or unusual cases in new and emerging areas. One of the roles of clinical ethicists is to act at the same time as both trusted organizational insider and as an objective neutral outsider. Clinical ethicists are best able to succeed in this capacity when they develop collaborative relationships with other service providers in the healthcare settings for example, risk management, pastoral care and social work. Fellows observed that this network of support included the JCB clinical ethics group as well as key professionals knowledgeable in areas of bioethics relevant to the specialized areas of health care. For example, one clinical ethicist had particular expertise in pediatric settings and was called upon often by colleagues when an ethical challenge concerned the care of neonates or children.
Fourth, the clinical ethicist's ability to see beyond the initial presenting problem was a crucial skill in the case consultation process. As the clinical ethicist entered into the situation the scope of inquiry often broadened and new and larger, and sometimes quite different, questions emerged. For example, when called in by staff for a consultation, the fellows often observed that upon discussion with the patient or family a different problem was brought to light. Fellows observed that ethicists that kept the dynamic nature of the consultation in mind usually had more successful consults.
Critical reflections
Christine Harrison challenges those engaged in bioethics to consider what "bioethics is" before contemplating its future [6]. The clinical ethics fellowship assisted the fellows in developing their own understanding of what clinical ethics is and the clinical ethicist's role, as well as acquiring the necessary knowledge, skills and character traits. The one-year practical learning experience in clinical ethics was perceived by the fellows as an excellent way for them to begin to understand what it means to be a clinical ethicist and to develop core competencies to succeed in that role. However, as the field is evolving quickly with new issues emerging, sometimes quite unexpectedly, it is unlikely that one would ever feel fully prepared to independently step into the position of clinical ethicist. The fellows in the second cohort learned this lesson first-hand, when Severe Acute Respiratory Syndrome (SARS) struck Toronto and dramatically transformed the work environment in the hospitals in which they served [12]. Rotations were in six-month segments with a shared work week between two hospitals, but due to SARS precautions which prohibited people from traveling between sites, fellows needed to limit their work to one hospital. Indeed, some of the fellows were not allowed into particular hospitals until infection control restrictions were lifted and were forced to continue their work from home as best they could. Even prior to SARS, fellows found that the disparate geographic location of multiple work settings made availability for consults difficult at times. Subsequently, full-time three-month block placements for fellows have been implemented at some hospital sites rather than the split workweek.
After the first year of the program, a position became available for a one-year senior clinical ethics fellowship. Laurie Hardingham accepted that position, and during the senior fellowship year, she worked in one teaching hospital, concentrating on ethics consultations, increasing educational opportunities for staff and strengthening the clinical ethics program in that hospital. She was also available to mentor and advise the new first year fellows, supporting the fellowship program. The senior fellowship allowed her to develop a greater understanding of how to integrate ethics throughout an organization and develop the ability to more effectively utilize organizational structures and resources in the clinical ethics program.
The hospital ethicists that the fellows assisted had many organizational commitments, were involved in numerous projects and could be called upon at a moment's notice for consultations. As the areas of focus for clinical ethics services varied significantly between hospital settings, fellows were required to review and research literature on many complex and different ethical, clinical and legal topics. To meet the demands of working in a fast-paced healthcare environment with rapidly changing needs, fellows were also faced with the challenges of being available, flexible and accommodating. Being introduced to several hospital settings at the beginning of each rotation presented the fellows with the additional tasks of quickly familiarizing themselves with and acclimatizing to new organizational rules and procedures, staff and institutional cultures. Being a fellow also brought in practical considerations such as taking leave from previously held positions, adjusting to a considerable reduction in pay and relocating to Toronto.
The fellows were exposed to stylistic and theoretical differences in the way clinical ethics was practiced when working with ethicists who entered the field through diverse academic and clinical backgrounds. The potential does exist for such differences to become a barrier to learning and building trust within the clinical ethicist/fellow relationship and the fellows who experienced this learned about developing working relationships with ethicists whose priorities differed. For example, when the hospital ethicists also had responsibilities as physicians or nurses in addition to clinical ethics responsibilities, the perspectives could differ on which activities receive attention first. Therefore, it is essential that support be made available in the form of advocacy and mediation for the fellows should such a conflict arise. In this program, such support is available through the program's coordinator at the Joint Centre for Bioethics.
Conclusions
Not unlike the field of bioethics itself, the Joint Centre for Bioethics Clinical Ethics Fellowship program is evolving with each successive year and will ultimately be judged by how well graduates are integrated into the healthcare community and the contributions they make to the field. The fellows concur that none of them would have felt sufficiently prepared to take on the considerable responsibilities, complex role demands and inevitable moral distress that are inherent in the position of clinical ethicist without the fellowship. Participation in the fellowship was instrumental in helping the fellows develop the necessary clinical ethics skills, knowledge and character traits required for them to assume a role as a clinical ethicist in a healthcare setting. As well, through the fellowship, they cultivated a support network for the future.
Since completing the fellowship, each of the first four fellows has obtained a position as a clinical ethicist in a healthcare setting. Because of their fellowship experiences, they embark on their new careers with a realistic picture of clinical ethics, demonstrated core competencies and a strong network of ethics support and expertise to draw upon in the future. Although other educational models for clinical ethicists exist, a clinical ethics fellowship that is applicable to individuals from a variety of backgrounds (i.e., not limited to clinicians or philosophers only) appears to be a viable educational option and one that ought to be further developed and more formally evaluated.
List of abbreviations used
JCB – The University of Toronto Joint Centre for Bioethics
SARS – Severe Acute Respiratory Syndrome
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
PC contributed substantially to the conception and design, analysis and interpretation of data, drafting of the article and revising it critically and gave final approval for the version to be published.
KF contributed substantially to the conception and design, analysis and interpretation of data, drafting of the article and revising it critically and gave final approval for the version to be published.
DG contributed substantially to the conception and design, analysis and interpretation of data, drafting of the article and revising it critically and gave final approval for the version to be published.
LH contributed substantially to the conception and design, analysis and interpretation of data, drafting of the article and revising it critically and gave final approval for the version to be published.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The authors wish to gratefully acknowledge Sue MacRae, Deputy Director, Joint Centre for Bioethics and Barbara Secker, Clinical Ethicist, Toronto Rehabilitation Institute for their editorial and substantive comments on an earlier draft of this paper.
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| 15533244 | PMC529455 | CC BY | 2021-01-04 16:31:54 | no | BMC Med Ethics. 2004 Nov 8; 5:6 | utf-8 | BMC Med Ethics | 2,004 | 10.1186/1472-6939-5-6 | oa_comm |
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BMC BiolBMC Biology1741-7007BioMed Central London 1741-7007-2-231552212310.1186/1741-7007-2-23Research ArticleAcetylation of insulin receptor substrate-1 is permissive for tyrosine phosphorylation Kaiser Christina [email protected] Stephen R [email protected] Section of Cell Biology, Department of Biology, Biovitrum AB, SE-112 76, Stockholm, Sweden2004 2 11 2004 2 23 23 7 7 2004 2 11 2004 Copyright © 2004 Kaiser and James; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Insulin receptor substrate (IRS) proteins are key moderators of insulin action. Their specific regulation determines downstream protein-protein interactions and confers specificity on growth factor signalling. Regulatory mechanisms that have been identified include phosphorylation of IRS proteins on tyrosine and serine residues and ubiquitination of lysine residues. This study investigated other potential molecular mechanisms of IRS-1 regulation.
Results
Using the sos recruitment yeast two-hybrid system we found that IRS-1 and histone deacetylase 2 (HDAC2) interact in the cytoplasmic compartment of yeast cells. The interaction mapped to the C-terminus of IRS-1 and was confirmed through co-immunoprecipitation in vitro of recombinant IRS-1 and HDAC2. HDAC2 bound to IRS-1 in mammalian cells treated with phorbol ester or after prolonged treatment with insulin/IGF-1 and also in the livers of ob/ob mice but not PTP1B knockout mice. Thus, the association occurs under conditions of compromised insulin signalling. We found that IRS-1 is an acetylated protein, of which the acetylation is increased by treatment of cells with Trichostatin A (TSA), an inhibitor of HDAC activity. TSA-induced increases in acetylation of IRS-1 were concomitant with increases in tyrosine phosphorylation in response to insulin. These effects were confirmed using RNA interference against HDAC2, indicating that HDAC2 specifically prevents phosphorylation of IRS-1 by the insulin receptor.
Conclusions
Our results show that IRS-1 is an acetylated protein, a post-translational modification that has not been previously described. Acetylation of IRS-1 is permissive for tyrosine phosphorylation and facilitates insulin-stimulated signal transduction. Specific inhibition of HDAC2 may increase insulin sensitivity in otherwise insulin resistant conditions.
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Background
The insulin receptor substrate (IRS) proteins represent key elements in insulin and insulin-like growth factor (IGF) actions, transducing pleiotropic effects on cellular function and regulating processes such as metabolism, growth, cell differentiation and survival [1]. At least four members (IRS 1–4) have been identified that differ with regard to tissue distribution, subcellular localization, developmental expression, binding to the insulin receptor, and interaction with Src homology 2 (SH2) domains. They are all structurally characterised by N-terminal pleckstrin-homology (PH) and phosphotyrosine-binding (PTB) domains, which are required for coupling to the activated insulin/IGF receptors, and a C-terminal region with multiple sites for tyrosine phosphorylation by the receptors. IRS proteins thus act as molecular adapters in recruiting, inter alia, a number of SH2-containing proteins binding to specific phosphorylated tyrosine residues. This leads to activation of different intracellular cascades [2], one of which is the PI 3-kinase signalling cascade implicated in mediating the metabolic effects of insulin [3].
The best-substantiated post-translational modification of IRS proteins, in addition to tyrosine phosphorylation, is phosphorylation of specific serine residues. Phosphorylation on these residues is associated both with inhibition of insulin-induced tyrosine phosphorylation of IRS proteins and with facilitation of the effects of insulin [4]. Phosphorylation catalysed by protein kinase C (PKC) isozymes [5,6], c-Jun N-terminal kinase (JNK) [7], inhibitor κB kinase (IKK) isozymes [8], mitogen activated protein kinases (MAPK) [9] and the mammalian target of rapamycin (mTOR) [10] are all associated with reducing the ability of insulin to stimulate tyrosine phosphorylation of IRS proteins and therefore may be part of the physiological and pathophysiological negative regulation of insulin signalling through the IRS pathway. Specific mechanisms explaining why serine phosphorylation leads to reduced tyrosine phosphorylation have not been completely identified, but candidates for this are reduced interaction of IRS proteins with the insulin receptor [11] and increased degradation of IRS [12,13]. Furthermore, phosphorylation of different residues can lead to different effects. Thus, phosphorylation of serine 307 in rat IRS-1 (serine 312 in human IRS-1) is associated with reduced insulin signalling [7] whilst phosphorylation of serine 302 has recently been suggested to facilitate insulin signalling [14], although this has been contested [15].
In addition to phosphorylation of different amino acid residues, insulin signalling through IRS proteins has been shown to be regulated by at least two other mechanisms. Prolonged signal transduction via phosphoinositide 3-kinase (PI3K), which generates the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate, has been shown to induce a state of insulin resistance in cells [16], in part through degradation of IRS-1 [17]. Thus, insulin signalling can be negatively regulated through modulation of IRS concentrations in cells, via degradation of the proteins in the proteasomal pathway [18-20]. The mechanism by which IRS proteins are degraded by the proteasome is not completely understood, but the N-terminal PH and PTB domains are required [21]. In addition, the sub-cellular localisation of IRS proteins may be important for appropriate insulin signalling. The sub-cellular localisation is not absolutely defined, with various lines of evidence pointing to potential places in the cell where the proteins can be found. In addition to the plasma membrane, IRS proteins have been associated with high-density pellets [22] implicating association with the cytoskeleton and recently also with the nucleus [23,24]. Thus, IRS proteins may be located to different parts of the cell where they carry out different functions.
Multiple histone acetyltransferases (HATs) and histone deacetylases (HDACs) control the state of histone acetylation and hence play a regulatory role in modulating the structure and function of chromatin [25]. About 20 HATs have been detected to date, grouped in three different classes on the basis of structural properties. They all have one structural motif in common, the so-called A-motif responsible for acetyl CoA recognition [25]. Several HATs also have non-histone substrates but it is not yet possible to identify putative acetylation sites within a protein simply by sequence analysis. Generally, acetylation affects DNA-binding, protein-protein interactions, protein stability, and protein localization [26]. The acetyl-mediated signals are reversed by HDACs that counteract the effects of HATs by deacetylating lysine residues on histone tails. In higher eukaryotes, HDACs can be subdivided into three distinct groups known as classes I, II, III, according to similarities of their sequences to those of yeast founding members [27]. To date, four enzymes, HDAC1, 2, 3 and 8, are the known members of class I deacetylases [28,29]. HDAC1 and 2 are the best characterised, and are chief constituents of the multiprotein transcriptional-repression complex Sin3/HDAC and the nucleosome remodelling deacetylase NuRD/Mi2/NRD complex [30]. Complexes that contain class I HDACs bind to numerous transcription factors, either directly, or indirectly through the nuclear-hormone corepressors NCOR and SMRT (silencing mediator for retinoid and thyroid hormone receptors). Although all class I and II HDACs can deacetylate histone tails, other cellular proteins can be specifically targeted by different HDACs as well, such as α-tubulin and importin-α [31]. Recent developments have shown that the class I enzymes are regulated by phosphorylation, by casein kinase II amongst others, which increases activity [32-34]. The fact that class II enzymes are phosphorylated has been known for longer, a reaction which is associated with re-localization of the enzymes to the cytoplasm through interactions with 14-3-3 proteins [35].
We now demonstrate that HDAC2 interacts with IRS-1 under conditions when the ability of cells to respond to insulin is compromised. As such, this interaction may constitute a new component of the negative regulation of IRS protein function. We also show that IRS-1 is acetylated, and that augmenting the acetylation level by treating cells with Trichostatin A (TSA, a non-specific inhibitor of HDACs) or with short inhibitory RNA oligonucleotides against HDAC2 partially restores normal responsiveness to insulin.
Results and discussion
Interaction between IRS-1 and HDAC2
In an attempt to elucidate the regulation of IRS-1, we investigated inter-molecular interactions between IRS-1 and potential binding partners using yeast two-hybrid screening through a human foetal brain plasmid cDNA library. The system we used was the sos recruitment system as described in the Methods section, which displays protein-protein interactions in the cytoplasm of yeast cells. In these experiments, full length human IRS-1 was used as bait. Two independent transformants from a screen of 4 × 105 cDNAs encoded the N-terminal portion of a 488 amino acid protein identified as histone deacetylase 2 (HDAC2, Figure 1A).
To map the interaction site of HDAC2 on IRS-1 we used a GAL4-based yeast two-hybrid system, where interactions take place in the nucleus of the yeast cell. Cells were transformed with vectors encoding full length HDAC2 and different truncation mutants of IRS-1. The truncations of IRS-1 that were used were the PH domain (residues 1–155), the PH-PTB domains (residues 1–578) and the PH-PTB-pre-C-terminal domains (residues 1–895). Using growth of yeast cells on selective medium as a readout for interaction between HDAC2 and IRS-1 showed that the interaction requires the C-terminal portion of the IRS-1 protein (Figure 1B). In order to confirm the interaction further in vitro, we used a coupled in vitro transcription/translation system in which full length IRS-1 and the HDAC2 N-terminal portion from the initial yeast two-hybrid screen were transcribed and translated in the presence of S35 methionine. IRS-1 was subsequently immunoprecipitated from the mixture and the proteins were resolved by SDS-PAGE. Gels were then subjected to autoradiography. Results showed that two radioactive protein bands were visible in the IRS-1 immunoprecipitates (Figure 1C) and their molecular weights corresponded to those of full length IRS-1 (approx 160 kD) and truncated HDAC2 (approx 35 kD). When the IRS-1 antibody was boiled prior to immunoprecipitation (Figure 1C lane 2) or omitted (Figure 1C lane 3), no radioactive proteins were observed, indicating that the interaction between the two proteins is specific and not due to non-specific interactions with immunoglobulins or beads. Thus, IRS-1 and HDAC2 proteins are able to interact with each other in cell-free systems.
To validate the interaction between IRS-1 and HDAC2 further and to ascertain whether IRS-1 and HDAC2 are associated in mammalian systems, we chose to work with MCF-7 cells (a human breast adenocarcinoma cell line), with a high endogenous expression of IRS-1 [36]. The cells were stimulated with IGF-1 or PMA (phorbol myristic acid; a PKC activator known to inhibit growth factor signalling [37]) for different time periods. Immunoprecipitations with IRS-1 antibody revealed that HDAC2 was co-precipitated to a larger extent in PMA-treated cells (Figure 2A). In addition, the interaction between IRS-1 and HDAC2 was more visible in cells under prolonged stimulation with IGF-1. Similar results were obtained during prolonged stimulation with insulin. Considering that the ability of cells to respond to insulin and IGF-1 is reduced after prolonged ligand stimulation or PMA treatment, these data indicate that IRS-1 and HDAC2 associate when responsiveness is low and intracellular serine phosphorylation is increased. Indeed, analysis of serine phosphorylation of IRS-1 after treatment of cells with insulin or phorbol ester showed that PMA treatment caused a significant increase in phosphorylation of IRS1 on serine 312 (equivalent to serine 307 in rat IRS-1), which has been associated with reduced phosphorylation on tyrosine residues by the insulin receptor (Figure 2B lanes 1,2 and 4), whereas insulin stimulation had no effect. In these experiments, cells were stimulated with insulin for 10 minutes and responsiveness was subsequently analysed by measuring tyrosine phosphorylation of IRS-1 (see Figure 4 and discussion below). Responsiveness of the cells to insulin was compromised after PMA treatment, thus confirming the apparent association of IRS-1 with HDAC2 under conditions of reduced cellular sensitivity to insulin.
To assess whether the interaction measured between IRS-1 and HDAC2 in vitro as described above occurs in vivo, we prepared lysates of liver tissues prepared from different mouse lines. The ob/ob mouse, which lacks functional leptin, was chosen as an insulin resistant animal model, and C57/bl6 was used as its genotype control. A PTP1B knockout mouse [38] was used as an insulin-sensitised animal model and balb/cJJ was used as its genotype control. IRS-1 was immunoprecipitated from liver lysates and western blotted for co-immunoprecipitation of HDAC2. The data showed that whilst a clear interaction between IRS-1 and HDAC2 was seen in livers from ob/ob mice (Figure 2C), no interaction was evident in the C57/bl6 control. In contrast, no interaction was evident in livers of PTP1B knockout animals, whilst the balb/cJJ genotype control demonstrated a measurable interaction. Taken together with the in vitro data, these results showed that IRS-1 and HDAC2 are able to interact with each other in the cytoplasmic compartment of cells and that the interaction occurs under conditions of reduced insulin sensitivity, both in mammalian cells and in animals. The cytoplasmic location of the interaction is interesting in view of the fact that HDAC2 is considered to be largely a nuclear protein. In our work with cells and tissues, we have utilised lysis methods that are designed to retain nuclei intact and thereby minimise cross-contamination of compartments [39,40]. Whilst we have not formally excluded the possibility of contamination of cytoplasmic extracts with nuclear lysate, thereby leading to the presence of HDAC2, we feel that the body of evidence indicates that cytoplasmic HDAC2 is interacting with cytoplasmic IRS-1 in our experiments. The yeast two hybid "Sos recruitment system" is built on the rescue of cell growth through the interaction of proteins in the cytoplasm, which is how we detected this interaction. Interestingly, it has recently been shown that histone deacetylase 1, another class I histone deacetylase, which was considered to be exclusively nuclear, is present in a cytoplasmic protein complex by virtue of interaction with a cellular phosphatase complex [41].
Lysine acetylation of IRS-1 and insulin signal transduction
The finding that HDAC2 binds to IRS-1 indicated that IRS-1 might be an acetylated protein in which acetylation might be a regulated post-translational modification of the protein. Indeed, the acetyl transferase Tip60 has been reported to bind to the PH domain of IRS-1 [42], suggesting the IRS-1 could be acetylated and deacetylated under different conditions. The lysine-acetylation status of IRS-1 was assessed by western blotting of IRS1 immunoprecipitated from MCF-7 cells after different treatments, using an antibody specific for acetylated lysine. Trichostatin A (TSA), which is a non-selective inhibitor of both class I and class II HDACs [43], was used as a positive control. Basal acetylation of the IRS1 protein was evident in unstimulated cells (Figure 3). Stimulation of cells with IGF-1 did not alter the level of acetylation although the basal signal was low and small effects cannot therefore be ruled out. PMA was also ineffective in altering the basal degree of acetylation of IRS1 whereas treatment of cells with TSA caused a very large increase in signal (Fig. 3). Our data therefore show that IRS-1 protein is acetylated on lysine residues, and the acetylation increases when HDAC activity is generally inhibited. This represents a heretofore-undescribed post-translational modification of IRS1 in addition to tyrosine/serine phosphorylation and ubiquitination previously described. TSA treatment did not induce phosphorylation of IRS1 on serine 312 (Fig 2B lane 3), nor did it modify the increase in serine 312 phosphorylation in the presence of PMA (lanes 1 and 2).
The regulation and function of proteins such as sterol regulatory element binding protein 1c (SREBP1c) [44] and p53 [45] has been shown to be altered by changes in acetylation. The alterations in lysine acetylation in IRS-1 induced by TSA raised the possibility that insulin signal transduction may be altered in cells after treatment with this compound. To assess the effects of changes in IRS-1 acetylation on insulin signalling, MCF-7 cells were treated with PMA, TSA and insulin in different combinations and immunoprecipitated IRS-1 protein was immunoblotted for the presence of phosphotyrosine. PMA alone and in combination with TSA did not increase tyrosine phosphorylation of IRS1 above basal, as expected (Fig 4 lanes 4–6). Furthermore, the ability of insulin to induce tyrosine phosphorylation of IRS-1 was reduced by 60% in cells pre-treated with PMA (Fig 4 lane 3) consistent with a state of insulin unresponsiveness. However, pre-treatment with TSA in the presence of PMA reduced this unresponsiveness, increasing insulin-stimulated tyrosine phosphorylation to 70% of control (Fig 4 lane 2). Thus, increases in IRS1 acetylation via TSA-mediated HDAC inhibition were able to restore insulin signalling significantly. This restoration occurred without reducing PMA-induced serine 312 phosphorylation of IRS-1 (Fig 2B lane 2), indicating that acetylation of IRS1 overcomes the inhibitory effects of phosphorylation of serine 312.
To assess the relative roles of altered intracellular protein acetylation and binding of HDAC2 to IRS-1 on insulin signalling, we treated cells with the general HAT inhibitor, desulfo coenzyme A (DesCoA, [46]) and examined HDAC2-IRS-1 interactions and insulin-stimulated tyrosine phosphorylation of IRS-1. The data showed that treatment with DesCoA induced HDAC2 to bind to IRS-1 to a similar extent to phorbol ester, which was coincident with reduced insulin-stimulated tyrosine phosphorylation of IRS-1 (Figure 5). In these experiments, interactions between HDAC2 and IRS-1 were apparently weaker in cells treated in the presence of TSA. This is not a consistent phenomenon, and occurs to varying degrees in our experiments (unpublished data). However, TSA has been reported to break other cellular HDAC-phosphatase complexes [41], so the effect here on HDAC2 and IRS-1 is not unprecedented. Treatment of cells with PMA and DesCoA did not lead to significantly greater effects, indicating that the two compounds share a common mechanism of reducing insulin signalling. Thus, inhibition of intracellular lysine acetylation accompanied by interactions between IRS-1 and HDAC2 leads to compromised insulin signalling, which can be overcome by inhibition of HDAC activity. Casein kinase II is an enzyme that has been shown to regulate the ability of HDAC2 to form oligomeric complexes both positively and negatively [32]. Interestingly, treatment with an inhibitor of casein kinase II (5,6-dichloro-1-β -D-ribofuranosylbenzimidazole) did not induce binding between IRS-1 and HDAC2 (Kaiser & James, unpublished) and had no effect on insulin signalling.
To ascertain if more distal insulin signalling was also enhanced, we examined the activation of protein kinase B (PKB) by western blotting, using an antibody against PKB phosphorylated on serine 474; this phosphorylation is induced in a PI3K-sensitive manner resulting in enhanced protein kinase activity. The data showed that PMA treatment reduced the activation of PKB by 50% (Figure 6 lane 3), whereas with pre-treatment with TSA, the response was 80% of control (Figure 6 lane 5). Thus, TSA-mediated increases in lysine acetylation of IRS-1 led to virtual restoration of PKB activation by insulin in PMA-treated cells. Interestingly, the PKB response in the presence of TSA and PMA (but no insulin, Figure 6 lane 4) showed significantly higher basal activation of PKB than in unstimulated cells. We speculate that this is due to the recently described ability of HDAC inhibitors, including TSA, to activate PKB through an unknown mechanism [47]. Thus, the increased response of cells to insulin in the presence of TSA (Figure 6 lane 5) may represent a summation of the effects of TSA alone and insulin. One candidate mechanism for the reported activation of PKB by HDAC inhibition is via increased acetylation of IRS-1 leading to enhanced basal PI3K activity and enhanced PKB activity. We could not, however, detect increases in basal tyrosine phosphorylation of IRS-1 in the presence of TSA without insulin (Figure 4 lane 4), suggesting that, if this is indeed part of the mechanism of activation of PKB by HDAC inhibition, it is beyond the limits of detection. Such a possibility is not without precedent. We have previously reported the ability of a non-specific protein tyrosine phosphatase inhibitor to increase PI3K-dependent glucose transport in muscle cells in culture without being able to detect changes in basal tyrosine phosphorylation of the insulin receptor or IRS-1 [48]. Thus, it remains possible that HDAC inhibition by TSA leads to enhanced PKB phosphorylation through small changes in IRS-1 phosphorylation.
A major functional response downstream of the PI3K arm of insulin signal transduction is increased glucose transport mediated by the GLUT4 transporter. We sought to examine the effects of TSA on glucose transport in rat L6 myotubes to see if the enhanced insulin signalling mediated by TSA treatment of cells translated into increased glucose uptake. We found that treatment of L6 myotubes with PMA resulted in increased basal glucose transport and had no effect on insulin-stimulated glucose transport (Kaiser & James, unpublished). Such effects are in line with data presented for rat epitrochlearis muscle [49] and indicated that L6 cells do not exhibit a clear insulin-resistance phenotype after PMA treatment, at the level of glucose transport. We also have similar observations in the human neuroblastoma cell line SHSY-5Y, which demonstrates insulin-stimulated glucose uptake [50]. Phorbol ester treatment of these cells increased basal glucose transport but in contrast to data in L6 cells, also inhibited insulin-stimulated glucose transport (Kaiser and James, unpublished). We have therefore not been able to distinguish an effect of TSA on GLUT4-mediated glucose transport owing to the large PMA-stimulated increases in insulin-independent glucose transport (presumably mediated by GLUT1), and are at present analysing other cells for their response to phorbol ester treatment. Interestingly, Takigawa-Imamura et al. [51] recently showed that several HDAC inhibitors increase glucose transport in muscle cells in culture. Although the treatment regimens with these inhibitors in these experiments were chronic, the data show that inhibition of HDAC activity enhances glucose transport. Molecular mechanisms behind this effect could be several, including enhanced insulin signalling through increases in intracellular protein acetylation.
TSA is an efficacious inhibitor of all class I and class II HDAC enzymes, with a potency in the low nanomolar range. To ascertain whether specific inhibition of HDAC2 activity is able to enhance insulin signalling in otherwise non-permissive conditions (PMA treatment), we used RNA interference to reduce HDAC2 activity specifically. MCF-7 cells were transiently transfected with a 21 base RNA duplex oligonucleotide against HDAC2 which reduced the HDAC2 protein content of the cells by approximately 70% (Figure 7). This was associated with a greater than three-fold increase in lysine acetylation of IRS-1. Furthermore, insulin-stimulated tyrosine phosphorylation of IRS-1 was increased 1.5-fold in RNAi-treated cells (Figure 7). A second RNAi oligonucleotide against HDAC2 was found to be much less efficient in silencing, exerting no effect on HDAC2 expression at 25 nM (corresponding to 80 pmol, see Methods section). Control experiments with this oligonucleotide at concentrations when HDAC2 expression was unaffected, showed that insulin-stimulated tyrosine phosphorylation of IRS-1 was not affected (data not shown), indicating that specific reductions in HDAC2 after RNAi treatment were the main cause of enhanced insulin signalling and IRS-1 acetylation. These data showed that specific reductions in HDAC2 activity in MCF-7 cells induced similar changes in IRS-1 regulation as treatment with TSA and that HDAC2 is an integral component of phorbol ester-induced insulin unresponsiveness in cells. The increase in lysine acetylation and tyrosine phosphorylation was arguably not as marked in RNAi-treated cells as in cells treated with TSA. An interpretation of these data could be that other members of the HDAC family are also involved in the processes leading to insulin resistance. We have found that HDAC1 does co-immunoprecipitate with IRS-1 from MCF-7 cells but its regulation is different, with no significant changes in the association by prolonged insulin stimulation or by PMA treatment of the cells (Kaiser & James, unpublished) suggesting that whatever the involvement of other HDACs, HDAC2 is central to the observed changes in insulin signalling.
The data we present here imply that treatment of insulin-resistant or diabetic animals with inhibitors of HDAC2 should increase insulin responsiveness. We attempted to assess the effects of TSA on insulin sensitivity in ob/ob mice. The animals were divided into two groups: vehicle (DMSO) and TSA (0.1 mg/kg) and treated subcutaneously for three days. At the same time as drug injection, all food was withdrawn from the animals and 4 hours later, blood was collected from the tail vein for blood glucose and plasma insulin analysis. On the third day, an insulin tolerance test (ITT) was performed 4 hours after administration of the drug. After 24 hours, fasting blood glucose tended to be lower in treated animals than vehicle controls, but after three days no difference was evident. Furthermore, we were unable to detect a change in insulin sensitivity after drug treatment during the ITT on day 3 (Kaiser, Warpman & James, unpublished). In addition, no changes in lysine acetylation of IRS-1 were observed, indicating that the lack of effect on insulin sensitivity could be due to the inability of TSA to work through the molecular mechanism of increasing IRS-1 acetylation. TSA is rapidly metabolised by liver cells in culture in two stages, initially by reduction to the imide followed by demethylation, leading to inactive metabolites [52]. It is therefore probable that the compound was rapidly metabolised by hepatic phase I metabolic processes in these experiments so that it was unable to exert pharmacodynamic effects on the animals. The poor bioavailability of TSA [53] has led to its discontinuation as a clinical candidate for the treatment of human disease and the possibility of testing the insulin sensitizing effects of HDAC inhibition must await the availability of a drug with better pharmacokinetics. Furthermore, HDACs are not redundant, but have specific expression patterns and functions. Therefore, it is of great importance to develop specific HDAC-inhibitors to be able to assess their respective contributions to increases in insulin sensitivity in vivo.
The mechanism whereby lysine acetylation of IRS-1 leads to increased tyrosine phosphorylation by the insulin receptor is not known. Time course experiments, in which cells were stimulated with insulin for one to ten minutes, showed that the kinetics of IRS-1 phosphorylation were the same, irrespective of pre-treatment of cells with TSA (Kaiser & James, unpublished). However, the IRS-1 tyrosine phosphorylation signal was greater at all times in cells treated with TSA, suggesting that lysine acetylation of IRS-1 simply increases the amount of phosphorylated IRS-1. It has recently been shown that lysine acetylation protects the transcription factor SREBP1C from ubiquitination and degradation via the proteasomal pathway by competing for the same lysine residues. IRS-1 has also been shown to be degraded via ubiquitination and subsequent proteasomal degradation [21]. We investigated the influence of lysine acetylation of IRS-1 on ubiquitination by blotting immunoprecipitates of IRS-1 from cells for the presence of ubiquitin (Fig 8). The data showed that in the absence of PMA, IRS-1 was only slightly ubiquitinated, whereas in cells treated with PMA, this was markedly increased. The molecular mass of both bands of the IRS-1 doublet increased after PMA treatment, spanning 132 kD to 145 kD, presumably due to the addition of ubiquitin molecules. TSA did not influence PMA-induced ubiquitination of IRS-1. These data therefore indicate that increases in IRS-1 phosphorylation after its lysine acetylation are not the result of increasing the concentration of the protein by preventing its degradation. Interestingly, a protein called PH domain interacting protein (PHIP) was recently described that selectively binds in vitro and constitutively associates in cells to the PH-domain of IRS-1 [54]. PHIP is not itself a substrate of the insulin receptor but rather a ligand of the IRS-1 PH-domain that serves to link IRS-1 to the insulin receptor and enhance its phosphorylation. PHIP contains two bromodomains located in tandem in the centre of the molecule [55]. Considering the fact that IRS-1 is acetylated and that bromodomains can interact specifically with acetylated lysine [56], the mode of interaction between PHIP and IRS-1 could be through the bromodomains, providing a molecular mechanism that explains why the increased acetylation of IRS-1, after TSA treatment, is accompanied by a higher level of tyrosine phosphorylation of IRS-1 despite the insulin resistant state. We have sought to test this hypothesis by blotting immunoprecipitates of IRS-1 for PHIP but have been unable to distinguish a specific band corresponding to PHIP using the antibodies that are available commercially.
Conclusions
In this study, we have identified a previously undescribed interaction between IRS-1 and HDAC2 in the cytosolic compartment of cells. The interaction is observed both in vitro and in vivo during conditions of compromised insulin signalling, as seen by reductions in insulin-stimulated IRS-1 tyrosine phosphorylation and PKB activation and increased phosphorylation of the negative regulatory phosphorylation site, serine 312. Our data indicate that it is the interaction with HDAC2 itself rather than its catalytic activity that is integral to the insulin unresponsiveness that ensues. Furthermore, our data show that IRS-1 is a lysine-acetylated protein, a previously unidentified post-translational modification of IRS-1, and that increases in the level lysine acetylation of IRS-1 result in improved insulin signal transduction. Increases in IRS-1 acetylation can be achieved pharmacologically (with TSA) or by ablation of HDAC2 specifically by use of RNAi. Out data therefore indicate that a new dimension to the physiology and pathophysiology of insulin sensitivity and insulin resistance involves changes in the degree of lysine acetylation of IRS-1 and that specific small molecule inhibitors of HDAC2 activity could represent novel therapeutics for the treatment of diseases that centre around insulin resistance, such as type 2 diabetes and obesity.
Methods
Yeast two hybrid screening
The CytoTrap™ (Stratagene) yeast two-hybrid system was used to discover protein-protein interactions in the cytoplasm of yeast cells. Interactions were detected by recruitment to the cell membrane of the human Sos (hSos) gene product, which activates the Ras pathway. The yeast strain used (cdc25H) harbours a temperature sensitive mutation in the cdc25 gene, the yeast homologue for hSos, which means that the cells can grow at 25°C but not at 37°C unless rescued with a protein-protein interaction. A human foetal brain plasmid cDNA library (Stratagene), harboured in the pMyr vector (with a myristylation signal to direct and anchor proteins in the membrane), was used as "prey" and the sub-cloned full length IRS-1 gene in the pSos vector was used as "bait". When prey and bait proteins interact the hSos is brought into close proximity to Ras and subsequently the yeast survive and are selected by growth at 37°C. The IRS-1/HDAC2 interaction rescued growth at 37°C in this way. The corresponding pMyr yeast plasmid was isolated and co-transformed with the pSos bait construct to perform false positive tests.
HDAC2 was full length cloned using RACE cDNA obtained from human heart tissue together with gene specific primers and the Advantage 2 polymerase mix (Clontech). With the purpose of mapping the interaction site of HDAC2 on IRS-1 we used the Matchmaker 3 yeast two-hybrid system (Clontech). This is a GAL4-based two-hybrid system that provides a transcriptional assay for detecting specific protein-protein interactions in yeast. Two nutritional markers and one enzymatic reporter gene were used to detect interactions. Different domains of IRS-1 (PH domain, residues 1–155, the PH-PTB domains, residues 1–578 and the PH-PTB-pre-C-terminal domains, residues 1–895, obtained by PCR) were sub-cloned into a "bait" vector (pGBKT7), fused to the DNA-binding domain of GAL4. Full length HDAC2 was sub-cloned into the "prey" vector (pGADT7), fused to the activation domain of GAL4. Cell growth on medium lacking the two nutritional markers was used as a readout of the interaction between the predator and prey.
In vitro transcription-translation
In order to confirm the IRS-1/HDAC2 interaction in vitro, we used a coupled transcription/translation system (Promega) comprising a rabbit reticulocyte lysate solution with RNA polymerase, nucleotides, salts, a ribonucleoside inhibitor, and [35S]-methionine (Amersham Biosciences) to allow detection of translated proteins. Since the prey vector pMyr already contains a T7 promoter, this was used directly in the system. However, the bait vector pSos lacks a T7 promoter and thus the IRS-1 gene was subcloned into a T7-containing vector (pGBKT7; Clontech) to permit transcription. The individually transcribed and translated proteins were mixed and co-immunoprecipitated with anti-IRS-1 antibodies (Upstate Biotechnologies) and subsequently analysed by polyacrylamide gel electrophoresis (4–12%). The gel was dried analysed by phosphorimagery.
Cell culture
The human breast adenocarcinoma cell line MCF-7 was cultured in a mixed medium of Dulbecco's Modified Eagle Medium with nutrient mixture F12 (Invitrogen) lacking phenol red with 10% Foetal Bovine Serum (Gibco). At near confluency, cells were starved of serum for 16 h and subsequently treated with IGF-1, insulin, PMA (phorbol myristic acid; Sigma) or TSA (Trichostatin A; Sigma), or combinations thereof, for different lengths of time as indicated in individual figures. Cells were harvested in hypotonic cell lysis buffer comprising 20 mM Hepes, pH 7.6, 20% glycerol, 10 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% NP40, 25 mM NaF, 25 mM β-glycerophosphate, 1 mM DTT, 1 mM Na-orthovanadate and protease inhibitors.
Western blot assays
Cell lysates were cleared by centrifugation at 16000 g for 10 min at 4°C, and protein content was determined using the Bradford method (BioRad). For immunoprecipitations, matched amounts of protein were incubated with primary antibody (amount used as recommended by the manufacturer or empirically determined) for 2 h at 4°C followed by addition of 20μl of protein A/G agarose suspension (Santa Cruz) for 1 h at 4°C with rotating tube. After washing (3 times with high salt (500 mM NaCl) and twice with isotonic buffer), beads were heated with SDS-PAGE sample buffer for 10 minutes at 70°C and proteins were resolved by 4–12% gradient SDS-PAGE. After blotting, membranes were blocked in 5% non-fat dried milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h prior to addition of the primary antibody. After incubation with secondary horseradish peroxidase-conjugated antibody, protein bands were visualised using enhanced chemiluminescence (ECL-plus detection kit, Amersham Biosciences).
Antibodies used were anti-IRS-1 (Upstate, cat. no 06–248); anti-HDAC2 (Santa Cruz, cat. no. sc-9959 and sc-6296); anti-phosphotyrosine (Santa Cruz, cat. no. sc-7020); anti-acetyl lysine (Cell Signalling, cat. no. 9681); anti-ubiquitin (Santa Cruz, cat. no. sc-6085 and sc-9133); anti-phospho-serine 307 IRS-1 (Upstate, cat. no. 07–247), HRP-conjugated anti-mouse IgG (Amersham Biosciences, cat. no. NA931V); HRP-conjugated anti-goat IgG (Dako cat. no. PO449) and HRP-conjugated anti-rabbit IgG (Upstate, cat. no. 12–348).
RNA interference
Double stranded RNA duplexes corresponding to amino acids from the C-terminal part of human HDAC2 (5'CAGCUCAGCAACCCCUGAAtt3') were annealed and transfected into human MCF-7 cells (Lipofectamine 2000 from Invitrogen was used as transfection agent): The effect of RNAi on HDAC2 expression and on insulin dependent IRS-1 tyrosine phosphorylation was measured after 48 hours. A second oligonucleotide (5'GGAGCAAAGAAAGCUAGAAtt3') was found to be non-silencing at a dose of 80 pmol, in contrast to the silencing oligonucleotide above, and was used in control experiments showing that no effect on IRS-1 phosphorylation or acetylation was observed (data not shown).
Animal experiments
Male 8-week old ob/ob mice were obtained from Bomhultsgard, Denmark and housed according to standard procedures. C57/bl6 genotype control mice were obtained from Scanbur BK AB (Sollentuna, Sweden). PTP1B knockout animals on a balb/cJJ background were purchased from McGill University, Montreal, Canada. Balb/cJJ genotype controls were obtained from Scanbur BK AB. In our hands, balb/cJJ mice are generally a healthy mouse strain that breeds well. In side-by-side experiments, the mice are more insulin sensitive than C57/bl6 mice whilst being less insulin sensitive than the PTP1B knockout animals on the same genetic background. The animals are somewhat smaller than C57/bl6 mice and have a relatively high body fat content.
For compound treatment experiments and insulin tolerance tests, animals were divided into two groups: vehicle (1% DMSO sub-cutaneous injection (s.c.), n = 15 and TSA 0.1 mg/kg s.c., n = 15) and subsequently treated s.c. at 09.00 h for three days. At the same time as injection, all food was withdrawn from the animals. Four hours later, blood was collected from the tail vein for blood glucose and plasma insulin analysis. On the third day, an insulin tolerance test (ITT) was performed 4 h after administration of the drug. ITT: insulin (actrapid 0.5 U/kg) was administered i.p. Following insulin administration, blood samples were collected after 15, 30, 60, 120 and 180 min from the tail vein for glucose analysis. Animals were then sacrificed and livers were dissected and immediately frozen in liquid nitrogen and stored at -70°C. All experiments were performed in accordance with permission from the local Swedish ethics committee and the company Pharmacology ethics review team.
For western blotting of liver proteins, frozen liver was powdered finely under liquid nitrogen using a pestle and mortar pre-cooled to -70°C. Powdered liver (1 g) was homogenized at 4°C using a Polytron in 3 ml of homogenisation buffer (4 mM EDTA, 50 mM NaF pH 8.0, 1 mM Na-orthovanadate, 1μM okadaic acid, 0.1% (v/v) 2-mercaptoethanol, with protease inhibitor cocktail). The homogenates were centrifuged at 13000 × g for 10 minutes at 4°C and the supernatant removed and used immediately for Western blot analysis or snap frozen in aliquots at -70°C until needed.
List of abbreviations
DesCoA: desulfo coenzyme A
HAT: histone acetyl transferase
HDAC: histone deacetylase
IGF: insulin-like growth factor
IRS: insulin receptor substrate
PH: pleckstrin homology
PKB: protein kinase B
PI3K: phosphoinositide 3-kinase
PMA: Phorbol myristic acid
PTB: phosphotyrosine binding domain
TSA: Trichostatin A
Authors' contributions
CK carried out all of the experimental procedures reported in this study and drafted the manuscript. SRJ conceived of the study, participated in the design of all the experiments and coordinated the study.
Acknowledgements
We are grateful to Ullrika Warpman for performing the studies examining the effects of TSA in vivo and to Ing-Marie Renström for performing glucose uptake analyses.
Figures and Tables
Figure 1 Analysis of the interaction between IRS-1 and HDAC2 in vitro. A: A human foetal brain plasmid cDNA library contained in the p-myr vector was transformed into yeast cdc25 h cells containing full length IRS-1 in the pSos vector. Cell growth at the non-permissive temperature (37°C) on galactose medium (GAL 1 promoter in the library vector) was observed only in the presence of both IRS-1 and HDAC2 from the library from two independent transformants, which both encoded human HDAC2. B: Full length HDAC2 and different sub-cloned domains of IRS- 1 were used in the Matchmaker-3 yeast two-hybrid system to map the interaction between HDAC2 and IRS-1. Growth on medium lacking two nutritional markers was analysed to confirm interactions between predator and prey. C: Recombinant human IRS-1 and HDAC2 were transcribed and translated in vitro individually using a rabbit reticulocyte lysate as described in the Methods section. Proteins were then mixed and IRS-1 was immunoprecipitated from the solution (lane 1). As controls for the immunoprecipitation, the IRS-1 antibody was denatured by boiling prior to immunoprecipitation (lane 2) or all antibodies were omitted and beads alone were used (lane 3). Proteins were resolved by SDS-PAGE, the gel was dried and immunoprecipitated proteins were analysed by phosphorimagery. Protein bands matched IRS-1 (approx 160 kD) and truncated HDAC2 (approx. 35 kD).
Figure 2 Analysis of conditions for interaction between IRS-1 and HDAC2 in mammalian cells and tissues. A: MCF-7 human breast carcinoma cells were treated with 10 ng/ml IGF-1 ("I") or 10 ng/ml PMA for the indicated times. IRS-1 was immunoprecipitated from cell lysates and western blots were analysed for co-immunoprecipitation of HDAC2. The upper gel shows presence of HDAC2 whilst the lower gel is a loading control for IRS-1. The data are representative of multiple experiments. Similar data are obtained if IGF-1 is exchanged for 100 nM insulin. B: MCF-7 cells were treated PMA (10 ng/ml) or TSA (150 ng/ml) for 4 hours prior to stimulation with or without insulin (100 nM) for 10 minutes, lysis and immunoprecipitation of IRS-1. Western blots were probed for the presence of phospho-serine 312 in IRS-1 (upper gel) and IRS-1 (lower gel). The histogram shows the means ± range for results for serine 312 phosphorylation from two independent experiments, normalised to the maximum phosphorylation signal, which was seen in cells treated with PMA and TSA. In separate control experiments, we have seen that the phosphorylation of serine 312 in cells is driven by PMA and that TSA does not contribute to the effect (data not shown). C: Liver tissues from ob/ob mice, c57/bl6 mice, PTP1B knockout mice and balb/cjj mice were prepared as described in the Methods section and IRS-1 was immunoprecipitated. Western blots were analysed for the presence of HDAC2 (upper gel) or IRS-1 (lower gel). Each lane is from one mouse and is representative of at least two other animals per group.
Figure 3 IRS-1 is a lysine acetylated protein. MCF-7 cells were treated with IGF-1 (10 ng/ml) for the indicated times, PMA (10 ng/ml) for 4 or 6 hours as indicated or TSA (150 ng/ml) for 4 hours prior to lysis and immunoprecipitation of IRS-1 Western blots were probed for the presence of acetyl lysine. Densitometric analysis of acetylated IRS-1 was performed and is displayed in the histogram. This experiment has been repeated one other time although the effects of TSA on acetylation of IRS-1 have been observed in many other experiments.
Figure 4 Increases in IRS-1 lysine acetylation enhance insulin signalling to IRS-1. MCF-7 cells were treated with PMA and/or TSA for 4 hours (concentrations as in Figure 3) prior to stimulation with 100 nM insulin for 10 minutes. IRS-1 was immunoprecipitated and blotted for the presence of phosphotyrosine. The graph summarises results from 3 experiments (± SEM) normalised to the response to insulin stimulation alone.
Figure 5 Inhibition of HAT activity inhibits insulin signalling. MCF-7 cells were treated with PMA, TSA and DesCoA (10 nM) in the indicated combinations for four hours prior to stimulation with insulin for 10 minutes. IRS-1 was immunoprecipitated and western blotted for the presence of IRS-1 itself, HDAC2 and phosphotyrosine. The graph shows average data for phosphotyrosine from three independent experiments (± SEM) normalised to the response to insulin alone.
Figure 6 Distal insulin signalling is enhanced by increased IRS-1 lysine acetylation. MCF-7 cells were treated with PMA, TSA and insulin as for Figure 4 and lysates were western blotted for the presence of PKB phosphorylated on serine 474. The graph shows average data from three independent experiments (± SEM) normalised to the insulin response alone.
Figure 7 Specific inhibition of HDAC2 enhances insulin signalling. MCF-7 cells were transfected with short inhibitory RNA oligonucleotides (80 and 160 pmol as indicated) for four hours and cultured a further 48 hours prior to treatment with PMA (10 ng/ml for 4 hours) and stimulation with insulin (100 nM) for 10 minutes. IRS-1 was immunoprecipitated and western blotted for the presence of HDAC2, phosphotyrosine, acetyl lysine and IRS-1 itself. The graphs show average data (± SEM) for 3–5 independent experiments, normalised to the insulin response alone. P-Y: phosphotyrosine.
Figure 8 IRS-1 ubiquitination is not altered by acetylation. MCF-7 cells were treated with PMA, TSA and insulin as described for Figure 4 and immunoprecipitated IRS-1 was western blotted for the presence of ubiquitin (upper panel) and IRS-1 (lower panel). The molecular mass of the immunoprecipitated ubiquitinated protein and IRS-1 protein is indicated.
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| 15522123 | PMC529456 | CC BY | 2021-01-04 16:02:56 | no | BMC Biol. 2004 Nov 2; 2:23 | utf-8 | BMC Biol | 2,004 | 10.1186/1741-7007-2-23 | oa_comm |
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BMC MedBMC Medicine1741-7015BioMed Central London 1741-7015-2-351544778810.1186/1741-7015-2-35Research ArticleNeoadjuvant or adjuvant therapy for resectable esophageal cancer: a systematic review and meta-analysis Malthaner Richard A [email protected] Rebecca KS [email protected] R Bryan [email protected] Lisa [email protected] of the Gastrointestinal Cancer Disease Site Group of Cancer Care Ontario's Program in Evidence-based Care. [email protected] University of Western Ontario, London Health Sciences Centre Division of Thoracic Surgery and Surgical Oncology, London, Ontario, Canada2 Princess Margaret Hospital, University of Toronto, Toronto, Ontario, Canada3 Department of Clinical Epidemiology & Biostatistics, McMaster University, Hamilton, Ontario, Canada2004 24 9 2004 2 35 35 13 11 2003 24 9 2004 Copyright © 2004 Malthaner et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Carcinoma of the esophagus is an aggressive malignancy with an increasing incidence. Its virulence, in terms of symptoms and mortality, justifies a continued search for optimal therapy. The large and growing number of patients affected, the high mortality rates, the worldwide geographic variation in practice, and the large body of good quality research warrants a systematic review with meta-analysis.
Methods
A systematic review and meta-analysis investigating the impact of neoadjuvant or adjuvant therapy on resectable thoracic esophageal cancer to inform evidence-based practice was produced.
MEDLINE, CANCERLIT, Cochrane Library, EMBASE, and abstracts from the American Society of Clinical Oncology and the American Society for Therapeutic Radiology and Oncology were searched for trial reports.
Included were randomized trials or meta-analyses of neoadjuvant or adjuvant treatments compared with surgery alone or other treatments in patients with resectable thoracic esophageal cancer. Outcomes of interest were survival, adverse effects, and quality of life. Either one- or three-year mortality data were pooled and reported as relative risk ratios.
Results
Thirty-four randomized controlled trials and six meta-analyses were obtained and grouped into 13 basic treatment approaches.
Single randomized controlled trials detected no differences in mortality between treatments for the following comparisons:
- Preoperative radiotherapy versus postoperative radiotherapy.
- Preoperative and postoperative radiotherapy versus postoperative radiotherapy. Preoperative and postoperative radiotherapy was associated with a significantly higher mortality rate.
- Postoperative chemotherapy versus postoperative radiotherapy.
- Postoperative radiotherapy versus postoperative radiotherapy plus protein-bound polysaccharide versus chemoradiation versus chemoradiation plus protein-bound polysaccharide.
Pooling one-year mortality detected no statistically significant differences in mortality between treatments for the following comparisons:
- Preoperative radiotherapy compared with surgery alone (five randomized trials).
- Postoperative radiotherapy compared with surgery alone (five randomized trials).
- Preoperative chemotherapy versus surgery alone (six randomized trials).
- Preoperative and postoperative chemotherapy versus surgery alone (two randomized trials).
- Preoperative chemoradiation therapy versus surgery alone (six randomized trials).
Single randomized controlled trials detected differences in mortality between treatments for the following comparison:
- Preoperative hyperthermia and chemoradiotherapy versus preoperative chemoradiotherapy in favour of hyperthermia.
Pooling three-year mortality detected no statistically significant difference in mortality between treatments for the following comparison:
- Postoperative chemotherapy compared with surgery alone (two randomized trials).
Pooling three-year mortality detected statistically significant differences between treatments for the following comparisons:
- Preoperative chemoradiation therapy versus surgery alone (six randomized trials) in favour of preoperative chemoradiation with surgery.
- Preoperative chemotherapy compared with preoperative radiotherapy (one randomized trial) in favour of preoperative radiotherapy.
Conclusion
For adult patients with resectable thoracic esophageal cancer for whom surgery is considered appropriate, surgery alone (i.e., without neoadjuvant or adjuvant therapy) is recommended as the standard practice.
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Background
Carcinoma of the esophagus is an aggressive malignancy that continues to kill more than 90% of people with the disease within five years [1]. The incidence of adenocarcinoma of the esophagus is rising faster than any other malignancy [2]. In 2001, there were at least 1,450 deaths due to esophageal cancer in Canada and many more people suffered because of the disease [3]. Its virulence, in terms of symptoms and mortality, justifies a continued search for optimal therapy.
Surgical esophagectomy remains the preferred treatment for clinically localized thoracic esophageal carcinoma [1,4-6]. Two randomized trials comparing surgery alone to radiation alone found surgery to be the better treatment for resectable cancer [5,6]. Fok et al randomly assigned 39 patients to surgery and 35 patients to 45 to 53 Gy radiation over four to five weeks [5]. The median survival time and five-year survival rate for surgery were 21.6 months and 16%, respectively, compared with 8.2 months and 7% for radiation (p < 0.05). Badwe et al compared 47 surgical patients to 52 patients undergoing 50 Gy radiation in 28 fractions plus 15 Gy boost in 8 fractions or 15 Gy brachytherapy [6]. Overall survival was better with surgery (odds ratio [OR], 2.74; 95% confidence interval [CI], 1.51 to 4.98; log-rank p = 0.002). The swallowing status was better in the surgery arm at six months after treatment (p = 0.03). Survival data from these two trials were pooled. The pooled results favoured surgery alone. There was no statistical heterogeneity (X2 = 0.02, p = 0.9) and a 52% relative increase in the risk of death at three years with radiotherapy compared with surgery alone (relative risk ratio [RR], 1.52; 95% CI, 1.23 to 1.86; p = 0.0007).
The failure of surgery alone is attributed to the systemic nature of the disease at the time of presentation [7,8]. Early and effective systemic chemotherapy and local radiotherapy, directed at micro-metastases and added to surgical resection, could lead to increased survival. Many clinical trials have evaluated the role of adjuvant therapy, both preoperatively and postoperatively, with conflicting results. Patients with cervical esophageal cancer are generally treated with chemoradiation, either preoperatively or postoperatively, in an attempt to avoid a laryngoesophagectomy and preserve the larynx. Although the majority of studies have been performed in squamous cell carcinomas, adenocarcinomas were included in some studies, but a distinction between the two histological subtypes was not made in this guideline report because previous studies have not consistently found that they respond differently to chemotherapy or radiation [9-17].
The large and growing number of patients affected, the high mortality rates, the geographic variation in practice, and the large body of good quality research evidence warrants a systematic review with meta-analysis.
Methods
This systematic review was developed by the Practice Guidelines Initiative (PGI) of Cancer Care Ontario's Program in Evidence-based Care (PEBC). Evidence was selected and reviewed by two members of the PGI's Gastrointestinal Cancer Disease Site Group (DSG) and two methodologists. Members of the Gastrointestinal Cancer DSG disclosed potential conflict of interest information.
This systematic review is a convenient and up-to-date source of the best available evidence on neoadjuvant or adjuvant therapy for resectable esophageal cancer. The body of evidence in this systematic review is primarily comprised of mature randomized controlled trial data; it forms the basis of a clinical practice guideline developed by the Gastrointestinal Cancer DSG published elsewhere (18). This systematic review and companion practice guideline are intended to promote evidence-based practice in Ontario, Canada. The PGI is editorially independent of Cancer Care Ontario and the Ontario Ministry of Health and Long-Term Care.
Literature search strategy
The MEDLINE (1966 through October (week 2) 2003), CANCERLIT (1983 to October 2001), Cochrane Library (2003, Issue 3), and EMBASE (to week 40, 2003) databases were searched with no language restrictions. "Esophageal neoplasms" (Medical subject heading (MeSH)) was combined with "chemotherapy, adjuvant" (MeSH), "radiotherapy, adjuvant" (MeSH), "immunotherapy, adjuvant" (MeSH), and each of the following phrases used as text words: "preoperative", "neoadjuvant", "chemotherapy", "radiotherapy", "radiation therapy", "irradiation", "immunotherapy", "chemoradiotherapy", "chemoradiation", and "hyperthermia". These terms were then combined with the search terms for the following study designs or publication types: practice guidelines, meta-analyses, and randomized controlled trials. Additionally, the conference proceedings of the 1997 to 2003 annual meetings of the American Society of Clinical Oncology (ASCO) and the 1999 to 2002 annual meetings of the American Society for Therapeutic Radiology and Oncology (ASTRO) were searched for reports of new or ongoing trials. Relevant articles and abstracts were reviewed, and the reference lists from these sources were searched for additional trials. This formal search was supplemented with published abstracts from thoracic surgery and oncology conferences, conversations with colleagues and experts in the field, and a review of textbooks related to esophageal oncology.
Study selection criteria
Articles were included in this systematic review if they were fully published reports, abstracts, or meta-analyses of randomized controlled trials (RCT) of neoadjuvant or adjuvant treatments compared with surgery alone or surgery plus another treatment in patients with resectable and operable thoracic esophageal cancer. Data on survival had to be reported. Other outcomes of interest were adverse effects and quality of life. Reports of carcinomas located in the cervical esophagus were excluded.
Synthesizing the evidence
Because diverse treatment strategies were evaluated, the eligible studies were grouped into 13 basic treatment approaches (Table 1), and each group was examined separately. Pooling was conducted using one-year mortality data for all meta-analyses except for the comparison of post-operative chemotherapy versus surgery alone, which was pooled at three years. Any time point selected for meta-analyses must be clinically credible and relevant but not so far along the survival curve that wide confidence intervals result from fewer patients contributing to the estimate. Since time points prior to the median will generally ensure that there are sufficient data to be credible, median survival times, weighted by the size of the treatment arms, were calculated to determine the time point for each meta-analysis as recommended in the literature [19]. Studies that did not provide values for survival at the time of pooling were not included in each meta-analysis, although they were included in calculating the weighted median survival time, if values for median survival were provided. All pooling was performed with Review Manager 4.2.1, available through the Cochrane Collaboration [Review Manager (RevMan) [Computer program]. Version 4.2 for Windows. Oxford, England: The Cochrane Collaboration, 2003]. Pooled results were expressed as mortality RR with 95% CI using the random effects model. An RR less than 1.0 favours neoadjuvant or adjuvant treatment, and an RR greater than 1.0 favours surgery alone. All analyses were made based on the intent-to-treat principle, except where only evaluable patient data were available.
Potential sources of heterogeneity and sensitivity analysis
Heterogeneity of study results was assessed using a visual plot of the outcomes and by calculating the X2 (Chi-square) statistic using a planned cut-off for significance of p < 0.05. Potential sources of heterogeneity were postulated a priori and included study quality assessed with the Jadad scale [20] (>2 versus ≤2), full article publication versus abstract publication, squamous cell versus adenocarcinoma, type of chemotherapy (cisplatin-containing versus others), type of surgery (transthoracic versus transhiatal), and radiotherapy dose (BED>48 versus BED<48). To facilitate comparison across trials, radiotherapy dose was converted to biological equivalent dose (same as biological effective dose) using the equation BED = nd (1+d/α/β), where n = number of fractions, d = dose per fraction, and it is assumed that α/β = 10 for tumour effect. Due to limitations inherent with this model, no allowances can be made for any time gaps in split-course treatments. These factors were used to explore any significant heterogeneity of results across the trials. The robustness of our conclusions was examined through subsequent sensitivity analyses using these factors. The sensitivity analysis results are not detailed, as they would not change the conclusions.
Results
Literature search results
Thirty-four randomized controlled trials were obtained. Of these, 30 were fully published reports [5,21-25,27-34,36,37,39,40,42-48,52,56-60], and four were available in abstract form only [35,41,51,53]. The four-arm trials by Fok et al [5] and Nygaard et al [24] contributed to multiple comparisons. Additionally, six meta-analyses were obtained, five fully published [26,38,49,50,55] and one abstract [54]. Literature search results appear in Table 1.
Outcomes
Preoperative radiotherapy and surgery versus surgery alone
Six randomized trials of preoperative radiotherapy and surgery versus surgery alone are presented in Table 2[5,21-25]. The radiotherapy regimens varied, using low to moderate doses ranging from 20 Gy in 10 fractions to 53 Gy in 20 fractions. Treatment was delivered between one to four weeks prior to surgery. Quality-of-life assessments were not conducted in any of the six trials. The Gastrointestinal Cancer DSG pooled the five trials that reported one-year mortality data [5,21,22,24,25] (Figure 1). No statistically significant difference in the risk of mortality with preoperative radiotherapy at one year compared with surgery alone was detected (RR, 1.01; 95% CI, 0.88 to 1.16; p = 0.87). No statistical heterogeneity was detected (X2 = 3.61, p = 0.46).
A published meta-analysis [26] using updated individual patient data on 1147 patients from five trials [21-25] detected a hazard ratio for death of 0.89 (95% CI, 0.78 to 1.01; p = 0.062) for preoperative radiotherapy compared with surgery alone. This meta-analysis included additional patients from the study by Wang et al. [23] with no description of why these patients were excluded from the published report of the trial (a total of 418 patients from this study were included in the meta-analysis versus 206 included in the trial report). The trial by Fok et al. [5] was not included in the published meta-analysis.
Postoperative radiotherapy and surgery versus surgery alone
Five randomized trials of surgery and postoperative radiotherapy compared with surgery alone are presented in Table 3[5,27-29,47]. Although all studies specified the absence of distant metastases as an inclusion criterion, Zieren et al. [29] and Teniere et al. [28] included patients with celiac node involvement (M1 disease). Fok et al. [27] included patients with positive margins and "a high chance of residual tumour". In the trials by Fok et al., Zieren et al. and Xiao et al., radiotherapy was delivered within six weeks postoperatively, while the trial by Teniere et al. specified within three months. The radiotherapy doses were higher than in the preoperative series. Of note, Fok et al. employed hypofractionation schedules using three fractions per week and 3.5 Gy per fraction to total doses of 49 Gy for patients with negative margins and 52.5 Gy for those with positive margins.
Only Zieren et al. assessed quality of life. The results indicated more rapid recovery of quality of life with surgery alone compared with postoperative radiotherapy. Three trials [28,29,47] demonstrated no significant difference in survival while another [27] found significantly shorter survival with postoperative radiotherapy and surgery compared with surgery alone.
The Gastrointestinal Cancer DSG pooled the five trials that reported one-year mortality data [5,27-29,47] (Figure 2). No significant difference in the risk of mortality with postoperative radiotherapy and surgery at one year compared with surgery alone was detected (RR, 1.23; 95% CI, 0.95 to 1.59; p = 0.11). No significant statistical heterogeneity was detected (X2 = 7.53, p = 0.11). The rate of local recurrence with radiotherapy was lower in three of the trials [27,28,47], but two trials [27,28] noted this benefit was achieved at the expense of increased morbidity.
Preoperative radiotherapy versus postoperative radiotherapy
One randomized trial evaluated preoperative radiotherapy versus postoperative radiotherapy with curative esophagectomy as part of a four-arm study [5]. Patients in this trial, performed between 1968 and 1981, received from 24 to 53 Gy preoperatively (n = 40) or 45 to 53 Gy postoperatively (n = 42). The median survival was 11 months for both groups. No difference in the survival rate was detected, but there was increased morbidity with preoperative radiotherapy. Quality of life was not assessed in this trial.
Preoperative radiotherapy and postoperative radiotherapy versus postoperative radiotherapy alone
Iizuka et al. [30] reported a randomized trial of preoperative and postoperative radiotherapy versus postoperative radiotherapy alone in 364 Japanese patients. In an analysis of 207 eligible patients (157 patients were excluded because of the extent of disease or operative complications), preoperative and postoperative radiotherapy was associated with a significantly higher mortality rate compared with postoperative radiotherapy alone (median survival was 394 days versus 648 days; p = 0.0069). The major postoperative complications were pneumonia (13.5% versus 9.7%) and leakage (11.5% versus 9.7%).
Preoperative chemotherapy and surgery versus surgery alone
Seven randomized trials of preoperative chemotherapy and surgery versus surgery alone are presented in Table 4[24,32-35,37,48]. Of these seven RCTs, six were available as fully published reports, and one was available as an abstract only [35]. Quality of life was not assessed in any of the trials. Additionally, three meta-analyses were obtained [38,49,50].
The Gastrointestinal Cancer DSG pooled the available data on preoperative chemotherapy with surgery versus surgery alone [24,32-34,37,48] (Figure 3). No significant difference in the risk of mortality at one year was detected (RR, 1.00; 95% CI, 0.83 to 1.19; p = 0.98). No statistical heterogeneity was detected (X2 = 8.26, p = 0.14).
The first meta-analysis, by Bhansali et al. [38], pooled data from 12 randomized trials of chemotherapy in a variety of combinations with radiotherapy with and without surgery, and no benefit for cisplatin-based chemotherapy was detected (OR, 0.96; 95% CI, 0.75 to 1.22; p > 0.10). This published meta-analysis included only four of the eight trials of preoperative chemotherapy versus surgery alone [24,31-33]. Trials that did not meet the inclusion criteria for this systematic review, such as trials involving patients with inoperable esophageal cancer and trials of combined chemoradiotherapy versus radiotherapy alone in the non-surgical management of esophageal cancer, were included in the Bhansali et al. meta-analysis.
The second meta-analysis, by Urschel et al. [49], pooled data from 11 RCTs (a total of 1,976 patients). These 11 RCTs were graded for quality using the Jadad scale [20]. Pooling detected no statistically significant difference between combination preoperative chemotherapy with surgery over surgery alone for survival at either one year (OR 1.00; 95% CI 0.76–1.30; p = 0.98), two years (OR 0.88; 95% CI 0.62–1.24; p = 0.45), or three years (OR 0.77; 95% CI 0.37–1.59; p = 0.48).
The third meta-analysis [50] was a Cochrane Review which pooled 11 RCTs (a total of 2,051 patients). Survival RRs were calculated at one, two, three, four, and five years, but a statistically significant difference in survival favouring preoperative chemotherapy was detected only at five years (RR = 1.44, 95% CI; 1.05–1.97; p = 0.02).
Preoperative and postoperative chemotherapy and surgery versus surgery alone
Two randomized trials of preoperative and postoperative chemotherapy and surgery versus surgery alone [31,36] (Table 5) were examined. Neither Roth et al. [31] (using a now out-dated combination of cisplatin, vindesine, and bleomycin) nor the largest North American trial as reported by Kelsen et al. [36] (using cisplatin and 5-FU) detected a statistically significant difference in overall survival. The Gastrointestinal Cancer DSG pooled these two trials (Figure 4). No significant difference in the risk of mortality with preoperative and postoperative chemotherapy and surgery compared with surgery alone was detected (RR, 0.99; 95% CI, 0.81 to 1.21; p = 0.93). No statistical heterogeneity was detected (X2 = 0.65, p = 0.42).
Postoperative chemotherapy and surgery versus surgery alone
Three randomized trials of postoperative chemotherapy and surgery compared with surgery alone are presented in Table 6[39-41]. All three trials used cisplatin-based regimens. Pouliquen et al. [39] found no improvement in the survival rate with postoperative chemotherapy. The patients were stratified into two groups: complete resections with or without nodal involvement and, palliative resections for positive margins or metastatic disease. Only the completely resected group was included in our analysis. Ando et al. [40] resected early (T1b) carcinomas and did not find any improvement in survival. In another study, reported in abstract form, Ando et al. [41] also found no survival benefit for postoperative chemotherapy in localized squamous cell carcinoma of the thoracic esophagus. Pouliquen et al. assessed quality of life and found that the duration of improved dysphagia was similar for both groups.
The Gastrointestinal Cancer DSG pooled the three-year mortality data for two trials [39,40] (Figure 5). The trial by Ando et al. [41] could not be included in the pooled analysis because the abstract did not report three-year survival. No significant difference in the risk of mortality at three years for postoperative chemotherapy compared with surgery alone was detected (RR, 0.94; 95% CI, 0.74 to 1.18; p = 0.59). There was no significant statistical heterogeneity (X2 = 0.08, p = 0.77).
Preoperative chemotherapy and radiotherapy and surgery versus surgery alone
Eight randomized trials of combined modality neoadjuvant chemotherapy and radiotherapy are presented in Table 7[24,42-46,51,53]. A ninth trial obtained [52] provided updated five-year data for another report [44]. None of the trials reported data on quality of life. In contrast to the other trials, the study by Walsh et al. [44,52] reported a significant overall increase in three-year survival with combined preoperative chemoradiation but was closed prematurely following an interim analysis. This study was criticized for the lack of preoperative staging using CT scans, premature closure, and an unusually poor survival rate in the surgery-alone arm.
The Gastrointestinal Cancer DSG pooled the one-year mortality data for the six trials with data available at one year [24,42-46] (Figure 6). No significant difference in the risk of mortality at one year for preoperative chemoradiation and surgery compared to surgery alone was detected (RR, 0.89; 95% CI, 0.76 to 1.03; p = 0.12). No significant statistical heterogeneity was detected (X2 = 1.67, p = 0.89).
The first meta-analysis, an abstract report by Fiorica et al. [54], pooled six RCTs comparing preoperative chemoradiation and surgery versus surgery alone. A systematic review, restricted to trials that included only patients with resectable esophageal carcinoma with no metastatic disease, obtained six RCTs. A significant difference in three-year mortality favouring neoadjuvant therapy with surgery versus surgery alone was detected (OR 0.53; 95% CI 0.31–0.92; p = 0.025). A conclusion was made that neoadjuvant chemoradiation and surgery significantly improved three-year survival compared to surgery alone in patients with resectable esophageal cancer but acknowledged that the magnitude of the benefit was relatively small. The authors recommend that research to determine the criteria that would identify patients likely to benefit from neoadjuvant chemoradiation be undertaken.
The second meta-analysis, by Urschel et al. [55], pooled nine RCTs, eight of which were included in this practice guideline [24,42-46,51,52]. The RCTs were graded for quality using the Jadad scale. This meta-analysis did not find a statistically significant difference in mortality at one year (OR 0.79; 95% CI 0.59–1.06; p = 0.12) or at two years (OR 0.77; 95% CI 0.59–1.05; p = 0.10). However, as in the meta-analysis by Fiorica et al. [54], a statistically significant difference was found at three years in favour of preoperative chemoradiation (OR 0.66; 95% CI 0.47–0.92; p = 0.016). The authors noted that the three-year survival benefit was most pronounced when chemoradiation was given concurrently (OR 0.45; 95% CI 0.26–0.79; p = 0.005) as opposed to sequentially (OR 0.82; 95% CI 0.54–1.25; p = 0.36).
To compare the results between the two published meta-analyses [54,55] with the trials included in this systematic review, the Gastrointestinal Cancer DSG pooled the data comparing neoadjuvant chemoradiation with surgery versus surgery alone at three years and obtained similar results. A significant difference in the risk of mortality at three years favouring neoadjuvant chemoradiation with surgery versus surgery alone was detected (RR = 0.87; 95% CI 0.80–0.96; p = 0.004). No statistically significant heterogeneity was detected (X2 = 6.59, p = 0.25).
Postoperative chemotherapy and radiotherapy versus surgery alone
No randomized trials have evaluated postoperative chemotherapy combined with radiation versus surgery alone.
Postoperative chemotherapy versus postoperative radiotherapy
One randomized trial evaluated postoperative chemotherapy versus postoperative radiotherapy following curative esophagectomy [56]. Patients in this Japanese trial received cisplatin and vindesine (n = 126) or radiotherapy at a dose of 50 Gy (n = 127). The median survival was 38 months for both groups. No difference in survival was detected (52% for chemotherapy versus 51% for radiotherapy at three years; log-rank p = 0.806). There were significantly more cases of decreased white blood cell counts (12 versus 3 for grade 3–4; p = 0.026), elevated blood urea nitrogen (26 versus 11 for grade 1–2; p = 0.018) and elevated creatinine concentrations (27 versus 9 for grade 1–3; p = 0.006) among patients randomized to chemotherapy compared with radiotherapy. Quality of life was not assessed in this trial.
Preoperative chemotherapy versus preoperative radiotherapy
Two randomized trials evaluating preoperative chemotherapy compared with preoperative radiotherapy were reviewed [24,57]. Kelsen et al. [57] randomly assigned 96 patients to preoperative radiotherapy or chemotherapy. Postoperative crossover therapy (i.e., postoperative radiotherapy for those who received preoperative chemotherapy and vice versa) was given to patients who were found to have unresectable or locally advanced disease. Only 11 of 48 chemotherapy patients and 9 of 48 radiotherapy patients did not receive additional postoperative treatment. Overall median survival was similar in both groups (10.4 months for chemotherapy versus 12.4 months for radiotherapy; p = 0.61), but the crossover design precluded proper analysis. In the four-arm trial by Nygaard et al. [24], preoperative chemotherapy was compared with preoperative radiotherapy, and the results demonstrated a significant difference in survival favouring preoperative radiotherapy (21% versus 3% at three years; p = 0.01). However, when compared to surgery alone, there was no benefit to either preoperative radiation or chemotherapy. Neither trial report included data on quality of life.
Preoperative chemoradiation versus preoperative radiotherapy
One randomized trial evaluated the role of preoperative bleomycin in addition to radiotherapy [58]. Seventy patients received preoperative chemoradiation with bleomycin and 63 patients received preoperative radiotherapy alone. The results demonstrated no significant difference in survival between the two groups (median survival was 25 weeks versus 26 weeks; survival rate was 25% versus 19% at two years; p = 0.56). There was also no benefit for bleomycin in the palliation of dysphagia. Quality of life was not assessed in this trial.
Postoperative immunotherapy in combination with radiotherapy or chemoradiation
One Japanese trial evaluated protein-bound polysaccharide (PSK) as an adjunct to postoperative radiotherapy or chemoradiation in resected esophageal cancer [59]. This trial involved 174 patients who were randomly assigned to four treatment groups. The three-year survival rates for radiotherapy, radiotherapy + PSK, chemoradiotherapy, and chemoradiotherapy + PSK were 43.3%, 45.5%, 33.5%, and 44.3%, respectively. There was no significant difference in survival when radiotherapy and radiotherapy + PSK were compared, or when chemoradiotherapy and chemoradiotherapy + PSK were compared (log-rank p = 0.19 for chemoradiotherapy versus chemoradiotherapy + PSK). Some patients randomized to PSK experienced adverse effects, including mild nausea, erythema, liver dysfunction and leukopenia, but there were no reports of toxicity that were definitely attributed to PSK. There was no assessment of quality of life.
Preoperative hyperthermia in combination with chemoradiation
One Japanese randomized trial, reported by Kitamura et al. [60], evaluated preoperative hyperthermia and chemoradiotherapy and surgery (n = 32) versus preoperative chemoradiotherapy and surgery (n = 34). Median survival was 36 months and 20 months, respectively. The results showed a significant improvement in the survival rate (50.4% versus 24.2% at three years; p-value not reported) and local tumour control with hyperthermia compared with control. It was reported that both adjuvant treatments were well tolerated and resulted in no postoperative complications. Quality of life was not assessed.
Adverse effects
Adverse effects were inconsistently reported (Tables 2,3,4,5,6,7). Most patients experienced treatment-related adverse effects associated with radiotherapy or chemotherapy.
Discussion
Most trials excluded patients with cancers located in the cervical esophagus, and therefore the interpretation of this review is limited to tumours in the more distal two thirds.
The options for neoadjuvant or adjuvant therapy for resectable thoracic esophageal cancer are many. On reviewing the results of randomized trials and meta-analyses, the Gastrointestinal Cancer DSG did not recommend neoadjuvant or adjuvant therapy based on the following:
Preoperative radiotherapy does not improve survival compared with surgery alone. Postoperative radiotherapy may, in fact, be harmful [27].
Preoperative chemoradiation does not appear to improve survival compared to surgery alone. Although the pooled analysis shows that all six studies are in the direction favouring preoperative chemoradiation at one year, the pooled estimate did not achieve statistical significance. When examining the individual trial results, only the trial by Walsh et al. [44] detected a statistically significant survival benefit, but this trial has been criticized for its methodology. The most recent trials, conducted by Bosset et al. [45] and Urba et al. [46], have five-year data available, and neither detected a statistically significant difference in survival between preoperative chemoradiation and surgery alone.
Preoperative cisplatin-based chemotherapy does not appear to improve survival. Four of the seven trials [24,32-34] detected a significant survival advantage favouring preoperative cisplatin-based chemotherapy. Kok et al. [35] reported a survival advantage for chemotherapy but only reported median survival results in abstract form. The two largest trials produced conflicting results [36,37]. Kelsen et al. [36] detected no survival advantage, while the MRC OE02 trial [37] detected a significant survival advantage for preoperative chemotherapy at two years. Although all chemotherapy protocols were cisplatin-based, the varying dosages, the number of cycles completed, and the other agents used contributed to clinical heterogeneity.
The available evidence from three randomized trials does not support the use of postoperative chemotherapy over surgery alone [39-41].
Two novel approaches, immunotherapy and hyperthermia, were studied by two groups in Japan [59,60]. Ogoshi et al. [59] detected no significant survival benefit for patients treated with PSK versus without PSK. Although Kitamura et al. [60] found a significant improvement in survival and local control favouring preoperative chemoradiotherapy with hyperthermia versus without hyperthermia, the Gastrointestinal Cancer DSG felt the results should be interpreted with caution until further confirmatory trials are conducted.
Examination of the results of randomized trials, including the pooled analyses, fails to support the use of preoperative or postoperative adjuvant treatment of any type at this time for patients with resectable carcinoma of the thoracic esophagus. Overall, the evidence does not support the use of neoadjuvant or adjuvant therapy for patients with resectable cancer of the lower two-thirds of the esophagus. Surgical resection alone should remain the standard for localized thoracic esophageal cancer. Patient staging information can be found in Appendix 1 (Additional file 1).
Future trials should continue to assess multi-modality treatments for this patient population. For clinicians seeking guidance on treatments for patients with non-resectable esophageal cancer, the role of radiotherapy alone and chemoradiation alone without surgery is addressed in another clinical practice guideline by the Gastrointestinal Cancer DSG, Combined modality radiotherapy and chemotherapy in the non-surgical management of localized carcinoma of the esophagus [61].
List of abbreviations used
In order of appearance:
Gy, Gray; OR, odds ratio; DSG, Disease Site Group; RR, (relative) risk ratio; PGI, Practice Guidelines Initiative; PEBC, Program in Evidence-based Care; MeSH, Medical subject heading; ASCO, American Society of Clinical Oncologists; ASTRO, American Society for Therapeutic Radiology and Oncology; RCT, randomized controlled trial; CI, confidence interval; X2, Chi-square; BED, biological equivalent (or effective) dose; PSK, protein-bound polysaccharide; MRC, Medical Research Council; PGCC, Practice Guidelines Coordinating Committee.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
RM, RW and LZ performed the initial literature search and created the initial drafts of the systematic review with input from other members of the Gastrointestinal Cancer DSG. RM, RW and BR created the final draft of this systematic review. All statistical analysis was performed by BR in consultation with RM and RW. Creation of the submitted manuscript was performed by BR and RM.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Supplementary Material
Additional file 1
Click here for file
Acknowledgements
Additional members of Cancer Care Ontario's Program in Evidence-based Care Practice Guidelines Initiative's Gastrointestinal Cancer Disease Site Group include: O. Agboola MD, M. Citron, F.G. DeNardi MD, S. Fine MD, B. Fisher MD, C. Germond MD, D. Jonker MD, K. Khoo MD, W. Kocha MD, M. Lethbridge, W. Lofters MD, R. Malthaner MD, M. Moore MD and V. Tandan MD. Please see the Program in Evidence-based Care's (PEBC) web site for a complete list of current and past Disease Site Group members.
Figures and Tables
Figure 1 Meta-analysis examining preoperative radiotherapy and surgery compared to surgery alone: mortality at one year. Overall risk ratio = 1.01 (95% CI, 0.88 to 1.16; p = 0.87)
Figure 2 Meta-analysis examining postoperative radiotherapy and surgery compared to surgery alone: mortality at one year. Overall risk ratio = 1.23 (95% CI, 0.95 to 1.59; p = 0.11)
Figure 3 Meta-analysis examining preoperative chemotherapy and surgery compared to surgery alone: mortality at one year. Overall risk ratio = 1.00 (95% CI, 0.83 to 1.19; p = 0.98)
Figure 4 Meta-analysis examining preoperative and postoperative chemotherapy and surgery to surgery alone: mortality at one year. Overall risk ratio = 0.99 (95% CI, 0.81 to 1.21; p = 0.93)
Figure 5 Meta-analysis examining postoperative chemotherapy and surgery compared to surgery alone: mortality at three years. Overall risk ratio = 0.94 (95% CI, 0.74 to 1.18; p = 0.59)
Figure 6 Meta-analysis examining preoperative chemoradiation and surgery compared to surgery alone: mortality at one year. Overall risk ratio = 0.89 (95% CI, 0.76 to 1.03; p = 0.12)
Table 1 Studies included in this systematic review.
Treatment Approach Number of Trials Reference Numbers Summary of Results
Randomized Controlled Trials
Preoperative RT v. Surgery Alone 6 5*,21–25† Table 2
Postoperative RT v. Surgery Alone 4 5*,27–29,47 Table 3
Preoperative RT v. Postoperative RT 1 5* -
Preoperative RT + Postoperative RT v. Postoperative RT 1 30 -
Preoperative CT v. Surgery Alone 6 24†,32–35‡,37,48 Table 4
Preoperative + Postoperative CT v. Surgery Alone 2 31,36 Table 5
Postoperative CT v. Surgery Alone 3 39–41‡ Table 6
Preoperative CRT v. Surgery Alone 6 24†,42–46,51–53‡ Table 7
Postoperative CT v. Postoperative RT 1 56 -
Preoperative CT v. Preoperative RT 2 24†,57 -
Preoperative CRT v. Preoperative RT 1 58 -
Postoperative Immunotherapy with RT or CRT v. RT or CRT 1 59 -
Preoperative Hyperthermia with CRT v. preoperative CRT 1 60 -
Meta-analyses
Preoperative RT v. Surgery Alone 1 26 -
Preoperative CT v. Surgery Alone 2 38,49,50 -
Preoperative CRT v. Surgery Alone 2 54‡,55 -
Note: CT indicates chemotherapy; CRT, chemoradiation; RT, radiotherapy; v., versus.
* The four-arm trial by Fok et al [5] contributed to three comparisons.
† The four-arm trial by Nygaard et al [24] contributed to four comparisons.
‡ Reports published in abstract form only [35,41,51,53,54].
Table 2 Randomized trials of preoperative radiotherapy (RT) and surgery versus surgery alone.
Study, year [Reference] Participants Number of patients Interventions Median Survival (Months) Survival Rate (%) Adverse Effects (Number of Patients)
1 yr 2 yr 3 yr 4 yr 5 yr
Launois et al. 1981 [21] 124 patients March 1973-June 1976 France, single centre, squamous cell 67 64 – 90 Gy preop RT + esophagectomy 4.5 46 20 15 14 10 perioperative mortality was 23% in both groups.
versus versus versus versus
57 esophagectomy (left thoracotomy) 8.2 (mean) 50 35 25 20 12
p = NS, but NR
Gignoux et al. 1987 [22] 229 patients [dates not reported] EORTC, 8 centres, squamous cell, no cervical lesions, no previous cancer, no previous treatment. 115 33 Gy preop RT + esophagectomy 12.3 55 24 20 17 10 tracheosophageal fistula, 2; bleeding, 1; esophagitis, 1; respiratory deaths, 6
versus versus versus versus versus
114 esophagectomy 12 (mean) 57 30 14 11 9 respiratory deaths, 8
No difference in survival (p = 0.94), but RT may delay local recurrence
Wang et al. 1989 [23] 206 patients June 1977-May 1985 China, single centre histology not reported < 65 years age, < 8 cm length no metastases 104 40 Gy preop RT + esophagectomy NR - - - - 35 leaks, 1; perioperative deaths, 5
versus versus versus versus versus
102 esophagectomy NR - - - - 30 leaks, 5; perioperative deaths, 5
No difference in survival (p > 0.05).
Nygaard* 1992 [24] 108 patients Jan 1983-Jan 1988 Scandinavia, multi centre squamous cell < 75 years of age, Karnofsky score > 50, T1, T2, Nx, M0 > 21 cm from incisors 58 35 Gy preop RT + esophagectomy 10 44 25 21 - - respiratory, 5; leaks, 2; postoperative deaths, 4
versus versus versus versus versus
50 esophagectomy 7 34 13 9 - - respiratory, 5; leaks, 2; postoperative deaths, 5
No difference in survival (p = 0.08).
Arnott 1992 [25] 176 patients 1979–1983 Scotland, single centre < 80 years, squamous cell adenocarcinoma, distal 2/3 esophagus 90 20 Gy preop RT + esophagectomy 8 40 22 13 9 9 respiratory, 10; postoperative deaths, 10
versus versus versus versus versus
86 esophagectomy (left thoracoabdominal) 8 40 28 23 21 17 respiratory, 5; postoperative deaths, 8; surgical, 2
No difference in survival (p = 0.40).
Fok* 1994 [5] 79 patients 1968–1981 Hong Kong, single centre Squamous cell, middle 1/3 esophagus 40 24–53 Gy preop RT + esophagectomy 11 42 34 24 10 10 respiratory, 20; postoperative deaths, 12; leaks 11
versus versus versus versus versus
39 esophagectomy (right thoracotomy, left neck, and abdomen) 22 58 36 24 16 16 respiratory, 15; postoperative deaths, 3; leaks, 7
No difference in survival.
*Patients randomized to four groups; data shown are for radiotherapy + surgery versus surgery alone.
Note: EORTC, European Organization for Research and Treatment of Cancer.
Table 3 Randomized trials of surgery and postoperative radiotherapy (RT) versus surgery alone.
Study, year [Reference] Participants Number of patients Interventions Median Survival (Months) Survival Rate (%) Adverse Effects (Number of Patients)
1 yr 2 yr 3 yr 4 yr 5 yr
Fok et al. 1993 [27] 130 patients July 1986-Dec 1989 Hong Kong, single centre squamous cell adenocarcinoma excluded leaks, respiratory failure, poor performance, metastases 65 esophagectomy + 49–52.5 Gy postop RT 8.7 34 18 16 16 - gastritis, 6; ulcer, 17; tracheo-esophageal fistulae, 1; strictures, 6
versus versus versus versus versus
65 esophagectomy (Lewis-Tanner or transhiatal or sternal split) 15.2 65 25 21 16 - gastritis, 3; ulcer, 1; tracheo-esophageal fistulae, 0; strictures, 6
Shorter survival with RT (p = 0.02). Better local control with RT (p = 0.06) but with more complications.
Teniere et al. 1991 [28] 221 patients Dec 1979-Dec 1985 France, multi centre squamous cell distal 2/3 esophagus 102 esophagectomy + 45–55 Gy postop RT 18 68 50 27 24 21 minor, 18; major, 4; death, 1
versus versus versus versus versus
119 esophagectomy (transhiatal or right thoracotomy with stomach or colon interposition) 18 73 51 29 22 19 none reported
No difference in survival (p-value not reported). Local or regional recurrence was lower with RT (70% versus 85%, p-value not reported).
Fok* 1994 [5] 79 patients 1968–1981 Hong Kong, single centre Squamous cell middle 1/3 esophagus 42 esophagectomy (one or two stage) + 45–53 Gy postop RT 11 48 17 17 12 10 respiratory 25; postoperative deaths 3; leaks 11
versus versus versus versus versus
39 esophagectomy (right thoracotomy, left neck, and abdomen) 22 58 36 24 16 16 respiratory 15; postoperative deaths 3; leaks 7
No difference in survival.
Zieren et al. 1995 [29] 68 patients (did not accrue entire sample size 68/160) June 1988-Dec 1991 Germany, single centre squamous cell excluded cervical location, metastases, other cancers, previous treatment 33 esophagectomy + 55.8 Gy postop RT 14 57 29 22 - - tracheo-esophageal fistulae, 1; skin, 18; strictures, 2
versus versus versus versus versus
35 esophagectomy (transhiatal or right thoracotomy with stomach interposition) 13 53 31 20 - - strictures, 1
No difference in survival (p-value not reported).
Xiao et al. 2003 [47] 495 patients 220 Midplane dose of 50–60 Gy in 25–30 fractions over 5–6 weeks NR - - - - 41 NR
versus versus versus versus
275 Surgery alone NR - - - - 32
p = 0.4474
Note: NR, not reported.
*Patients randomized to four groups; data shown are for surgery + radiotherapy versus surgery alone.
Table 4 Randomized trials of preoperative chemotherapy (CT) and surgery versus surgery alone.
Study, year [Reference] Participants Number of patients Interventions Median Survival (Months) Survival Rate (%) Adverse Effects (Number of Patients)
1 yr 2 yr 3 yr 4 yr 5 yr
Nygaard* et al. 1992 [24] 106 patients Jan 1983–Jan 1988 Scandinavia, multi centre squamous cell < 75 years of age Karnofsky score > 50 T1, T2, Nx, M0 > 21 cm from incisors 56 cisplatin 20 mg/m2 × 5 days × 2 cycles bleomycin 10 mg/m2 × 5 days × 2 cycles + esophagectomy 7 31 6 3 - - respiratory, 3; leaks, 3; postoperative deaths, 6; hematologic, 1; alopecia, 1
versus versus versus versus versus
50 esophagectomy (laparotomy and right thoracotomy) 7 34 13 9 - - respiratory, 5; leaks, 2; postoperative deaths, 5
No difference in survival (p-value not reported).
Schlag 1992 [32] 46 patients dates not reported Germany, single centre squamous cell < 68 years of age Karnofsky > 70 Stage I, II, III 22 cisplatin 20 mg/m2 × 5 days × 3 cycles 5-fluorouracil 1 g/m2 × 5 days × 3 cycles + esophagectomy 7.5 20 - - - - vomiting, 11; alopecia, 10; fever, 2; bone marrow suppression, 5; renal, 2;
versus versus versus versus versus
24 esophagectomy (abdominothoracic or thoracoabdominocervical with gastric or colon interposition) 5 32 - - - - not reported
No difference in survival (p = 0.91).
Maipang et al. 1994 [33] 46 patients Aug 1988–Dec 1990 Thailand, single centre squamous cell < 75 years of age ECOG 1, 2. Stage I, II, III distal 2/3 esophagus 24 cisplatin 100 mg/m2 × 1 day × 2 cycles vinblastine 3 mg/m2 × 4 days × 2 cycles bleomycin 10 mg/m2 × 5 days × 2 cycles + esophagectomy 17 58 31 31 - - hematologic, 15; vomiting, 15; alopecia, 14; hepatic, 3; lung, 1; urologic, 8; perioperative deaths, 4
versus versus versus versus versus
22 esophagectomy (laparotomy, right thoracotomy with gastric or colon interposition) 17 85 40 36 - - none reported
p = 0.186 Early survival better in surgery alone group.
Law et al. 1997 [34] 147 patients Dec 1989–Jan 1995 Hong Kong, single centre squamous cell exclude non regional nodes, tracheal involvement, metastases 74 cisplatin 100 mg/m2 × 1 day × 2 cycles 5-fluorouracil 500 mg/m2 × 5 days × 2 cycles + esophagectomy 16.8 60 44 38 28 28 Anemia, 47; neutropenia, 43; thrombocytopenia, 12; renal, 24; vomiting, 34; electrolytes, 21; leaks, 3; pulmonary, 10; respiratory failure, 14; perioperative deaths, 5
versus versus versus versus versus
73 esophagectomy (transhiatal or Lewis-Tanner) 13 50 31 14 14 - pulmonary, 11; respiratory failure, 22; perioperative deaths, 6
p = 0.17 Responders to CT lived longer but non-responders had lower median survival than controls (p = 0.03). Lower local recurrence with CT.
Kok et al. 1997 [35] [abstract] 160 patients 1990–1996 Netherlands, multi-centered Squamous cell 74 cisplatin 80 mg/m2 × 1 day × 2 cycles, etoposide 100 mg IV × 2 days + 200 mg/m2 PO × 2 days × 2 cycles + esophagectomy Note: CT responders received an additional 2 cycles of CT prior to surgery while non-responders received only 2 cycles 18.5 toxic deaths, 1; alopecia, 67; renal, 10
versus versus versus versus
74 esophagectomy (transhiatal). 11 none reported
Not reported but median survival favoured CT (p = 0.002).
MRC OE02 2002 [37] 802 patients Mar 1992 to June 1998 United Kingdom, multi-centered Resectable esophageal cancer 67% adenocarcinoma, 33% squamous or undifferentiated. 400 cisplatin 80 mg/m2 × 1 day × 2 cycles 5-fluorouracil 1 g/m2 × 4 days × 2 cycles + esophagectomy 16.8 59 43 35 28 26 postoperative complications, 41%; postoperative deaths, 10%
versus versus versus versus versus
402 esophagectomy 13.3 54 34 27 20 15 postoperative complications, 42%; postoperative deaths, 10%
Significant improvement in survival with chemotherapy HR = 0.79 (95% CI 0.67 to 0.93; p = 0.004)
Ancona et al. 2001 [48] 94 47 5-FU 1000 mg/m2 CI d1-5 + Cisplatin 100 mg/m2 d1 25 75 55 44 42 34 Gr. 3–4 neutropenia; 10 pts.
versus versus versus versus versus
47 Surgery alone 24 75 55 41 38 22 NR
Note: NR, not reported.
* Patients randomized to four groups; data shown are for chemotherapy + surgery versus surgery alone.
Table 5 Randomized trials of preoperative chemotherapy (CT) and postoperative chemotherapy (CT) versus surgery alone.
Study, year [Reference] Participants Number of patients Interventions Median Survival (Months) Survival Rate (%) Adverse Effects (Number of Patients)
1 yr 2 yr 3 yr 4 yr 5 yr
Roth et al. 1988 [31] 39 patients 19 cisplatin 120 mg/m2 × 1 day × 1 cycle vindesine 3 mg/m2 × 4 days × 2 cycles bleomycin 10 U/m2 × 4 days × 2 cycles + esophagectomy + cisplatin 120 mg/m2 q 6 wks × 6 months + vindesine 3 mg/m2 q 12 wks × 6 months 9 50 28 28 - - alopecia, 17; vomiting, 2; pneumonia, 1; sepsis, 1; neurological, 1; respiratory failure, 1; renal, 1; leaks, 1; chylothorax, 3; pulmonary embolus, 1; wound infection, 1
Nov 1982–May 1986 NCI, single centre squamous cell Stage I, II, III versus versus versus versus versus
20 esophagectomy (transthoracic with cervical or thoracic anastomosis) 9 35 15 8 - - leaks, 3; chylothorax, 1; pulmonary embolus, 1; pneumonia, 1; strictures, 1; empyema, 1; subphrenic abscess, 1
No difference in survival (p = 0.34). Survival advantage in responders and if less than 10% weight loss.
Kelsen et al. 1998 [36] 467 patients Aug 1990 to Dec 1995 North America, multi-centered Resectable esophageal cancer 55% adenocarcinoma 45% squamous cell 233 cisplatin 100 mg/m2 × 1 day × 3 cycles 5-fluorouracil 1 g/m2 × 5 days × 3 cycles + esophagectomy + cisplatin 75 mg/m2 × 1 day × 2 cycles if responded 14.9 59 35 23 19 18 minor, 49; major, 53; toxic deaths, 9; neutropenia, 68; mucositis, 58; postoperative deaths, 10
versus versus versus versus versus
234 esophagectomy 16.1 60 37 26 21 20 minor, 67; major, 57; postoperative deaths, 13
No survival difference.
Note: NCI, National Cancer Institute
Table 6 Randomized trials of surgery and postoperative chemotherapy (CT) versus surgery alone.
Study, year [Reference] Participants Number of patients Interventions Median Survival (Months) Survival Rate (%) Adverse Effects (Number of Patients)
1 yr 2 yr 3 yr 4 yr 5 yr
Pouliquen et al. 1996 [39] 120 patients total 62 had curative resections (no residual disease) France, 15 centres July 1987–Mar 1992 Excluded tracheal fistula, >30% liver metastases, brain metastases, node negative resections 24 esophagectomy + cisplatin 100 mg/m2 × 1 day × 6–8 cycles 5-fluorouracil 1000 mg/m2 × 5 days × 6–8 cycles 20 83 34 32 18 17 For 120 patients: tracheoesophageal fistulae, 9; sepsis, 5; infections, 11; pulmonary, 13; gastrointestinal, 26; neurologic, 9; neutropenia, 11; thrombocytopenia, 9; renal, 15; deaths, 4.
versus versus versus versus versus
38 esophagectomy 20 70 44 32 20 12 tracheoesophageal fistulae, 8; sepsis, 4; infections, 9; pulmonary, 12; gastrointestinal, 18; neurologic, 1; neutropenia, 3; thrombocytopenia, 5; renal, 1; no deaths.
This analysis based only on complete resections. No difference in survival (p-value not reported).
Ando et al. 1997 [40] 205 patients Japan, multicenter Dec 1988–July 1991 Resectable T1b, < 75 years 105 esophagectomy + cisplatin 70 mg/m2 × 1 day × 2 cycles vindesine 3 mg/m2 × 2 days × 2 cycles 57 90 67 58 58 48 anemia, 2; neutropenia, 13; vomiting, 13; renal, 8; diarrhea, 2; infection, 1.
versus versus versus versus versus
100 esophagectomy (laparotomy and right thoracotomy with 3 field radical lymphadenectomy with gastric or colon interposition). 47 90 67 54 48 45 none reported
No difference in survival (p = 0.60). Note: 36% unable to complete chemotherapy due to complications.
Ando et al. 1999 [41] [abstract] 242 patients 120 esophagectomy + cisplatin 80 mg/m2 × 2 cycles 5-fluorouracil (800 mg/m2 × 5 days × 2 cycles NR - - - - 51 Grade 3 or 4 hematologic or non-hematologic toxicities were limited in the chemotherapy group.
Japan, multicenter Jul 1992–Jan 1997 versus versus versus versus
122 esophagectomy NR - - - - 61
No difference in survival (p = 0.30)
Table 7 Randomized trials of preoperative chemoradiation (CRT) and surgery versus surgery alone.
Study, year [Reference] Participants Number of patients Interventions Median Survival (Months) Survival Rate (%) Adverse Effects (Number of Patients)
1 yr 2 yr 3 yr 4 yr 5 yr
Nygaard* et al. 1992 [24] 103 patients Jan 1983–Jan 1988 Scandinavia, multi centre squamous cell, < 75 years of age, Karnofsky score > 50, T1, T2, Nx, M0 > 21 cm from incisors 53 cisplatin 20 mg/m2 × 5 days × 2 cycles; bleomycin 5 mg/m2 × 5 days × 2 cycles + 35 Gy sequential radiotherapy + esophagectomy 7 39 23 17 - - leaks,2; respiratory, 10
versus versus versus versus versus
50 esophagectomy (laparotomy and right thoracotomy) 7 34 13 9 - - respiratory, 5; leaks, 2; postoperative deaths, 5.
No difference in survival (p = 0.30).
Le Prise et al. 1994 [42] 86 patients (stopped early after 104/150 patients entered) Jan 1988–April 1991 France, single centre squamous cell, < 70 years of age, < 15% weight loss excluded poor performance, metastases, tracheoesophageal fistula 41 cisplatin 100 mg/m2 × 1 day × 2 cycles 5-fluorouracil 600 mg/m2 × 4 days × 2 cycles + 20 Gy concurrent RT + esophagectomy 11 47 27 19 - - Neurological, 1; hematological, 7; renal, 2; tracheo-esophageal fistulae, 3; infections, 4; effusions, 2; deaths, 3; pulmonary embolism, 1; respiratory failure, 1.
versus versus versus versus versus
45 esophagectomy 11 47 33 14 - - tracheoesophageal fistulae, 5; infections, 7; effusions, 3; deaths, 3.
No difference in survival (p = 0.56 at one year).
Apinop et al. 1994 [43] 69 patients Thailand, single centre Jan 1986–Dec 1992 squamous cell carcinoma Mid to distal 1/3 esophagus, operable 35 cisplatin 100 mg/m2 × 1 day × 2 cycles 5-fluorouracil 1000 mg/m2 × 8 days × 2 cycles + 40 Gy concurrent radiotherapy + esophagectomy 9.7 49 30 26 24 24 leaks, 1; toxic deaths, 2; respiratory, 2; esophageal perforation, 1; cardiovascular, 2; electrolytes, 2
versus versus versus versus versus
34 esophagectomy (right thoracotomy) 7.4 39 23 20 19 10 leaks, 2; respiratory, 2; cardiovascular, 1
No overall survival difference (p = 0.40 for median survival). Responders had improved survival (p = 0.001).
Walsh et al. 1996 [44] 113 patients (closed early after 113/190 patients) May 1990–Sept 1995 Ireland, single centre adenocarcinoma < 76 years of age excluded poor performance, metastases, other cancers, previous chemotherapy or radiotherapy 58 cisplatin 75 mg/m2 × 1 day × 2 cycles; 5-fluorouracil 15 mg/kg × 5 days × 2 cycles + 40 Gy concurrent RT + esophagectomy 16 52 37 32 - - gastrointestinal, 4; hematologic, 2; cardiac, 15; toxic deaths, 1; respiratory, 28; leaks, 2; recurrent laryngeal nerve palsy, 1; chylothorax, 1
versus versus versus versus versus
55 esophagectomy (transhiatal, or Lewis-Tanner, or abdominal and left thoracotomy) 11 44 26 6 - - leaks, 2; recurrent laryngeal nerve palsy, 1; chylothorax, 1; respiratory, 32; cardiac, 13
Preoperative chemoradiation + surgery prolongs survival compared with surgery alone (p = 0.01). Inferior results in surgery alone arm.
Bosset et al. 1994 [45] 282 patients Jan 1989–June 1995 France, multi centre squamous cell < 70 years of age < 15% weight loss < WHO status 2 resectable Exclude tracheal fistula, T3N1, T4N0, T4N1 143 cisplatin 80 mg/m2 × 3 days × 2 cycles + 37 Gy concurrent radiotherapy + esophagectomy 18.6 69 48 39 35 33 vomiting, 37; neutropenia, 3; toxic deaths, 1; postoperative deaths, 17; respiratory failure, 6; sepsis,7
versus versus versus versus versus
139 esophagectomy (right thoracotomy + cervical anastomosis) 18.6 67 43 37 34 32 sepsis, 2; postoperative deaths, 5 Note: Trial stopped early 282/320 due to increased mortality in CRT group.
No difference in overall survival (p = 0.78).
Urba et al. 2001 [46] 100 patients 1989–1994 Michigan, single centre 25% squamous cell 75% adenocarcinoma 50 cisplatin 20 mg/m2 × 5 days × 2 cycles vinblastine 1 mg/m2 × 4 days × 2 cycles 5-fluorouracil 300 mg/m2 × 21 days + 45 Gy concurrent radiotherapy +esophagectomy 17.6 72 42 30 25 20 grade 3/4 granulocytopenia, 38; grade 3/4 thrombocytopenia, 15; neutropenic fever, 19; red blood cell transfusion, 8; feeding tube, 31; perioperative deaths, 1
versus versus versus versus versus
50 esophagectomy (transhiatal with cervical anastomosis) 16.9 58 38 16 14 10 perioperative deaths, 2; anastomotic leaks, 7 versus 5
No difference in overall survival (p = 0.15).
Burmeister et al. 2002 [51] 256 randomized 128† Cisplatin 80 mg/m2 d1 + 5-FU 800 mg/m2 d2-5 + RT 35 Gy in 15 fractions 22 NR NR NR NR NR
versus versus versus versus Treatment related mortality 4.6%
128† Surgery alone 19 NR NR NR NR NR
Lee J-L et al. 2003 [53] [abstract] 102 March 1999 – May 2002 Stage II/III resectable esophageal SCC 52 Cisplatin 60 mg/m2 IV d1, 5FU 1,000 mg/m2 IV d2-5, cisplatin 60 mg/m2 IV d22 + RT 45.6 Gy, 1.2 Gy bid d1-28 + surgery 3–4 weeks post RT 28.2 NR NR NR NR NR NR
versus versus versus versus versus
50 Surgery alone 27.3 NR NR NR NR NR NR
p = 0.67 p = NS
Note: NR, not reported; NS, not significant.
*Patients randomized to four groups; data shown are for chemotherapy + radiotherapy + surgery versus surgery alone.
† number of patients randomized into each treatment arm estimated from total number of patients.
==== Refs
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| 15447788 | PMC529457 | CC BY | 2021-01-04 16:03:34 | no | BMC Med. 2004 Sep 24; 2:35 | utf-8 | BMC Med | 2,004 | 10.1186/1741-7015-2-35 | oa_comm |
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Biomed Eng OnlineBioMedical Engineering OnLine1475-925XBioMed Central London 1475-925X-3-361549406810.1186/1475-925X-3-36Book ReviewReview of, "Biostatistics and Epidemiology" by S. Wassertheil-Smoller Schneck Daniel J [email protected] Virginia Tech, Blacksburg, Virginia 24061-0219, USA2004 19 10 2004 3 36 36 Wassertheil-Smoller S: Biostatistics and Epidemiology 3rd edition. Springer-Verlag; 2004. 243pp. Indexed. ISBN 0-387-40292-613 10 2004 19 10 2004 Copyright © 2004 Schneck; licensee BioMed Central Ltd.2004Schneck; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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If you need to know anything basic about Biostatistics and Epidemiology, this book belongs in your library! Unlike most science and technology books – which, I have concluded after nearly 42 years in this business, tend to be written less to teach and communicate information than to impress the reader with how much the author knows about the subject matter – this one, refreshingly and quite successfully, ensures that the reader comes away with a better understanding of the topics of Biostatistics and Epidemiology, as a result of the authors' genuine desire to convey information in a clear, concise, relevant, understandable, and very readable way.
In nine Chapters and nine Appendices, spanning 243 pages (which include ample references and suggested further readings), the indexed material covers a wide range of fundamental principles that form the basis for such subjects as: (1) The Scientific Method; (2) Probability Theory; (3) Statistics; (4) Epidemiology; (5) Screening Methods and Techniques; (6) Clinical Trials; (7) Quality of Life; (8) Genetics; and (9) Biomedical Ethics. A unique feature of the way the book is written, is that individual Chapters can be read out-of-sequence with no loss of continuity. The reader can skip around, omit certain sections, and still glean what he or she needs to know without feeling cheated. That's not easy to do, but this author does it well.
I found the material to be written at a very comfortable cognitive level – aimed mainly at medical, upper-level-college, and graduate students – and sequenced in a logical manner, thus making it easy to follow and totally user-friendly. The author has a special talent for reducing complicated concepts to an easily understandable level. For example the Appendix dealing with Genetic Principles gives the best introductory synopsis of this topic that I have seen anywhere; and the "middle Chapters" that address "Mostly About" – Statistics (Chapter 3), Epidemiology (Chapter 4), Screening (Chapter 5), Clinical Trials (Chapter 6), Quality of Life (Chapter 7), and Epidemiology (Chapter 8), are gems!.
What are especially nice are the many relevant, easy-to-understand and practical examples, that the author judiciously and effectively intersperses with the theoretical background material; and, the Chapter Summaries that conclude most of them. There is a very well-balanced give-and-take between theory and practice. I also liked the wonderful (sometimes witty) little parables and anecdotes that give the book a charming personality. In all, I would offer my compliments to the author for a "job well done!"
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Biomed Eng OnlineBioMedical Engineering OnLine1475-925XBioMed Central London 1475-925X-3-391550423510.1186/1475-925X-3-39ResearchMultimodal pressure-flow method to assess dynamics of cerebral autoregulation in stroke and hypertension Novak Vera [email protected] Albert CC [email protected] Lukas [email protected] Ary L [email protected] Lewis A [email protected] Chung-Kang [email protected] Division of Gerontology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA2 Margret and H. A. Rey Institute for Nonlinear Dynamics in Medicine and Cardiovascular Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA2004 25 10 2004 3 39 39 25 8 2004 25 10 2004 Copyright © 2004 Novak et al; licensee BioMed Central Ltd.2004Novak et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
This study evaluated the effects of stroke on regulation of cerebral blood flow in response to fluctuations in systemic blood pressure (BP). The autoregulatory dynamics are difficult to assess because of the nonstationarity and nonlinearity of the component signals.
Methods
We studied 15 normotensive, 20 hypertensive and 15 minor stroke subjects (48.0 ± 1.3 years). BP and blood flow velocities (BFV) from middle cerebral arteries (MCA) were measured during the Valsalva maneuver (VM) using transcranial Doppler ultrasound.
Results
A new technique, multimodal pressure-flow analysis (MMPF), was implemented to analyze these short, nonstationary signals. MMPF analysis decomposes complex BP and BFV signals into multiple empirical modes, representing their instantaneous frequency-amplitude modulation. The empirical mode corresponding to the VM BP profile was used to construct the continuous phase diagram and to identify the minimum and maximum values from the residual BP (BPR) and BFV (BFVR) signals. The BP-BFV phase shift was calculated as the difference between the phase corresponding to the BPR and BFVR minimum (maximum) values. BP-BFV phase shifts were significantly different between groups. In the normotensive group, the BFVR minimum and maximum preceded the BPR minimum and maximum, respectively, leading to large positive values of BP-BFV shifts.
Conclusion
In the stroke and hypertensive groups, the resulting BP-BFV phase shift was significantly smaller compared to the normotensive group. A standard autoregulation index did not differentiate the groups. The MMPF method enables evaluation of autoregulatory dynamics based on instantaneous BP-BFV phase analysis. Regulation of BP-BFV dynamics is altered with hypertension and after stroke, rendering blood flow dependent on blood pressure.
cerebral autoregulationHilbert-Huang transformnonlinear dynamicstime-frequency analysisstrokeValsalva maneuver
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Background
Noninvasive assessment of cerebral vasoregulation is a major challenge in medical diagnostics and post-stroke care. Dynamic autoregulatory mechanisms adapt cerebral perfusion to spontaneous variations in intracranial and systemic pressure within a few heartbeats. Decline of cerebral blood flow that occurs with normal aging is further potentiated by presence of risk factors for cerebrovascular disease such as hypertension. Cerebral autoregulation is damaged by acute stroke, rendering cerebral blood flow dependent on blood pressure (BP) [1-3]. The duration of post-stroke autoregulatory impairment and the degree of recovery after stroke are not known. Stroke is more common in older subjects, but the consequences of stroke in younger subjects may last for decades. It is not known if cerebral autoregulation is impaired in subjects with minor chronic infarcts and good neurological outcome. This study, employing a new signal analysis method, addresses the unresolved issue of whether the dynamics of cerebral autoregulation are altered in younger subjects with minor chronic stroke.
Continuous monitoring of BFV using transcranial Doppler ultrasound enables assessment of dynamic autoregulation from spontaneous BP and BFV fluctuations [4], and during interventions inducing a sudden BP reduction, such as the VM, thigh cuff deflation, and the sit-to-stand test [5-7]. The VM induces a sudden increase in intrathoracic and cerebrospinal fluid pressure that is associated with rapid declines in BP and BFV. The BFV response to intracranial pressure changes precedes peripheral BP responses. The end of straining is followed by a BP increase ≈30 mm Hg above baseline, associated with an increase in BFV and cerebrovascular resistance [8,9]. BP and BFV fluctuations evoked by the VM are transient and highly nonstationary. Rapid changes in vascular tone that act to adjust perfusion pressure during these fluctuations may yield a nonlinear pressure/flow relationship. These short and nonstationary time series present a methodological challenge since they are not suitable for analysis using traditional analytic techniques based on Fourier transform analysis [4,10] or Volterra-Wiener moving average modeling [11] which assume signal linearity and stationarity.
Accordingly, we: 1) introduce a new method, multimodal pressure flow analysis (MMPF), based on the Hilbert-Huang transformation [12], to quantify the relationships between two nonstationary signals; 2) apply the MMPF method to the assessment of dynamic autoregulation using the instantaneous BP-BFV phase relationships during the VM; 3) compare pressure/flow dynamics in younger subjects with a minor chronic stroke to that of normotensive and hypertensive subjects without stroke, and 4) compare the MMPF method with standard indices of autoregulation in the stroke and non-stroke groups.
Methods
Subjects
Studies were conducted at the Autonomic Nervous System Laboratory at the Department of Neurology at The Ohio State University and at the SAFE (Syncope and Falls in the Elderly) Laboratory at the Beth Israel Deaconess Medical Center at Harvard Medical School. All subjects signed informed consent, approved by the Institutional Review Boards. Subjects in the stroke and non-stroke groups were recruited from the Neurology Stroke Service and through advertisement. Demographic characteristics are summarized in Table 1.
Table 1 Demographic characteristics and baseline blood pressure and blood flow velocities in MCAs
Group Normotensive Hypertensive Stroke
Men/Women 9/6 8/12 5/10
Age (yrs) 40.2 ± 2.0 49.9 ± 2.0 53.1 ± 1.6**
Race W/AA 14/1 15/5 14/1
Stroke side, R/L -- -- 4/11
Baseline values
mean BP (mm Hg) 84.2 ± 2.2 102.1 ± 3.0 97.2 ± 2.4***
mean BFV MCAR (cm/s) 53.1 ± 3.4 60.7 ± 3.7 59.5 ± 4.3
mean BFV MCAL (cm/s) 53.0 ± 4.3 58.5 ± 3.5 57.2 ± 3.8
EtCO2 (mm Hg) 37.5 ± 1.9 35.4 ± 1.3 36.2 ± 1.1
Demographic characteristics, age and baseline values (mean ± SE, ** p < 0.003, p < 0.0001), gender and race (W = white, AA = African American) and the stroke side (R/L = right/left), BP = mean blood pressure, BFV = mean blood flow velocity in the right (MCAR) and left (MCAL) middle cerebral artery, EtCO2 = end tidal carbon dioxide
Normotensive group
15 healthy normotensive subjects (age 40.2 ± 2.0 years, mean ± SE).
Hypertensive group
20 patients with essential hypertension, controlled by antihypertensive medications (age 49.9 ± 2.0 years).
Stroke group
15 subjects with the first minor ischemic stroke (18.3 ± 4.5 months after acute onset) (age 53.1 ± 1.6 years) including 9 subjects treated for hypertension (stroke-hypertensive) and 6 subjects who were normotensive (stroke-normotensive) with no BP treatment. Stroke subjects had a documented infarct on MRI or CT affecting <1/3 of the vascular territory with a minor neurological deficit (Modified Rankin Score scale <3). The side of the lesion was determined by neurological evaluation and confirmed with MRI and CT. The lesion was in the right hemisphere in 4 and in the left hemisphere in 11 subjects. The infarct types and locations were as follows: small vessel infarcts in 13 subjects and large vessel infarcts in 2 subjects. Cortical infarcts were found in 10 subjects and subcortical infarcts in 5 subjects. Normal carotid Doppler ultrasound study was required for participation. Patients with hemorrhagic strokes, clinically important cardiac disease including major arrhythmias, diabetes and any other systemic illness were excluded. All subjects were carefully screened with a medical history, physical and laboratory examination. Subjects with hypertension (with or without stroke) were treated with antihypertensive agents from the following categories (diuretics, beta-adrenergic blockers and angiotensin-converting enzyme inhibitors). Antihypertensive medications were gradually tapered over 3 days and discontinued for 3 days prior to the study. Anticoagulants and other medications that did not affect cardiovascular or autonomic nervous system function were allowed.
Experimental protocol
VM
After instructions and several practice sessions, each subject rested for 5 minutes in the supine position. The subject was then asked to take a breath and expire forcefully through a mouthpiece with a small air-leak, maintaining for 15 seconds a pressure of 40 mm Hg monitored on a pressure gauge connected to the mouthpiece. All data were continuously acquired over the 5 minute period during which BP returned to baseline. The VM was repeated twice and the signal showing the more prominent VM oscillation by visual inspection was selected.
Data acquisition, processing and analysis
The experiments were done in the morning or > 2 hours after the last meal. The electrocardiogram was measured from a modified standard lead II or III using a Spacelab Monitor (SpaceLab Medical Inc., Issaquah, WA). Beat-to-beat BP was recorded from a finger with a Finapres device (Ohmeda Monitoring Systems, Englewood CO), which is based on a photoplethysmographic volume clamp method. During the study protocol, BP was verified by arterial tonometry. With finger position at the heart level and temperature kept constant, the Finapres device can reliably track intraarterial BP changes over prolonged periods of time [13]. Respiratory waveforms were measured with a nasal thermistor. CO2 was measured from a mask using an infrared end tidal volume CO2 monitor (Datex Ohmeda, Madison WI). The right and left MCAs were insonated from the temporal windows, by placing the 2-MHz probe in the temporal area above the zygomatic arch using a transcranial Doppler ultrasonography system (MultiDop X4, DWL Neuroscan Inc, Sterling, VA). Each probe was positioned to record the maximal BFV and fixed at a desired angle using a three-dimensional positioning system attached to the light-metal probe holder. Special attention was given to stabilize the probes, since their steady position is crucial for reliable, continuous BFV recordings. BFV and all cardiovascular analog signals were continuously acquired at 200 Hz and exported at 50 Hz for off-line post-processing. Data were visually inspected and occasional extrasystoles and outlier data points were removed using linear interpolation. Fourier transform of the Doppler shift (the difference between the frequency of the emitted signal and its echo (frequency of reflected signal) was used to calculate BFV. BFVs in the MCA correlate with invasive measurements of blood flow with xenon clearance [14], laser Doppler flux [15] and positron emission tomography [16]. Since MCA diameter is relatively constant under physiological conditions, BFV can be used for blood flow estimates [17].
Multimodal pressure-flow method
To quantify the dependency between cerebral blood flow and systemic pressure, we developed a novel computational procedure, called multimodal pressure-flow (MMPF) analysis. The MMPF analysis implemented the Hilbert-Huang transformation [12] technique to measure the coupling between two nonstationary signals. This method was motivated by the fact that the original BP and BFV signals are recorded over time. However, different subjects vary in the amount of time spent in each stage of the VM. Therefore, it is essential to find an alternative coordinate system for both BP and BFV signals that allows for a meaningful, non-time dependent cross referencing of these two signals. Since the complete VM cycle can be treated as a full cycle of BP oscillation, the oscillatory phase of the BP modulation during the VM can serve as such a useful coordinate system.
To implement this approach, we first need to calculate how BP phase changes as a function of time. Then we can map the original time-varying BP and BFV signals to the new axis of reference, namely BP oscillatory phase. To precisely calculate the BP phase, we need to extract the characteristic (dominant) BP oscillation induced by the VM. We applied the empirical mode decomposition (EMD) technique developed by Huang et al. [12] The EMD algorithm decomposes complex signals such as BP and BFV into multiple empirical modes. Each mode represents the frequency-amplitude modulation at a specific time scale. Figure 1A shows the original BP waveform, which is modulated by multiple frequencies corresponding to the systolic peak, dicrotic notch, heart rate, respiratory frequency and BP fluctuations induced by the VM. Figure 1B shows the decomposed empirical modes (1–10) of the original BP signal. Empirical modes 1–5, corresponding to the faster frequencies, were removed from the signal. The remaining lower frequency BP signal, termed the "residual BP" or BPR (shown as thick curve in Figure 1A), was used to identify the maximum and minimum values during the VM. The empirical mode best representing the dominant BP profile during the VM, denoted as BPVM (mode 6 in this example, plotted as the thick line in Figure 1B), was visually identified and used for subsequent phase analysis. We followed the same procedure to obtain the residual BFV signal, denoted as BFVR.
Figure 1 Schematic diagram showing Hilbert-Huang decomposition of the original blood pressure (BP) signal into the empirical modes corresponding to amplitude-frequency modulation for different time scales. Panel A shows the profile of the BP waveform over the course of the VM: I- indicates the beginning of the maneuver, II- the duration of straining, III-the end of straining and IV- the BP overshoot above baseline. (Note that the transient BP decrease in phase III is due to inspiration.) Panel B shows the empirical modes for each component frequencies and their corresponding amplitudes were detected from the signal (mode 1–10). Empirical modes corresponding to the faster frequencies (modes 1–5) were removed from the original BP and BFV signals. The empirical mode corresponding to the characteristic BP profile induced by the VM (BPVM – mode 6 in this example) was used to obtain phase information. Modes 7–10 reflect BP modulations at slow frequencies. Similarly, the empirical mode corresponding to the characteristic BFV profile was extracted from the raw BFV waveform (not shown).
In the next step of the MMPF analysis, we applied the Hilbert transform to the BPVM signal to calculate its instantaneous phases. This phase was then used as a reference coordinate both for BP and BFV signals. From this point on, the term phase refers to the phase of the BPVM oscillation during the VM. Unlike the Fourier transform, the Hilbert transform does not assume that signals are composed of superimposed sinusoidal oscillations of constant amplitude and frequency. Real-world biological fluctuations, such as BP and BFV, are not stationary and are better described by analytical methods that can quantify variations of amplitude and frequency.
Mathematically, the first two steps of the MMPF algorithm can be summarized in the following way: Any complex signal s(t) can be represented as the superimposition of more basic (simpler) components: S(t) = ∑k Sk (t), where Sk are empirical modes that fulfill certain criteria of the original signal [12]. For each empirical mode, its Hilbert transform is defined as:
where the Cauchy principal value is taken in the integral. Instantaneous amplitude, Ak (t), and instantaneous phase, φk (t), can be calculated by
The BPVM profile from the first peak at the beginning of the VM to the subsequent BPVM maximum forms a complete phase cycle from 0–360° over 30–40 seconds. We assigned the first peak at the beginning of the VM to phase 0°, the BPVM minimum during the VM to phase 180° and the subsequent BPVM maximum to phase 360°. To quantify the relationships between BP and BFV signals, we measured the BP-BFV phase shift, defined as the difference between the phase at the BPR minimum (maximum) value and the phase at the BFVR minimum (maximum) value. We have calculated phase values for all data points in this interval and, in principle, we can measure the phase shift at all point of the VM cycle. However, for simplicity, we only calculated the phase shift at the minimum and maximum of these two signals for statistical analysis. Since these BP-BFV phase shifts reflect dynamical changes in peripheral and cerebral vascular tone over the course of the VM, they can be used as a sensitive index of cerebral autoregulation dynamics in normal and pathological conditions.
Autoregulation indices
We also assessed autoregulation using a standard index, calculated using the second-order differential equation model proposed by Tiecks et al[5] This model assumes a linear flow-pressure relationship and a constant cerebral perfusion pressure over the course of a sudden BP reduction, such as occurs during thigh cuff deflation. This technique can be also applied to the sudden BP decline during the VM. The autoregulation index ranges from 0 = "no autoregulation" to 9 = "the fastest autoregulation;" value 5 reflects "normal autoregulation." We also calculated the "rate of autoregulation" (RoR) using the slope of the linear regression fitted to the original BP and BFV waveforms signals during the period between the baseline and the BP minimum (descending slope) and between BP minimum and maximum (ascending slope).
Statistical analysis
We used one-way analysis of variance for between-group comparisons of baseline, minimum and maximum BPR and BFVR values and phases. Two-way analysis of variance was used for side-to-side comparisons of BFV between groups (JMP-5.0 SAS Institute, Cary, NC). For the group comparisons, we used the BFVR in the right and left MCAs for the normotensive and hypertensive groups compared to the BFVR in the stroke-side and non-stroke side MCA in the stroke group. Age was different between the groups (p < 0.003). However, age and stroke subtypes had no significant effects when included as co-variants in the analysis. Data are presented as mean ± SE.
Results
Figure 2A shows representative raw BP and BFV waveforms (right and left MCAs) during the VM for a normotensive 31 year old man. The BPR and BFVR signals are superimposed on the raw waveforms. For comparison, we show similar signals (the right, non-stroke side, MCA and the left, stroke side, MCA) for a 48 year old woman with the left temporal stroke in Figure 2B. The BPR and BFVR for the right (non-stroke side) MCA and for the left (stroke side) MCA are shown in the bottom three panels as a function of the phase. With normal autoregulation, the BFVR changes precede BPR changes over the course of the VM. Therefore, the phase at the BFVR minimum is smaller than the phase at the BPR minimum. (Phase at the BFVR minimum for the right MCA = 129° and the left MCA = 112° vs. the phase at BPR minimum = 178°.) In contrast, with post-stroke cerebral autoregulation, the phase at the BFVR minimum is similar to the phase at the BPR minimum, suggesting that BFV is dependent on blood pressure. (Phase at the BFVR minimum for the non-stroke MCA = 173° and for the stroke MCA = 180° vs. the phase at BPR minimum = 185°).
Figure 2 Panel A shows blood pressure (BP) and blood flow velocity (BFV) waveforms from the right and left MCAs (MCAR and MCAL respectively) during the VM for a normotensive subject (top 3 panels). The duration of the VM straining is indicated by a horizontal line. The thick black line indicates the BPR and BFVR that reflect the characteristic VM oscillation. Bottom 3 panels show BPR and BFVR in the MCAR and MCAL. Arrows indicate phases at the BPR and BFVR minima. With normal autoregulation, BFVR minimum preceded BPR minimum. Panel B shows BP and BFV waveforms for a subject with a left temporal infarct (MCAR = non stroke-side MCA, MCAL = stroke side MCA) (top 3 panels). Horizontal line indicates duration of the VM. Black thick line indicates the BPR and BFVR obtained from the BP and BFV raw waveforms. Bottom 3 panels show BPR and BFVR in the non-stroke side MCA and in the stroke-side MCA expressed as a function of BPVM phase. Arrows indicate that the phase at BFV minimum was similar to the phase at BP minimum.
Pressure flow relationship at the BFVR minimum
Figure 3A shows the group averages of BFVR values and the phases at the BFVR minimum and maximum values for the right MCA in the normotensive and hypertensive groups and for the non-stroke side MCA in the stroke group. Figure 3B shows the BFVR values and the phases for the left MCA in the normotensive and hypertensive group, and the stroke side MCA in the stroke group. Mean BPR values and corresponding phases are shown in panel C. The phase at BFVR minimum was different between groups for the right (non-stroke side) (p = 0.0005) and left (stroke side) (p = 0.004) MCAs. In the normotensive group, the phase at BFVR minimum was shorter than the phase at BPR minimum. In the stroke group, the phase at BFVR minimum was similar to the phase at BPR minimum. The phase at BFVR minimum was greater in the non-stroke (p = 0.002) and stroke (p = 0.03) MCAs, compared to the normotensive group. In the hypertensive group, the phase at BFVR minimum was also greater for the right (p = 0.0007) and left (p = 0.006) MCAs compared to the normotensive group. No significant differences were found between the stroke and hypertensive groups.
The phase at BPR minimum was similar between groups (normotensive group = 172.7 ± 3.9°, hypertensive group = 178.6 ± 2.0°, stroke group = 178 ± 1.9°). Average BPR values were higher in the stroke and hypertensive groups compared to the normotensive group at baseline (p = 0.001), at BPR minimum (p = 0.054) and at BPR maximum (p = 0.0001) (Figure 3C). Average BFVR values at baseline, at BFVR minimum and maximum, and BFVR change from baseline were not different among groups. Average BPR change and percent change from baseline to BPR minimum and maximum were not different. Average BFVR change and percent change from baseline to BFV minimum and maximum were also not different.
Figure 3 Panel A shows the phase and corresponding residual blood flow velocity (BFVR) values at baseline, BFVR minimum and BFVR maximum for the right MCA for the normotensive -●- and - -▼- hypertensive groups and for - O- the non-stroke side MCA in the stroke group. Panel B shows the phase and corresponding BFVR values for the left MCA in the normotensive and hypertensive groups and for the stroke side MCA in the stroke group. BFVR phase was significantly greater in the stroke and hypertensive groups compared to the normotensive group for BFVR minimum and maximum in both MCAs (between groups phase comparisons *** p < 0.005, ** p < 0.01). Panel C shows the phase and corresponding residual blood pressure (BPR) values for the BPR minimum and maximum (between groups BPR values comparisons +++ p < 0.001, mean ± SE).
Pressure flow relationship at the BFVR maximum
In the normotensive group, the phase at BFVR maximum preceded the phase at BPR maximum (Figure 3). The phase at BFVR maximum was different between groups for the right (non-stroke side) (p = 0.009) and left (stroke side) (p = 0.003) MCAs. In the stroke group, the phase at BFVR maximum was similar to the phase at BPR maximum. The phase was greater for the non-stroke side (p = 0.008) and stroke side (p = 0.03) MCAs, compared to the normotensive group. In the hypertensive group, the phase at BFVR maximum was also greater for the right (p = 0.005) and left (p = 0.009) MCAs compared to the normotensive group. The phase at BPR maximum was similar among groups (normotensive group = 354 ± 4.7°, hypertensive group = 360.1 ± 2.2° stroke group = 364.1 ± 4.3°).
BP-BFV phase shifts
Figure 4A summarizes the differences between the phases at BPR and BFVR minimum and between the phases at BPR and BFVR maximum for the right MCA in normotensive and hypertensive groups and for the non-stroke side MCA in the stroke group. The BP-BFV phase shifts at the minimum points were smaller in the stroke and hypertensive groups compared to the normotensive group (p = 0.009). The BP-BFV phase shifts at the maximum points were smaller in stroke and hypertensive groups compared to the normotensive group (p = 0.03). Figure 4B shows the phase shift for the left MCA in normotensive and hypertensive groups and for the stroke-side MCA in the stroke group. The BP-BFV phase shifts at the minima and maxima were also smaller in the stroke and hypertensive groups compared to the normotensive group (p = 0.0002). The BP-BFV phase shifts at the maxima were smaller in the stroke and hypertensive groups compared to the normotensive group (p = 0.008). In the stroke group, the BP-BFV phase shift at the maxima was greater compared to the phase shift at the minima for the non-stroke MCA (p = 0.02). The BP-BFV phase shifts at the minima and maxima for the stroke MCA did not reach statistical significance (p = 0.08). The BP-BFV phase shifts between the stroke and non-stroke MCA at the minima and maxima were not different. In the normotensive group, the phase difference between BPR and BFVR minima was about 60 degrees corresponding to a time difference of about 3.6 seconds. In contrast, in the stroke and hypertensive groups, the time difference between BFV and BP phases was < 0.5 second.
Figure 4 Panel A shows the phase shift between BPR minimum and BFVR minimum and the phase shift between BPR maximum and BFVR maximum for the right MCA for the □ normotensive and hypertensive groups and ■ for the non-stroke side MCA in the stroke group. Figure 4B shows the phase shift between BPR minimum and BFVR minimum and the phase shift between BPR maximum and BFVR maximum for the left MCA for the normotensive, and hypertensive groups and for the stroke side MCA in the stroke group. Phase shift was greater in the normotensive compared to other groups (between group comparisons *** p < 0.005, ** p < 0.01 *p < 0.05, mean ± SE).
Autoregulation indices
The standard autoregulation index was not different between groups and between MCAs in both hemispheres (Table 2). The rate of autoregulation (RoR) of BFV responses to BP reduction and increases during the VM were also not different (Table 2).
Table 2 Autoregulation Indices
Variable Normotensive Hypertensive Stroke
MCA side Right Right Non-stroke-side
ARI 5.7 ± 3.3 6.1 ± 2.6 6.1 ± 2.6
RoR – descending slope 0.7 ± 0.1 1.1 ± 0.6 0.6 ± 0.2
RoR – ascending slope 0.5 ± 0.1 1.6 ± 1.0 0.6 ± 0.2
MCA side Left Left Stroke-side
ARI 5.9 ± 3.2 5.9 ± 2.6 5.1 ± 1.9
RoR-descending slope 0.6 ± 0.1 0.8 ± 0.4 0.5 ± 0.2
RoR-ascending slope 0.5 ± 0.1 1.5 ± 1.0 0.6 ± 0.2
Autoregulation index (ARI) and the rate of autoregulation (RoR) for the right and left MCAs in the normotensive and hypertensive groups and for the non-stroke and stroke side MCA in the stroke group. RoR – descending slope of the linear portion of the BP and BFV reduction between the baseline and BP minimum. RoR – ascending slope of the linear portion of the BP and BFV increase between BP minimum and maximum during the VM. ARI and RoR were not significantly different between the groups. Data are presented as mean ± SE
Stroke-normotensive and stroke-hypertensive subjects
In a subset analysis, we separated stroke-normotensive (N = 6) and stroke-hypertensive subjects (N = 9) and compared them to the non-stroke normotensive and hypertensive groups. For the non-stroke side MCA, the phases at BFVR minima (p = 0.01) and maxima (p = 0.04) were greater in the stroke-normotensive group compared to the normotensive group. For the stroke side MCA, the phases at BFVR minima (p = 0.04) and maxima (NS, p = 0.08) were greater in the stroke-normotensive group compared to the normotensive group. The phases at BFVR minima and maxima were not different between the stroke-hypertensive and hypertensive groups for both MCAs. No significant differences were found between the stroke and non-stroke side MCA.
Discussion
This study introduces a new technique, based on the Hilbert-Huang transformation [12,18], termed multimodal pressure-flow analysis, for assessing the relationships between systemic blood pressure and cerebral blood flow changes associated with provocative maneuvers. Development of this method was motivated by the facts that 1) that the duration of the VM stages and resulting BP and BFV responses vary over time and among subjects, and 2) these types of time series are short and nonstationary, and therefore, not suitable for analysis using standard Fourier transform and autoregressive type approaches. We implemented the MMPF method to evaluate the dynamics of cerebral autoregulation using the instantaneous systemic BP and MCA BFV phase relationships during the VM. The frequency and corresponding BPR and BFVR amplitudes were computed for each data sample to construct a continuous phase diagram. The BPR and BFVR profiles were similar over the course of the VM, but the phase relationships were different. The autoregulation indices, calculated using the standard methods, did not differentiate the groups.
Cerebral vasoregulation compensates for rapid BP and BFV transitions over the course of the VM. A sudden and parallel increase in intrathoracic [19] and cerebrospinal fluid [8] pressures is associated with a rapid decline in BP and an initial increase of cerebrovascular resistance, resulting in a rapid decline in BFV. With active autoregulation, cerebrovascular resistance diminishes, enabling BFV to recover in the face of falling perfusion pressure, and BFV responses in the MCA precede the systemic BP changes. Therefore, in healthy controls, the phases corresponding to the BFVR minimum and maximum were smaller than BPR phases, reflecting an active vasoregulatory process. With delayed or impaired autoregulation, BFV becomes synchronized with blood pressure. In the stroke group, the BFVR and BPR phase diagrams were similar, suggesting that cerebral blood flow was entrained by systemic BP. In the stroke and hypertensive groups, BFV phases at the BFVR minimum and maximum were greater compared to the normotensive group. The BP-BFV phase shifts were smaller in the stroke and hypertensive group, compared to healthy controls.
Methods evaluating dynamic cerebral autoregulation that use the Fourier transform-based coherence and transfer functions assume a linear relationship between stationary signals. Coherence, phase and gain derived from the transfer function of spontaneous BP and BFV fluctuations have been used to assess autoregulation. These analyses have shown a significant phase lead of cerebral BFV with respect to systemic BP [4,20,21]. However, assumptions about signal stationarity and a linear flow/pressure relationship are not met for the short nonstationary time series from the VM, and therefore transfer function gain was not evaluated. The joint time-frequency distributions [22], such as the new MMPF developed here, that make no assumptions about signal characteristics and can reliably track simultaneous changes of spectral powers and frequencies, are better suited for these short nonstationary signals. The variable time delay between BP and BFV suggests that the relationship between the cerebral blood flow and systemic pressure is not linear.
Previous studies indicated bilateral impairment of the dynamics of pressure autoregulation after an acute [2,3] and subacute ischemic stroke [23]. Dynamic indices of autoregulation that were calculated from spontaneous BP and BFV fluctuations were altered, with no significant difference between autoregulatory indices in the affected and unaffected hemispheres. No significant differences were found in autoregulatory indices between large vessel anterior and posterior circulation infarcts and lacunar infarcts [2]. In patients with large hemispheric strokes, head elevations from 0° to 45° induced reductions in arterial BP, BFV, intracranial and perfusion pressures [1]. We have reported [24] that cerebral vasomotor responses to hypocapnia and hypercapnia were diminished following minor chronic infarctions. Baseline BFVs in the MCAs were similar between the stroke and the non-stroke groups, but differed during head-up tilt. BFV declined in the stroke side MCA during the head-up tilt. Side-to-side BFV differences were the most prominent in stroke-normotensive subjects with lower BP during head-up tilt compared to stroke-hypertensive subjects and non-stroke groups. The present study has confirmed bilateral impairment of autoregulation dynamics in stroke-normotensive and stroke-hypertensive subjects and also in a non-stroke hypertensive group. The BP-BFV phase relationships suggest that the autoregulatory responses were delayed in both hemispheres and that the BFV responses were dependent on perfusion pressure. About one third of stroke patients are hypertensive upon hospital admission. Hypertension and stroke may exert similar pathophysiological effects on vascular compliance, sympatho-vagal interactions [25] and blood pressure regulation [26,27]. Increased vascular stiffness, impaired vasodilatation and shift of the autoregulatory responses toward higher BP values may also affect the timing of autoregulatory responses in both hemispheres.
There are several limitations of this study: 1) The MMPF method was implemented for the VM, which is widely used for clinical autonomic testing. The VM allows noninvasive evaluation of pressure autoregulation, and testing can be completed in less than 5 minutes. However, the VM requires active patient cooperation, and may not be advisable in acute stroke settings where change in intracranial pressure should be avoided. 2) This study evaluated a population of younger subjects with minor stroke. A larger cohort is needed to determine the effects of ischemic stroke subtypes on the dynamics of autoregulation. 3) Age was different between groups; however, it had no significant effect on BP-BFV relationship in our analysis. Aging and cardiovascular risk factors exert significant but distinct effects on regulation of cerebral blood flow. The vasomotor reactivity to hypercarbia declines with aging and hypertension, while the dynamics of pressure regulation can be preserved [7]. This effect may be in part due to a shift of the autoregulatory range toward higher blood pressure values.
Conclusions
Multimodal pressure-flow analysis is a new method that enables evaluation of short nonstationary time-series not suitable for Fourier–based techniques. The MMPF method provides high time and frequency resolution and permits construction of instantaneous phase diagrams on a beat-to-beat basis. This method may be particularly useful as a complementary measure of cerebral autoregulation for the short and nonstationary time series acquired during provocative interventions such as the VM. Application of this method reveals that the regulation of BP-BFV dynamics is altered in both hemispheres after minor stroke, rendering blood flow dependent on blood pressure. Hypertension without stroke is also associated with delayed BP-BFV dynamics.
List of abbreviations
BP = blood pressure
BPR = residual BP calculated by MMPF method
BPVM = empirical mode of BP corresponding to the dominant VM oscillation
BFV = blood flow velocity in the MCA
BFVR = residual BFV calculated by MMPF method
EMD = empirical mode decomposition
HHT = Hilbert-Huang Transform
MCA = middle cerebral artery
MMPF = multimodal pressure-flow analysis
VM = Valsalva maneuver
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
VN – designed the study, conducted the experiments, participated in the analysis and wrote the first draft of the manuscript. ACY – contributed to the MMPF development and analysis; LL – conducted the data and statistical analysis. ALG – contributed to the method application, data interpretation and manuscript preparation. LAL – contributed to data interpretation and manuscript preparation. CKP – designed and developed MMPF method, contributed to data analysis and manuscript preparation.
Acknowledgements
The authors thank Mitul Vyas, BS for technical assistance. This study was supported by the American Heart Association grant 9930119N; The Ohio State University General Clinical Research Center, grant NIH MO1-RR00034; the Beth Israel Deaconess Medical Center General Clinical Research Center, NIH grant MO1-RR01302; NIH grant 1R01 NS045745-01A2; the Older Americans Independence Center, NIH Grant 2P60 AG08812-11; the NIH/NCRR Research Resource for Complex Physiologic Signals, grant P41 RR13622; the G. Harold and Leila Y. Mathers Charitable Foundation; and NIA Program Project Grant AG04390.
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| 15504235 | PMC529459 | CC BY | 2021-01-04 16:37:31 | no | Biomed Eng Online. 2004 Oct 25; 3:39 | utf-8 | Biomed Eng Online | 2,004 | 10.1186/1475-925X-3-39 | oa_comm |
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Ann Clin Microbiol AntimicrobAnnals of Clinical Microbiology and Antimicrobials1476-0711BioMed Central London 1476-0711-3-231550929810.1186/1476-0711-3-23ResearchEffect of Ampicillin on the kinetics of colonization of Streptococcus pneumoniae and Lactobacillus fermentum in the respiratory tract of mice Cangemi de Gutiérrez Rosa [email protected] Viviana [email protected] Marta [email protected] Clara [email protected]ías María Elena [email protected] Facultad de Bioquímica, Química y Farmacia. Universidad Nacional de Tucumán. Tucumán. Argentina2 CERELA-CONICET. Chacabuco 145. 4000. Tucumán. Argentina2004 27 10 2004 3 23 23 15 6 2004 27 10 2004 Copyright © 2004 de Gutiérrez et al; licensee BioMed Central Ltd.2004de Gutiérrez et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Ampicillin was selected to further study the effect of this antibiotic on the colonization capability of S. pneumoniae and L. fermentum intranasally inoculated in a mice experimental model. The sensitivity of S. pneumoniae and L. fermentum to antibiotics was evaluated by different "in vitro" techniques. The results showed that both microorganisms have a typical pattern of sensitivity to antibiotics in these assays. The "in vivo" experiments showed that the treatment with Ampicillin increased the number of lactobacilli and neumococci in the groups of mice treated only with one of the microorganisms. In those mice treated with Lactobacillus, challenged later with neumococci and treated with Ampicillin, the pathogen in lung decreased on the 4th day, disappearing completely after on. The histological studies showed that the antibiotic treatment decreased the inflammatory response produced by the pathogen at the lung and trachea levels.
S. pneumoniaeL. fermentumprobioticsAmpicillinantimicrobialscolonization
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Introduction
Respiratory tract infections are commonly caused by Streptococcus pneumoniae. Extensive antibiotic use for these infections as well as misuse for viral respiratory infections has led to increased penicillin resistance amongst the streptococci [1-6]. Even though there is a very broad description of the pattern of sensibility to antibiotics of this pathogen and other potentially pathogenic microorganisms, there are a small number of publications referred to the lactobacilli sensitivity to these types of compounds [7-10]. The antibiotic treatment modifies the stability of the normal or indigenous microbiota, producing the dominance of certain microorganisms able sometimes to produce a secondary infection [11]. There are a lot of approaches trying to restore the normal microbiota, or to avoid modifications of the different ecosystems to prevent infections, both for human and animal application. One of the main research areas related to the restoration of the indigenous flora is the application of probiotic microorganisms [12-14].
Lactic Acid Bacteria (LAB) has been widely used to restore the ecologic equilibrium of different areas [12-14], mainly for the gastrointestinal tract. In the last decade, there is a lot of other research areas referred to the potential application of probiotics in the respiratory tract mainly as vaccine vectors [15-17]. They could be applied for the protection against pathogenic microorganisms as S. pneumoniae which is a frequent nasopharynx colonizer. In previous papers, the isolation and identification of the microorganisms of the normal microbiota of the respiratory tract of mice was reported by our research group. Also, the evolution from the moment they were born up to two months age was published [18]. In the isolated microorganisms, the probiotic or beneficial characteristics were studied, selecting some strains of the genus Lactobacillus that shared some properties [19]. From them, a strain of Lactobacillus fermentum was selected by the beneficial probiotic properties. Later the optimal dose to produce a transitory colonization or permanence [20,21] and the protection exerted against S. pneumoniae [22] intranasally inoculated was determined.
The objective of the present paper was to study the effect of specific antimicrobial agents against the growth of S. pneumoniae and L. fermentum by "in vitro" assays. Also, to study the effect of Ampicillin, orally administered in a mice experimental model, against the intranasal inoculation of S. pneumoniae, L. fermentum or both microorganisms. The "in vivo" assays were complemented with histological and cytological studies to determine if there was some type of general response of the animals to the treatment.
Materials and methods
Microorganisms and culture media
L. fermentum was isolated from the respiratory tract (pharynx) of adult BALB/c mice [18,20-22]. The mutants resistant to Rifampicin (RR) were obtained to differentiate the inoculated strains from the normal microbiota. The conditions of storage and culture were described previously [18,20-22]. S. pneumoniae A6 serotype was isolated from human pneumonia-suffering subjects, and identified by standard techniques. The serotypification was performed at the "Servicio de Bacteriología Clínica, Instituto Nacional de Enfermedades Infecciosas-ANLIS "Dr Carlos G. Malbran, Buenos Aires, Argentina by Quellung technique. Pathogenicity in mice was increased by inoculating S. pneumoniae intraperitoneally [22]. The pathogen was stored in 25% glycerol added to BHI broth (Brain-Heart Infusion) at -70°C.
Antibiotics assayed
those antibiotics broadly used for the treatment of Gram Positive microorganisms affecting the respiratory tract were studied by determining the Minimal Inhibitory Concentration: penicillin, ampicillin, ceftriazone, ceftazidine, claritromicine, tetracicline, rifampicin, ciprofloxacin, aztreonam, cloramphenicol and imipenen.
Quantitative determination of the bactericidal activity of antibiotics against lactobacilli
The Minimal Inhibitory Concentration Method (MIC) recommended by the National Committee for Clinical Laboratory Standards (NCCLS) was used, replacing the culture media by MRS agar as basal media [23], because Lactic acid bacteria are not able to grow in the recommended Muller Hinton agar. The reference strains used were Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 29213.
Antibiotic sensitivity of S. pneumoniae. Method of diffusion and dilution in agar
The behavior of the pathogenic microorganisms with antimicrobial substances was determined by the qualitative and quantitative techniques of diffusion and dilution in agar (MIC), according to the National Committee for Clinical Laboratory Standards, 2001 (NCCLS). The antibiotics assayed for the qualitative method were oxacillin, vancomycin, chloramphenicol, tetracycline, rifampicin, ciprofloxacin, trimetroprim-sulphametoxazol, claritromycin. The antibiotics assayed for the quantitative method were penicillin, ampicillin, ceftriazone and claritromycin. The reference strain used was S. pneumoniae ATCC 49619.
"In vivo" assays
Each experimental group (or assay) included 24 to 30 animals. Four to six mice were killed in each experimental day. Each one of the "in vivo" experiments was performed twice, and their data used to calculate the media and Standard Deviation. The protocols used were accepted by the Animal Ethics committee of CERELA.
L. fermentum and Ampicillin assay
groups of adult (two months old) male BALB/c mice were intranasally inoculated with 4 doses of a RR L. fermentum strain every 12 h. (50 μl of a suspension containing 1 × 109 CFU/ml). Later, Ampicillin was administered by the oral way in 4 doses of 100 mg/Kg/day, fractionated in two daily doses (1.5 mg/dose/mice) every 12 h. The experimental protocol is schematized in Fig 1a.
Figure 1 Experimental protocols, doses of microorganism and time schedule applied to perform the "in vivo" assays: Fig 1a: Administration of L. fermentum (empty arrows) and Ampicillin (A). Fig 1b: Administration of S. pneumoniae (red arrow) and Ampicillin (A). Fig 1c: Administration of L. fermentum, S. pneumoniae and Ampicillin. The days when mice were sacrificed are indicated with black crosses.
S. pneumoniae and Ampicillin assay
BALB/c mice were intranasally inoculated with a dose of 50 μl of a suspension of S. pneumoniae in saline solution (1 × 109 CFU/ml). The antibiotic was administered by the oral way in 4 doses of 100 mg/Kg/day, following the same scheme than before. The experimental protocol is schematized in Fig 1b.
L. fermentum + S. pneumoniae + Ampicilin assay
Mice were intranasally inoculated with RR L. fermentum (4 doses of 50 μl of a suspension of 1 × 109 CFU/ml), then challenged with S. pneumoniae A6 by the same way (50 μl of a suspension of 1 × 109 CFU/ml) and later treated with Ampicillin in four doses every 12 hours as before, as shown in Fig 1c.
All the experimental groups were sacrificed by cervical dislocation on days 2, 4, 7 and 10 post antibiotic administrations. The samples from nasopharynx and pharynx were obtained with a cotton swab. Trachea, bronchia and lung were aseptically removed and homogenized with a Teflon pestle. The number of microorganisms was determined by the successive dilutions method with peptone water, and plated in MRS agar-Rifampicin. The plates were incubated for 24 h. at 37°C in microaerophilic environment. The identification of microorganisms was performed by macroscopic, microscopic characteristics, Gram staining and biochemical tests.
Histological studies
the samples for histological assays were obtained from the higher trachea, located at the neck basis, bronchia in the area where the main bronchia are divided, and lung in the terminal bronchiole area and alveolar wall. They were fixed with 10% paraformaldehyde and stained with Hematoxylin-eosin and Ramon and Cajal technique [24]. They were analyzed by light microscopy.
Cytological technique
The left lobule of the lung was used to perform the cytological slices. The organ was first immersed in saline solution, then fixed in methanol for 3 min, and afterwards stained with the Romanovsky method (Giemsa stain-Merck,Germany) used routinely in the lab [25].
Statistical techniques
The numbers in the figures represent the mean and Standard Deviation of the results obtained in the two set of experiments performed for each assay. The Student's t test was applied to determine the differences statistically significant of the data.
Results
Antimicrobial sensitivity
L. fermentum
The MIC results for L. fermentum (ug/ml) are as follows: penicillin, ampicilin, ceftriazone, ceftazidine, rifampicin, ciprofloxacin: ≥100 ug/ml, tetraciclin, imipenem and aztreonam: 10 ug/ml, cloramphenicol 1 ug/ml, claritromicine: 0.1 ug/ml. Comparing these values with those obtained for the MIC of Enterococcus spp and Streptococcus spp, type strains recommended by NCCLS, L. fermentum would be resistant to penicillin, ampicillin, ceftriazone, rifampicin and ciprofloxacin.
S. pneumoniae
The results of the antimicrobial sensibility of S. pneumoniae are shown in Table 1 and 2, demonstrating by the two methods applied that S. pneumoniae is sensitive to all the antibiotics assayed by both, the disc diffusion assay, and the MIC.
Table 1 Sensitivity of S. pneumoniae to different antimicrobials by the disc diffusion assay
Antibiotic Concentration (ug) Diameter (mm) Results
R(1) S(2)
Oxacillin 1 - ≥20 24
Vancomycin 30 - ≥17 22
Chloramphenicol 30 ≤20 ≥21 24
Tetracycline 30 ≤18 ≥23 32
Rifampicin 5 ≤16 ≥19 28
Ciprofloxacin 5 ≤13 ≥17 29
Trimetoprim+Sulphametoxazol 1.25 + 23.75 ≤15 ≥19 26
Claritromicin 15 ≤16 ≥21 25
(1) Resistant, (2) Sensitive.
Table 2 Sensitivity of S. pneumoniae to antimicrobials by the Agar dilution method (MIC)
Antibiotic MIC (ug/ml) Break point Sensitive Results (ug/ml)
Penicillin - ≤0.12 ≤0.02
Ampicillin - ≤0.25 ≤0.10
Ceftriazone ≥2 ≤0.50 ≤0.10
Claritromicin ≥1 ≤0.25 ≤0.02
"In vivo" assays
from the results obtained in "in vitro" sensitivity assays, that are predictive, the antibiotic Ampicillin was selected to be used in "in vivo" assays at the recommended dose for human beings (equivalent to the one applied to respiratory tract infections therapy).
Mice treated with L. fermentum (1 × 107 CFU/ml/dose) and Ampicillin
Mice inoculated intranasally with four doses of lactobacilli (determined previously as the dose needed to obtain colonization of the respiratory tract) and treated with Ampicillin showed an increased colonization of lactobacilli during all the days studied in nasal and pharynx exudates (p < 0.01), with values of 107–8 CFU/organ on days 1 and 4, and lower values on the following days. The higher number were in nasal and pharynx exudates. In the control mice without antibiotic, Lactobacillus numbers were around 106 CFU on the first days of the assay. On the 7th and 10th days with antibiotic treatment, Lactobacillus were still present in all the organs, except in lung, while in the mice without antibiotics they were only present on nasal and pharynx exudates on day 7 disappearing on day 10 (upper right Figure 2). The results obtained are shown in Figure 2.
Figure 2 Colonization of L. fermentum in the respiratory tract of mice obtained after the administration of Ampicillin. The figures express the number of L. fermentum in nasal instillations, pharynx, trachea, bronchia or lung from mice inoculated intranasally with L. fermentum (4 doses of 107 CFU/mouse) and treated with 4 doses of Ampicillin. The inserted figure shows the number of lactobacilli obtained from control mice without antibiotic. (*) indicates differences statistically significant between the two groups of mice
Mice challenged with S. pneumoniae (1 × 107 CFU/ml) and treated with Ampicillin
Mice challenged with the pathogen and treated with the antibiotic showed that neumococci were present a longer time in all the organs of the respiratory tract, (as shown in Figure 3) compared with the respective mice without antibiotic treatment (upper right figure 3). The differences statistically significant (p < 0.01) between the two groups of mice are indicated in the figure.
Figure 3 Colonization of S. pneumoniae in the respiratory tract of mice obtained after the administration of Ampicillin. The figures express the number of S. pneumoniae in nasal instillations, pharynx, trachea, bronchia or lung from mice, challenged with S. pneumoniae (108 CFU/mouse) and treated with 4 doses of Ampicillin. The inserted figure shows the number of pathogens obtained from control mice without antibiotic. (*) indicates differences statistically significant between the two groups of mice
Mice treated with L. fermentum, S. pneumoniae and Ampicillin
The colonization of Lactobacillus in this group was similar to the one obtained in the experimental group treated with Lactobacillus and antibiotics without the pathogen (not showed results) In the case of S. pneumoniae, the previous treatment with Lactobacillus, reduced significantly the number of pathogenic microorganism in the lung, as showed in Fig 4. It was present only on the first day, being completely cleared on the following days studied.
Figure 4 Colonization S. pneumoniae in mice inoculated intranasally with L. fermentum (4 doses of 1 × 107 CFU/dose), challenged with S. pneumoniae (1 × 108 CFU/mice) and treated with 4 doses of Ampicillin compared with mice treated only with the pathogen and antibiotics. The figures express the number of pathogen in lung.
Histological and cytological studies
The histological studies showed that in all the organs of the respiratory tract there are not significant structural modifications. Only a moderate leukocyte exudation is observed in the alveolar region in the mice treated with S. pneumoniae and Ampicillin These results are resumed in Table 3. The administration of Lactobacillus produces a stimulation of the lung macrophages, and a lymphocytic infiltration at the trachea level, as resumed in Table 3 and Figure 5a. Mice treated with lactobacilli, S. pneumoniae and antibiotic decreased the inflammatory response produced by the pathogen (picture 5b) compared with the pattern produced by the pathogen and antibiotic, represented in Fig 5c.
Table 3 Histological modifications of the respiratory tract of mice on day 4 of the experiment.
Organ control L.f.+ampicillin(1) S.p.+ampicillin(2) L.f.+S.p.+ampicillin(3)
Bronchia Cilindric epithelia Conserved Conserved Conserved
Bronchioli Cubic epithelia Conserved Without extension of the interalveolar wall Conserved
Lungs Alveolus and duct with plane epithelia. Regular duct and alveolus. Alveolar light with macrophages Regular exudation of mononuclear lymphocytes close to the interaveolar duct. Slight areas with a scarce increased lymphocytic density
Mice were inoculated with: L. fermentum + ampicillin (1), S. pneumoniae + ampicillin (2) and L. fermentum + S. pneumoniae + ampicillin (3).
Figure 5 Light microscopy photographs (200×) of histological slices stained with hematoxylin-eosin from mice intranasally treated with L. fermentum, (5a) S. pneumoniae (5b) or both (5c) and later with Ampicillin. Fig 5a: regular ducts (long arrow) and alveolus (short arrow) (100×), Fig 5b: regular mononuclear exudation close to the interalveolar duct (100×). Fig 5c: Increased lymphocytic density (100×).
The cytological studies showed an increased number of activated macrophages by Lactobacillus, neumococci, or both. The addition of the antibiotic produces an activation of the non-specific line of defense represented by the alveolar macrophages, as shown in Table 4.
Table 4 Cytology of lung impressions stained with Giemsa on day 4 post inoculation in mice treated with L. fermentum+S. pneumoniae+ampicillin
Normal Macrophages G1 Macrophages G2 Macrophages Lymphocytes Neutrophils Other cells
Control Mice 26 +/- 2 13 +/- 4 10 +/- 2 25 +/- 4 9 +/- 3 17 +/- 5
L. fermentum + ampicillin 14 +/- 4 30 +/- 5 28 +/- 4 14 +/- 3 9 +/- 2 5 +/- 4
S. pneumoniae+ampicillin 18 +/- 3 25 +/- 5 30 +/- 2 7 +/- 4 12 +/- 2 8 +/- 5
L.f + S.p. + ampicillin 20 +/- 2 30 +/- 2 25 +/- 3 13 +/- 2 6 +/- 2 6 +/- 3
Discussion
One of the main goals of our research is to go further into the mechanisms involved in the probiotic effect in the respiratory tract [26,27]. Having in mind that we have available a mice experimental model, our interest was focused in the knowledge of the effect produced by the antibiotics more frequently used to treat the respiratory infections on the kinetics of colonization of both microorganisms, either separately or combined. We were also interested in the effect produced by antibiotics on the microbial colonization by the potential use of probiotics together with antibiotics as therapeutic agents and for the restoration of the normal microbiota.
Our results showed that the administration of Ampicillin by the oral route and Lactobacilus intranasally increases their colonization in the respiratory tract. These results support the use of antibiotics together with probiotics, which would help in having a higher colonization of the protective microorganisms. A paper published by Dielemen et al [28] demonstrated that the administration of Lactobacillus GG prevents recurrence of colitis in transgenic rats after the treatment with Vancomycin and Imipenem, showing that our hypothesis has been proved in the intestinal tract.
Coincidently with these results, the treatment with Ampicillin, even though the S. pneumoniae strain used was sensitive to Ampicillin in the "in vitro" test, in the experiments performed in mice produces an increase in the number of the pathogenic microorganism that are also present a longer time in the tract. The pathogen produces a lymphocytic infiltration al the tracheal level, together with an increased inflammatory response evidenced in the lung cytological studies.
Why the response to the pathogen is different in "in vitro" assays than in "in vivo" experiments?. Referred to the behavior of Lactobacillus, there is a broad discussion based on the different results obtained through the application of "in vitro" test, and the differences found in "in vivo" assays. Even though the first one can be used as screening and to predict some characteristics or properties assayed in experimental animals models, one must consider that not always they are coincident [29].
In those experiments performed in mice treated with Lactobacillus, neumococci and Ampicillin, the final results is that the pathogen was cleared faster from the lung supporting the combined use of lactobacilli and antibiotics in the prevention of the infections produced by this pathogen. These results are also demonstrated by the histological slides, (Fig 5c) where the effect produced by the pneumococci decreased by the concomitant use of lactobacilli and antibiotics, when compared with the damage produced by the pathogen administration (Fig 5b). The histological modifications produced by the pathogen in mice previously protected with lactobacilli is lower compared to that obtained when the pathogen alone is inoculated into mice, [20] which produces congestion zones and edema in the terminal bronchiolar area.
The activation of lung or alveolar macrophages produced by the administration of L. fermentum, S. pneumoniae, or both, indicates the highly active non-specific branch of the immune system, which is an important first line of defense against microbial invasion in the lower airways infection. This activation could also help in the clearance of the neumococci observed on the 4th day of the experiments. These results do not agree with those from one infections produced by Pseudomonas aeruginosa, that does not produce an activation of the Pulmonary Alveolar Macrophages [30]. The activation of the immune system at the respiratory tract is taking more relevance, mainly by some researchers who study this way of administration of different antigens and vaccines [15-17].
More studies must be undertaken trying to elucidate which are the mechanisms involved in the protection exerted by lactobacilli in the respiratory tract, and also which are the reasons of the increased colonization of the pathogen and lactobacilli by the effect of the Ampicillin treatment.
Authors' contributions
Rosa Cangemi de Gutiérrez, carried out the experimental and microbiological assays in animals
Viviana Santos, performed the histological and cytological studies
Marta Cecilia, participated in the interpretation of antibiotic assays
Clara Silva, carried out the in vitro sensibility test
María Elena Nader-Macías: conceived the study and participated in its design, evaluation of the results and writing of the manuscript
Acknowledgements
This paper was supported by CONICET (PIP 395) grants.
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| 15509298 | PMC529460 | CC BY | 2021-01-04 16:38:17 | no | Ann Clin Microbiol Antimicrob. 2004 Oct 27; 3:23 | utf-8 | Ann Clin Microbiol Antimicrob | 2,004 | 10.1186/1476-0711-3-23 | oa_comm |
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Int J Health GeogrInternational Journal of Health Geographics1476-072XBioMed Central London 1476-072X-3-231547947810.1186/1476-072X-3-23ResearchUsing GIS technology to identify areas of tuberculosis transmission and incidence Moonan Patrick K [email protected] Manuel [email protected] Teresa N [email protected] Joseph [email protected] Denise [email protected] Kenneth C [email protected] Gerry [email protected] Karan P [email protected] Stephen E [email protected] Department of Medicine, 3500 Camp Bowie Blvd. University of North Texas Health Science Center at Fort Worth, Fort Worth, Texas 76107, USA2 School of Public Health, 3500 Camp Bowie Blvd. University of North Texas Health Science Center at Fort Worth, Fort Worth, Texas 76107, USA3 Department of Microbiology and Immunology, 15355 Lambda Drive. University of Texas Health Science Center at San Antonio South Texas Center for Biology in Medicine Bldg, Room 2.100.04, San Antonio, TX 78245, USA4 Department of Geography, 1704 W. Mulberry. University of North Texas, P.O. Box 305279 Denton, Texas 76203, USA5 Bureau of Laboratories, Texas Department of Health Austin, Texas 78756, USA6 Tarrant County Public Health Department, 1101 S. Main St. Fort Worth, Texas 76104, Suite 1600, USA2004 13 10 2004 3 23 23 6 8 2004 13 10 2004 Copyright © 2004 Moonan et al; licensee BioMed Central Ltd.2004Moonan et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Currently in the U.S. it is recommended that tuberculosis screening and treatment programs be targeted at high-risk populations. While a strategy of targeted testing and treatment of persons most likely to develop tuberculosis is attractive, it is uncertain how best to accomplish this goal. In this study we seek to identify geographical areas where on-going tuberculosis transmission is occurring by linking Geographic Information Systems (GIS) technology with molecular surveillance.
Methods
This cross-sectional analysis was performed on data collected on persons newly diagnosed with culture positive tuberculosis at the Tarrant County Health Department (TCHD) between January 1, 1993 and December 31, 2000. Clinical isolates were molecularly characterized using IS6110-based RFLP analysis and spoligotyping methods to identify patients infected with the same strain. Residential addresses at the time of diagnosis of tuberculosis were geocoded and mapped according to strain characterization. Generalized estimating equations (GEE) analysis models were used to identify risk factors involved in clustering.
Results
Evaluation of the spatial distribution of cases within zip-code boundaries identified distinct areas of geographical distribution of same strain disease. We identified these geographical areas as having increased likelihood of on-going transmission. Based on this evidence we plan to perform geographically based screening and treatment programs.
Conclusion
Using GIS analysis combined with molecular epidemiological surveillance may be an effective method for identifying instances of local transmission. These methods can be used to enhance targeted screening and control efforts, with the goal of interruption of disease transmission and ultimately incidence reduction.
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Background
The application of molecular analysis to identify specific Mycobacterium tuberculosis strains (TB), in combination with traditional surveillance, has yielded insights into tuberculosis transmission [1]. These insights together with a downward trend in tuberculosis in the United States have resulted in the Center for Disease Control and Prevention re-evaluating the TB elimination strategy, and recommending that testing be targeted at specific high risk populations [2,3]. The Institute of Medicine (IOM) also recommended the development of more effective methods for identifying persons with recently acquired infections as an important component of new strategies to limit the spread of tuberculosis [4]. While a strategy of targeted testing and treatment of persons most likely to develop tuberculosis is attractive, it is uncertain how best to accomplish this goal.
Persons with molecularly clustered tuberculosis isolates are assumed to be in the same chain of recent tuberculosis transmission [5,6]. Limited studies have been conducted to evaluate whether these clusters occur in predefined geographical areas [7-11]. If so, then geographically based screening and treatment could be an effective method for TB control programs to identify high risk populations. In this study we seek to determine if we can identify geographical areas with on-going tuberculosis transmission by linking Geographic Information Systems (GIS) technology with ongoing molecular surveillance.
Methods
This cross-sectional analysis was performed on data collected on all persons newly diagnosed with culture positive tuberculosis at the Tarrant County Health Department (TCHD) between January 1, 1993 and December 31, 2000. The TCHD serves the western portion of the Fort Worth-Dallas metropolitan area and includes a population of approximately 1.5 million [12]. The Fort Worth-Dallas metropolitan area is the ninth largest in the U.S. [13]. This study is part of the recent collaborative project sponsored by the Center for Disease Control and Prevention National Tuberculosis Genotyping and Surveillance Network for studying the molecular epidemiology of tuberculosis [14]. All data and materials; including isolates, isolate genotypes, demographic factors, and addresses; were collected prospectively. Moreover, one of the stated objectives of the National Tuberculosis Genotyping and Surveillance Network was to characterize places involved in potential TB transmission [15].
All positive isolates obtained from persons residing in Tarrant County were sent to the Texas Department of Health (TDH) for DNA fingerprinting. Only persons whose M. tuberculosis strains were typed by the Texas Department of Health Mycobacteriology Laboratory were analyzed. Clinical isolate IS6110-based RFLP analysis and spoligotyping analyses were utilized to identify patients infected with the same strain using published methods [16,17]. RFLP analysis using IS6110 RFLP is a powerful tool for discerning one strain of M. tuberculosis from another when there are greater than 6 copies of IS6110 however, a secondary typing method is needed to help differentiate strains with 6 or fewer IS6110 copies [18]. For this project, isolates were considered to be clonally related (i.e., genotypically clustered) if they had identical IS6110 patterns containing seven or more bands, or they had identical IS6110 patterns containing six or fewer bands and identical spoligotypes. A geographic cluster was defined as two or more patients with molecularly related TB strains living in Tarrant County, TX. The proportion of cases due to ongoing transmission was estimated allowing one source case per cluster (i.e. n-1 method) [5].
Any patient who did not have both spoligotyping and RFLP analysis of IS6110 performed on their M. tuberculosis isolate, and/or did not live within Tarrant County at the time of collection was excluded from the geographical analysis. Each eligible patient participated in a standard interview as part of their routine initial medical evaluation. Interview data collected included current and past employment, housing, alcohol and illicit drug use, incarceration history, sexual orientation, and psychiatric history. Persons paid daily for work were considered sporadically employed; others were employed or unemployed. Homelessness was defined as being without a permanent address for more than 3 days since 1991. If persons had a history of homelessness, paid rent by the day, or lived with a non-spousal roommate without paying rent, they were considered unstably housed. Alcoholism was defined by admission of daily consumption of three or more ounces of an alcoholic beverage; a history of alcohol-related conditions including cirrhosis, hepatitis, alcohol withdrawal seizures; or incarceration for alcohol use. Illicit drug use was defined by admission of use or documentation of being under the influence of an illicit drug. Persons were classified as having a history of incarceration if they had spent more than 24 hours in any criminal justice facility since 1991. Patients born in the U.S. or one of its territories were considered American-born; all others were considered foreign-born. All patients received HIV testing and counseling as part of standard clinical practice at the time of diagnosis. HIV status was determined from these tests.
Residential address at the time of diagnosis of tuberculosis, including zip code, were geocoded using ArcView, 4.0, Geographic Information System Software, (ESRI, Redlands, CA). After geocoding, automatically and interactively, 94% of the cases were correctly matched. The numbers of cases were then aggregated by zip code and, for each zip code, an average of the total population reported for the US Census 2000 and US Census 1990 was used to calculate incidence. Population information was retrieved from the US Census Bureau [20] and the North Central Texas Council of Governments (NCTOG) [21]. The US Census Bureau website provides census data aggregated to certain boundaries (e.g.) block groups, blocks, census tracts, zip codes, counties, and states. The NCTOG is a collection of local governments in the Dallas Fort Worth area, provides demographic and GIS data for the region. The demographic data provided has been directly extracted from the US Census. Zip-code level boundaries were established for incidence comparison purpose using zip code tabulation areas (ZTCAs) [21].
The three-dimensional analysis was performed using Inverse Distance Weighting (IDW) [22]. Interpolation is the estimation of values for points in an area not actually sampled. There are many different types of interpolation, with IDW being the simplest interpolation method. A neighborhood about the interpolated point is identified and a weighted average is taken of the observation values within this neighborhood. The weights are a decreasing function of distance. The simplest weighting function is inverse power: w(d)= 1/dp with p > 0. For p = 1, the interpolated function is "cone-like" in the vicinity of the data points [22]. The resulting "cone" shows the clustering of data around the center point of a geographical area.
Statistical analysis was performed utilizing SAS V.8 statistical software (SAS Institute, Cary, NC). Patients with genotypically clustered and unique strains (non-clustered) were compared regarding each categorical risk variable by using odds ratio as a measure of association. Risk categories included patient demographics, and tuberculosis risk factors such as homelessness, HIV-infection, incarceration, and foreign birth. Because members of the clustered cases are assumed to be related, generalized estimating equations (GEE) analysis [23] was performed to determine factors associated with infection of genotypically and geographically clustered strains of M. tuberculosis, and to derive maximum likelihood odds ratio and 95% confidence intervals for all variables. Age was the only continuous variable. Age statistics were analyzed by comparing the means between groups. A 95% confidence interval for the mean age difference was calculated by using the normal approximation, and an independent sample two-tailed student's t-test was used to assess the statistical significance of the mean age difference. The institutional review board of the University of North Texas Health Science Center at Fort Worth approved this investigation.
Results
From January 1, 1993 to December 31, 2000, there were 991 incident cases of tuberculosis in Tarrant County, Texas; M. tuberculosis was isolated from 828 (83.6%) cases. Of the 828 cases with a positive culture, 527 (63.6%) had viable clinical isolates for molecular analysis. Persons excluded because of no viable clinical isolation of M. tuberculosis did not differ statistically from those with those with viable isolates by age (p = 0.49), gender (p = 0.57), or location (p = 0.64). Two hundred and ninety-two (55.4%) patients met the criteria for molecular clustering. These patients were categorized into 48 clusters varying from 2 to 95 patients per group. In nine instances (1.7%), patients had an identical low copy RFLP pattern but did not have spoligotyping conducted, and were therefore classified as missing data (Figure 1). The proportion of cases attributable to ongoing transmission (allowing one source case per cluster i.e. n-1 method) is estimated to be (292 -48)/518 = 47%.
Figure 1 Derivation of study population, Tarrant County, Texas 1993 – 2000
The mean age for the entire population was 44.7 ± 17.3 (SD), 44.1 ± 16.6 (SD) for those genotypically clustered, and 48.5 ± 17.6 (SD) for those with unique strains. Patients that were genotypically clustered differ significantly with age when compared to patients with unique strains, [p = 0.005]. One hundred and seventy-one (32.4%) patients were African-American, 165 (31.3%) were Caucasian, 109 (20.7%) were Hispanic, and 82 (15.6%) were Asian. African-Americans with tuberculosis were significantly more likely to have a clustered strain [OR = 2.7, 95% CI = 1.8, 4.0]. Alternatively, Asians [OR = 3.9, 95% CI = 2.3, 6.0], and Hispanics [OR = 1.9, 95% CI = 1.2, 2.9] were significantly more likely to have a unique strain. Three hundred and twenty-nine (67.4%) of the patients were males, and of these, 214 (65.0%) had clustered strains; 78 of 159 females (49.1%) had clustered strains. Males were more likely than females to have a strain that matched at least one other person in Tarrant County [OR = 1.9, 95% CI = 1.2, 2.8]. Persons with previous experience of homelessness were strongly associated with clustering suggesting a high rate of on-going transmission among this population. [OR = 12.4, 95% CI = 2.9, 52.1] (Table 1).
Table 1 Selected factors associated with genotypic clustering *Within Clustering, CI = confidence interval; OR = Odds Ratio
N(%)* OR 95% CI p-value
Homelessness 33 (11.3) 12.4 2.9, 52.1 <0.001
Living in Zip Code 1 41 (14.0) 6.2 2.4, 16.1 <0.001
American born 235 (80.5) 5.3 3.5, 7.9 <0.001
African-American 123 (42.1) 2.7 1.8, 4.0 <0.001
Male gender 214 (73.3) 1.9 1.3, 2.8 0.001
Living in Zip Code 2 40 (13.7) 1.9 1.0, 3.6 0.038
Living in Zip Code 3 9 (34.6) 0.3 0.1, 0.7 0.03
Three hundred and twenty-one (65.7%) patients were born in the United States. Of those, 235 (73.2%) had clinical isolates that matched the isolate from at least one other person living in Tarrant County. One hundred and sixty-seven patients were born outside of the United States. Of those, 57 (34.1%) clinical isolates that matched the isolate from at least one other person living in Tarrant County. U.S. born individuals were significantly more likely to be genotypically clustered than foreign-born counterparts [OR = 5.3, 95% CI 3.5, 7.9]. The birth country of foreign-born patients varied. Of those born outside of the U.S, 77 (46.1%) were born in Latin America, 47 (28.1%) in Southeast Asia, 14 (8.4%) in Sub-Saharan Africa, 12 (7.2%) in Pacific Asia, 11 (6.6%) in South Asia, and 6 (3.6%) in Europe.
Evaluation of the spatial distribution of number of cases within zip-code boundaries displayed a distinct geographical distribution of disease. The average incidence for the entire county during the study period was 5.9 cases per 100,000. Zip code 1 recorded the highest incidence of 94.3 cases per 100,000 populations, followed by zip code 2 with an average incidence of 55.2 cases per 100,000 population (Figure 2). These areas are characterized by low socioeconomic status, high unemployment rates, homelessness, drug use, and poor quality housing conditions. To examine how molecular clustering varies spatially by zip code, a map of percent molecular clustering at the zip-code level was produced (Figure 3). This map displayed the number of genotypically clustered cases divided by the total number of cases reported in that zip code. The map demonstrated that molecularly clustered disease is not homogenously distributed throughout the county.
Figure 2 Average incidence of Tuberculosis by zip code Tarrant County, Texas (1993 – 2000). Specific zip codes of interest are labeled in green.
Figure 3 Percent of patients genotypically cluster by zip code Tarrant County, Texas (1993 – 2000). Percent genotypically clustered cases = number of genotypically clustered cases/ total number of cases × 100
GIS analysis demonstrated that the areas with the highest incidence also have the highest proportion of persons with genotypically clustered isolates. A strong preponderance of clustering occurred in the urban center of Tarrant County. The highest proportion of persons with molecular clustered TB isolates (80.4% clustered) occurred in the same zip code with the highest incidence. Similarly, zip code 2 on the southeast border of zip code 1, recorded the second highest proportion of persons with molecular clustered TB isolates with 76.6% of all reported cases clustered. Cases reported in zip code 1 were more than six times as likely [OR = 6.2, 95% CI = 2.4, 16.1] than any other zip code to have isolates that match at least one other person living in Tarrant County.
In zip code 3, we observed a morbidity that was more than triple the county average (22.3 cases per 100,000). Unlike other high morbidity areas, zip code 3 had a strong preponderance of unique strain distribution. In this zip code, 17 out of 26 (65.4%) patients had isolates that did not match any other patient in Tarrant County. Cases reported in this zip code were 70% less likely [OR = 0.3, 95% CI = 0.1, 0.7] to have a clustered strain, suggesting that the high rates of tuberculosis did not result from local on-going transmission.
Discussion
The number of cases in the United States is at its lowest point in history, with 15,075 cases reported in 2002 [24]. The role of treatment of LTBI in tuberculosis elimination is of increasing importance. The IOM recommended developing improved methods for identifying persons with recently acquired infections as an important component of strategic tuberculosis elimination in the United States [4]. This study uncovered geographical links to on-going tuberculosis transmission enhancing traditional public health surveillance. We found that by combining molecular strain characterization with GIS analysis that risk of on-going transmission was geographically focal (p = 0.003) with significant clustering of cases occurring in 3 of 59 zip codes. This demonstrated that the current methods of surveillance of contacts of persons with tuberculosis were not completely effective in interrupting disease transmission in these zip code areas.
The use of molecular strain characterization methods in conjunction with traditional surveillance has led to the recognition of a number of risk factors associated with on-going transmission, and has identified numerous outbreaks of tuberculosis undetected by conventional approaches [25-27]. These studies have demonstrated the importance of non-household location based transmission, such as homeless shelters, and social settings such as bars and crack houses [26-29]. Urban centers have traditionally had higher rates of tuberculosis than rural areas [30,31]. Population density, poverty and overcrowding appear in most areas to be major factors for disease transmission [32]. We found similar risks in our population. Location factors, specifically where patients reside at the time of diagnosis, were found to be significantly associated for certain zip codes in Tarrant County. Cases in urban zip codes 1 [OR = 6.2; 95% CI = 2.4, 16.1] and 2 [OR = 1.9; 95% CI = 1.3, 2.8] were strongly associated to infection with a clustered strain as compared to the rest of the county. These zip codes are in the urban center of the county. Zip code 1 is also the site of the largest homeless shelter in the county. Inverse Distance Weighting of this area graphically (Figure 4) demonstrates the three-dimensional result of the interpolation, representing the burden of disease in this area. The resulting "cone" shows the geographic clustering of cases around a particular point of the zip code, the physical location of homeless shelter.
Figure 4 Three-dimensional analysis using Inverse Distance Weighting Interpolation, Tarrant County, Texas (1993 – 2000). Inverse power: w(d)= 1/dp with p = 1. Zip code 1 consists of 264.89 acres.
These finding are similar to those reported in Los Angeles where locations, specifically homeless shelters were identified as important sites of tuberculosis transmission [33]. Similarly, in Houston, locations, specifically bars, were as important as persons in uncovering epidemiological and genotypical links in outbreak investigations [34]. The authors of both of these studies suggested measures to reduce tuberculosis transmission should be based on locations as well as personal contacts [33,34].
We identified that 55% of our patients were clustered and 47% attributable to ongoing community transmission. This differs from a study conducted in a high incidence area of South Africa, where 72% of cases were clustered and 58% attributable to ongoing community transmission [11]. Our lower percentage of clustering and attributable on-going transmission may be related to a much lower reported overall morbidity or the effects of differences in programmatic interventions, such as contact investigation, targeted screening efforts or DOT completion rates.
Although the majority of the tuberculosis morbidity within the developed world is strongly influenced by imported tuberculosis from high prevalence countries [35,36], the rates at which these individuals transmit disease to the general population remain low. We found that foreign-born cases were significantly more likely to have a unique strain [OR = 6.4, 95% CI = 4.1, 9.8] indicating that immigrants were less likely to be source of ongoing transmission of TB in Tarrant County. In a San Francisco based study, investigators identified only two instances of a foreign-born individual transmitting the disease to the native population [37]. Similarly, only 1.8% of transmission from infectious Somali immigrants was to the native population in the Netherlands over the period from 1992 to 1999 [38]. Historically, tracking these populations of foreign born to assess transmission has been difficult. GIS provides another approach for evaluating this issue. As this study illustrates, identifying geographical areas of increased incidence with a high percentage of unique strains may improve local surveillance methods to locate hard to reach foreign-born populations before transmission occurs.
There are some limitations to this research approach. This is based on secondary data, which includes variables collected from a cross-sectional period of time. Although each case is an incident case at the time of diagnosis, under this cross-sectional design, exposure and disease outcomes are assessed simultaneously. In addition patients with tuberculosis may have moved shortly before their diagnosis. However, this should not cause systematic error (bias) or result in an association of clustering with specific locations, because these events would be expected to produce a random misclassification. Also persons exposed within certain zip codes may go on to reside elsewhere and later develop the disease, and result in an underestimate of the morbidity and that may be reflected in calculating associations. Finally, genotyping results were not available for a proportion of TB cases in this study. Some unique isolates might have clustered if some of the missing isolates had been available or if other cases with the same strain moved or are located outside the study area [39]. We therefore believe that estimates of the degree of clustering and the size of clusters are conservative.
When using this approach TB control programs must select the appropriate geographical boundary to examine transmission in their area. For example, using zip codes may be too large a boundary in very populated metropolitan areas. Census block groups may provide greater resolution in determining localized transmission.
Nor are the molecular techniques used without limitation. Patients are clustered according to their isolates having the same genotype. While IS6110 RFLP is recognized as the most discriminatory method for genotyping M. tuberculosis isolates, the discriminatory ability of the technique decreases when there are fewer than 6 IS6110 insertions in the genome. In this case, spoligotyping was used for further strain discrimination. However, it is still possible that some isolates classified as being the same strain based on identical genotypes may represent distantly related, but distinct, strains. Moreover, demonstration that particular patients have the same strain supports, but does not irrefutably prove, direct transmission between these patients as opposed to another source of infection. Conversely, strains continue to evolve, and the resulting genotypic differences over time can result in assigning isolates from cases of direct transmission to distinct strain lineages. Given that a small minority of the isolates had fewer than 6 IS6110 bands (18.2%) or differed by the presence or absence of one band in an otherwise conserved pattern (3.7%), we believe that estimates of the degree of clustering and the size of clusters are conservative.
Conclusion
Using GIS analysis combined with molecular epidemiological surveillance can be an effective method for identifying tuberculosis transmission not identified during standard contact tracing methods. The application of these methods can be utilized in countries where contact tracing is routinely performed. These methods can enhance targeted screening and control efforts, with the goal of interruption of disease transmission and ultimately incidence reduction. This study demonstrates that using existing health data, GIS can identify previously undetected TB transmission. These results were used to design new targeted screening efforts [40]. Studies of these efforts are ongoing to demonstrate if identifying focal areas for targeted screening has utility in reducing TB transmission.
List of abbreviations
CI Confidence Interval
GEE Generalized Estimating Equations
GIS Geographic Information Systems
HIV Human Immuno-deficiency Virus
IDW Inverse Distance Weighting
NCTCG North Central Texas Council of Governments
OR Odds Ratio
RFLP Restriction Fragment Length Polymorphism
TB Tuberculosis
TCHD Tarrant County Health Department
TDH Texas Department of Health
Authors' Contributions
Study concept and design: PM, MB, SW
Acquisition of data: PM, TQ, KJ, DD, GB, SW
Analysis and interpretation of data: PM, MB, TQ, JO, SW
Drafting of the manuscript: PM, SW
Critical revision of the manuscript for important intellectual content: SW, PM, JO, TN
Statistical expertise: KS, MB, PM
Obtained funding: TQ, SW
Administrative, technical or material support: GB
Funding/Support
This work was supported in part by the Centers for Disease Control and Prevention, National Tuberculosis Genotyping and Surveillance Network Cooperative Agreement U52/CCU600497-18, and Tuberculosis Epidemiologic Studies Consortium 200-2001-00084.
Acknowledgments
We are grateful to Drs. Wendy Cronin and Marco Marruffo for their thoughtful review of the manuscript, and to Curtis Denton and Deanna Sanchez for their assistance in creating maps.
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| 15479478 | PMC529461 | CC BY | 2021-01-04 16:39:01 | no | Int J Health Geogr. 2004 Oct 13; 3:23 | utf-8 | Int J Health Geogr | 2,004 | 10.1186/1476-072X-3-23 | oa_comm |
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Int J Health GeogrInternational Journal of Health Geographics1476-072XBioMed Central London 1476-072X-3-261553325310.1186/1476-072X-3-26ResearchVisualization and exploratory analysis of epidemiologic data using a novel space time information system AvRuskin Gillian A [email protected] Geoffrey M [email protected] Jaymie R [email protected] Melissa J [email protected] Andrew M [email protected] Jerome O [email protected] BioMedware Inc., 516 N. State St., Ann Arbor, MI 48104, USA2 Department of Environmental Health Sciences, School of Public Health, University of Michigan, Ann Arbor, MI 48109–2029, USA2004 8 11 2004 3 26 26 9 9 2004 8 11 2004 Copyright © 2004 AvRuskin et al; licensee BioMed Central Ltd.2004AvRuskin et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Recent years have seen an expansion in the use of Geographic Information Systems (GIS) in environmental health research. In this field GIS can be used to detect disease clustering, to analyze access to hospital emergency care, to predict environmental outbreaks, and to estimate exposure to toxic compounds. Despite these advances the inability of GIS to properly handle temporal information is increasingly recognised as a significant constraint. The effective representation and visualization of both spatial and temporal dimensions therefore is expected to significantly enhance our ability to undertake environmental health research using time-referenced geospatial data. Especially for diseases with long latency periods (such as cancer) the ability to represent, quantify and model individual exposure through time is a critical component of risk estimation. In response to this need a STIS – a Space Time Information System has been developed to visualize and analyze objects simultaneously through space and time.
Results
In this paper we present a "first use" of a STIS in a case-control study of the relationship between arsenic exposure and bladder cancer in south eastern Michigan. Individual arsenic exposure is reconstructed by incorporating spatiotemporal data including residential mobility and drinking water habits. The unique contribution of the STIS is its ability to visualize and analyze residential histories over different temporal scales. Participant information is viewed and statistically analyzed using dynamic views in which values of an attribute change through time. These views include tables, graphs (such as histograms and scatterplots), and maps. In addition, these views can be linked and synchronized for complex data exploration using cartographic brushing, statistical brushing, and animation.
Conclusion
The STIS provides new and powerful ways to visualize and analyze how individual exposure and associated environmental variables change through time. We expect to see innovative space-time methods being utilized in future environmental health research now that the successful "first use" of a STIS in exposure reconstruction has been accomplished.
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Background
Geographic Information Systems are beneficial tools in modelling static representations of reality; however they fall short in their ability to handle time. The ability to store, visualize, and analyze both the temporal and spatial dimension of data continues to be a challenging task. Over the past decade, there have been several attempts to include time enabled capabilities into GIS. [1] and [2] proposed amendment vectors to extend the vector data model to the time dimension, while others enhanced the grid data model to represent snap-shots of raster data at different time intervals [3]. Although temporal extensions exist, e.g. [2] commercial GIS packages do not properly support temporal aspects of spatial data [4].
The importance of GIS for medical research and epidemiology has long been recognized [5-7], and GIS is frequently used for retrospective exposure reconstruction [8-10]. However the application of GIS to risk and exposure assessment has historically focused on the hazard as the object of interest – such as the locations of contaminated industrial sites with high concentrations of carcinogens – instead of the individual [3]. More recently exposure assessment using GIS has targeted individuals in their present homes, but relatively little attention has been placed on individual exposure reconstruction involving residential histories and past activities. This in large part is due to the poor ability of current GISs to handle multitemporal geographic information and the movement of individuals within the context of putative exposure sources whose locations and output change through time. Consequently, there have been few attempts to expand on the 'static map' to provide a more accurate view of exposure.
The ability to effectively represent, query, and model the temporal dimension is expected to significantly enhance researchers' abilities to undertake environmental health research with georeferenced data. Studying an individual's exposure over time is a key factor in determining risk, particularly for diseases with long latency periods such as cancer [3], because individual exposure to environmental contaminants (eg carcinogens) can change as people move through space over time. Exposure assessment characterizes the concentration of potential toxins, as well as the frequency and duration of contacts between individuals and those toxins. Therefore, accurate exposure assessment requires estimation of variation in contaminant concentration as well as changes in geographic proximity to contaminant sources over time. This requires models that can account for residential histories and how residential location influences ambient contaminant concentrations as well as exposure opportunities.
In this research we applied a STIS to visualize and analyze data from a bladder cancer case-control study. The objective of the epidemiologic research project is to identify a range of factors that have contributed to bladder cancer incidence in Michigan, with the focus on spatial and spatiotemporal patterns of exposure to naturally occurring arsenic in drinking water. Cases are recruited from the Michigan State Cancer Registry and diagnosed in the years 2000–2003. Controls are frequency matched to cases by age (± 5 years), race, and gender, and recruited using a random digit dialing procedure from an age-weighted list. To be eligible for inclusion in the study, participants must have lived in the eleven county study area for at least the past five years and had no prior history of cancer (with the exception of non-melanoma skin cancer). The goal is to enroll 1400 participants in total. This is an ongoing five year project and only some preliminary spatiotemporal datasets, visualization tools, and results are shown here. Conclusive results will not be available for a few more years, until data has been collected and analyzed for all 1400 participants. The STIS is being developed at BioMedware, in Ann Arbor Michigan with funding from the National Institutes of Environmental Health Sciences and the National Cancer Institute. In this paper STIS is used to visualize and analyze data from a bladder cancer case-control study but it can also be used for health/environment interactions or marketplace sales trends. More information about the STIS and a free 30 day download can be evaluated at .
Results and discussion
Data from a case-control study of bladder cancer in south eastern Michigan was used to evaluate the efficacy of the STIS for documenting and visualizing space-time relationships between cases, controls and putative risk factors. Lifetime exposure to arsenic in drinking water (an element that has been associated with bladder cancer at high levels [12,13]) was reconstructed for each individual by incorporating spatiotemporal information about residential mobility (every address inhabited since birth), occupational history (every full time job since the age of 16), drinking water patterns, and concentration of arsenic in drinking water.
Space time information system
The motivation for this system comes from the idea that the 'what and where' of conventional GIS needs to be extended to the 'what, where, and when' of reality and spatiotemporal modelling. Based on similar spatiotemporal approaches (e.g. [4], [18], [19]), objects are implemented using the space time model: {object, space-time coordinate, attributes} where object identifies the modelled entity (e.g. person X); space-time coordinate is a spatiotemporal location which may be a space-time point (e.g. latitude, longitude, altitude, date, movement model) or a space-time polygon (e.g. polygon centroid, polygon boundary, date, movement model); and attributes are observations on objects (e.g. income).
Within the space time coordinate, in addition to the well known descriptors (e.g. latitude, longitude), we also specify a movement model that defines how the object moves through space as a function of time. Among the simplest of movement models is an instantaneous displacement such that the object ceases to exist at one location and immediately reappears at another location. We use this simple model to describe residential histories.
Morphing describes how the shape of geographic features (such as lines and polygons) changes through time. Here an object is comprised of multiple vertices changing shape through time by the addition, deletion and movement of vertices. This is called network morphing (for lines) and polygon morphing (for polygons). Morphing can be gradual, in which case the change in the object's shape occurs over a defined time interval; or it can be abrupt. In our research we utilize this approach to model cadastral systems and the realignment of administrative and political boundaries. This allows us to track, for example, how municipal water districts change through time, and to then estimate arsenic exposure from drinking water for individuals on municipal water supplies.
Attributes are observations on variables describing the modelled entity and its environment (e.g. case/control identifier, population size, ethnicity, etc.) Our data model assumes observations occur at discrete times at which the attributes of an object are quantified. Attribute change models describe how the values of attributes change between observation times. The simplest attribute change model is a step function that updates an attribute's value when a new observation is made on that attribute. More complex change functions that obtain values from nearby locations are used to interpolate values through space and time for both categorical and continuous data [14]. These include techniques from the field of geostatistics that provide a probabilistic framework for space-time interpolation by building on the joint spatial and temporal dependence between observations [15]. In this research we use the step function approach to model, for example, change in arsenic concentration in potable water when an individual's water supply source is switched from one source of supply to another. We also use geostatistics to model how arsenic concentration in ground water changes spatially and as a function of geology (described in [16]).
Study data
We reconstructed individual exposures by incorporating spatiotemporal data on residential mobility (where people have lived throughout their lives), water supplies (private well, city well water, or city surface water), and drinking water habits. Only locations in which the participants have lived or worked for longer than one year were collected and geocoded. Data about diet, smoking, and medical history were also collected by a phone interview or written questionnaire. A point file (where each point represents a participant) was then imported into the STIS along with associated database files containing attribute information such as address and primary source of drinking water. Table 1 is an example of the drinking water and residential mobility database. Even though information for only three participants is shown, seven different addresses and nine different sources of drinking water are represented. (Street addresses are not shown to protect participant's identity). Therefore, a change in address or primary source of water warrants a new row in the database.
Table 1 Part of database of participant addresses and water source information Information for four participants is shown. For each change in address or primary source of water a new row is entered in the database. Therefore there are 16 rows in this sample database.
Year moved in Year moved out Sample ID City Primary Source of Water
9/12/1935 1/1/1953 1 Swartz Creek Private well
1/1/1956 1/1/1958 1 Swartz Creek Private well, softener
1/1/1958 1/1/1963 1 Swartz Creek Private well
1/1/1963 1/1/1974 1 Swartz Creek Private well, reverse osmosis
1/1/1974 1/1/1990 1 Swartz Creek new private well
1/1/1990 1/1/2002 1 Swartz Creek Community Supply
1/1/2002 1/1/2004 1 Swartz Creek Community Supply, softener
1/1/1976 1/1/1990 2 Livonia Community Supply
1/1/1990 1/1/2004 2 Brighton CS (township well and treatment plant)
1/1/1953 1/1/1961 3 Jackson Community Supply
1/1/1961 1/1/1971 3 Jackson Well (30 ft)
1/1/1971 1/1/1984 3 Michigan Centre Private well
1/1/1984 1/1/1993 3 Vandercook Lake Private well
1/1/1993 1/1/2004 3 Horton Well (280 feet)
1/1/1943 1/1/1958 4 Ferndale Community Supply
1/1/1982 1/1/2004 4 Waterford Community Supply
Other point files were imported including present and historical data on industries and contaminated sites in the study area. A township map and water supply boundary map were imported as polygons. In addition to temporal changes in attributes such as township population, source of community's water supply, and number of people served, town boundaries and water supply boundaries changed with time. New towns were incorporated, community systems expanded their borders, and occasionally, communities were combined and town boundaries dissolved. All of these temporal changes were handled using attribute change models and morphing.
Importing spatiotemporal datasets
We imported shapefiles describing the above data using the STIS data import facility that allows the variables to be time stamped. The user is prompted to import vector information into a new geography or an existing geography (if new information is to be added to an already existing geographic layer the latter will be chosen). The user must tell the system whether the data is (1) a time slice (similar to a collection of GIS static maps) where changes take place at specified times for all objects in the dataset, or (2) a time series where data varies asynchronously and objects move or change attributes at different times. For example Census data are time slice data – attributes remain constant for a decade (1980–1990) and then all attributes are updated with the next decade's census information (1990–2000). On the other hand, data associated with tracking residential histories are time series data, with household moves occurring at different times for each individual. The system imports data at temporal granularities varying from seconds to years; and the data may then be analyzed at these different time scales.
Visualization procedures
Being able to visualize changes in boundaries and attribute values over time is an effective approach to better understanding and exploring data. Because time is a dimension of the data rather than an attribute all views of the data are easily animated. Analogous to a static GIS, attributes of data are visualized by specifying colour, shape, and size of graphical elements (e.g. symbols). However, in contrast to a GIS, the STIS easily facilitates visualization of changing polygon shapes and attribute values over time by animating maps, histograms, and tables simultaneously. Valuable information that might be lost in an atemporal GIS is captured and can become the focus of analysis in the STIS. There are four major visualization views – maps, graphs (histograms, scatter plots, box plots), tables, and time plots.
(1) The map view displays spatial data and the user interacts with the maps by zooming, panning, selecting, and querying. The added feature of the STIS is the animation toolbar. It is employed to show individuals changing place of residence through time; arsenic-emitting industries being founded, operating, and going out of business; municipal water supply districts growing and coalescing; and attribute values, such as arsenic concentrations, changing through time.
(2) In the STIS histograms, scatter plots, and box plots are also animated over time. An individual or group of individuals (e.g. cases vs. controls) may be selected at one point in time and the user can explore how that selection's values change through time. For example, we used this feature to explore how individual arsenic exposure changed over a participant's lifetime. We also used it to compare estimated arsenic burdens for the cases to those of the control population.
(3) Table views also are animated, as the given value of a variable (such as the arsenic concentration in a municipal water supply) will change through time. Tables thus show how data values change over time by updating a given objects value when it increases or decreases.
(4) The time plot graphs time on the x-axis and the value of a variable, such as estimated arsenic exposure, on the y-axis. Objects of interest, such as cases and controls, then map into this bivariate time plot to explore time dependencies in arsenic exposure. Unlike the other views, the time plot is not animated because it already shows the entire time range of the data on the x-axis.
A novel feature of the STIS is the ability to time-link visualization windows. Maps, statistical graphics, and tables may be time-linked so that all of the views are synchronized to the same point in time. Animating the time-linked windows then displays the views simultaneously changing through time. We use this feature to display the changing residential locations of the cases and controls along with the locations and emission volumes of arsenic-producing industries. All of this is done within the context of municipal water districts whose boundaries morph and whose arsenic concentrations are dynamic. While this map visualization is occurring we observe how the frequency distributions of modelled arsenic exposure are changing for the cases relative to that of the controls. Participants (cases or controls) are thus easily evaluated and compared to other participants in terms of their residential histories, and population-level characteristics, such as the mean and dispersion for arsenic exposure estimates, may be compared statistically as they evolve over time.
Statistical and Cartographic Brushing is employed to link together the views associated with a given dataset. This is made possible by using unique identifiers (such as the participant ID's of the cases and controls, or the names of the municipal water districts) to link together corresponding values on the maps and statistical graphics. Statistical brushing is used to select objects (such as the points on a scatter plot) and to then highlight the corresponding objects on maps and other statistical graphics. Cartographic brushing occurs when objects are selected on a map, and their corresponding values on the statistical graphics are highlighted. We used statistical brushing to select participants with high arsenic exposures, and to then identify their locations on maps of their residential histories. We use cartographic brushing to explore possible associations between proximity to arsenic emitting industries and the local densities of cases relative to the controls.
Application of visualization procedures
We first investigate changes in the water supply systems (Figure 1). It is clear that over a 50 year interval (from 1935–1995) private well owners and some community ground water systems replaced their private wells or ground water systems with a purchased surface water system (hooking up to a larger system such as the Detroit Sewer and Water System). Visualizing this information over time is valuable as it shows areas that historically might have been associated with high arsenic levels. It also is used to help assign arsenic concentrations to previous residences. For some public ground or surface water systems historic arsenic concentrations have been recorded. For participants on such water supply systems we therefore can directly assign water source arsenic concentrations. Historic arsenic concentrations for well water supplies often are not available, and for these we interpolate arsenic concentration values using geostatistical procedures that account for values in nearby wells, spatial covariance in these values, and their dependency on predictors such as groundwater geology [16].
Figure 1 Change in water supply systems over 50 years (1935, 1965, 1995) Over the years many towns in Oakland County and Genesee County begin to purchase surface water (from Detroit).
Visualizing the movement of bladder cancer cases and controls through time is crucial in our analysis of arsenic exposure and how it relates to the incidence of bladder cancer. Figure 2 presents participants at three different time points (1960, 1982, 2001). A case is represented by a circle and a control by a square. In 1960 there were two cases and one control. By 1982 four more cases and two more controls moved into the study area and in 2001 the same number of cases and controls remain in the area. Note that one case and one control have moved residences. The animated map thus informs us regarding the residential mobility of the cases and controls. Spatial and temporal subsets of these populations can then be selected and statistically analyzed and summarized using other visualization windows and statistical methods.
Figure 2 Participant movement over 20 years Cases (circles) and controls (squares) continue to move in, out, and around the study area. In 1960 there were two cases and one control. By 1982 four more cases and two more controls moved into the study area and in 2001 the same number of cases and controls remain in the area however one case and one control have moved addresses.
Analysis of arsenic exposure
In this analysis we are interested in the temporal variability in arsenic exposure in cases versus controls as well as clusters of high arsenic values. Arsenic exposure was calculated by multiplying arsenic concentration (μg/L) by home consumption of water and beverages made with water (L/day) at each residence and for each change in water consumption. Data regarding water and beverage consumption was obtained via survey [17]. We utilize the box plot to look at means and interquartile ranges through time (Figure 3 for 1988). The windows are time linked and show cases (on the left) and controls (on the right). A more evenly distributed exposure to arsenic in the case subset is indicated by the large interquartile, and 1.5X interquartile range.
Figure 3 Box plot of arsenic exposure in 1988 for cases (left) and controls (right) The median is the black line that bisects the box. The upper and lower quartiles, the medians of the upper and lower halves of the data, are the edges of the black box. The "whiskers" on the box, the bars at the top and bottom, are 1.5X the interquartile range.
The time plot is another visualization method and provides information over the entire time range (Figure 4 x-axis equals time, y-axis represents arsenic exposure). This graph shows general trends in this preliminary dataset. In the early 1960's arsenic exposure was actually greater for controls (bottom graph) than for cases (upper graph). We also notice that the highest arsenic value (51 μg/L) occurred for a control in 1964 and lasted until the end of the study period. The highest value for a case (38 μg/L) occurred later in the study period (1990). All records are linked to the map view and an investigation of geographical clustering can occur in tandem with the temporal analysis of the time plot.
Figure 4 Time graph of arsenic exposure in cases (top) and controls (bottom) Notice the increase in arsenic for both sets after 1951. The increase in arsenic is much larger for controls and remains high for at least two individuals.
In addition to the graphical analysis we employed statistical clustering methods to identify spatial clusters of homes with high arsenic concentrations in their water supplies. The Univariate Local Moran is a statistical method used to detect local spatial autocorrelation by decomposing Moran's I into contributions for each location. Here, each location refers to an arsenic value sampled at the home of each participant. Moran's I is a weighted correlation coefficient that is used to determine whether neighbouring areas are more similar than would be expected under the null hypothesis. In this study the local Moran statistic is used to detect where there are statistically significant clusters of high (or low) arsenic values in participants' drinking water. Data regarding arsenic in drinking water was collected at the kitchen tap of each participant from their present residence. Water samples were stored on ice, acidified with 0.2% trace metal grade nitric acid, and refrigerated until analysis. Water samples were subsequently analyzed for arsenic using an inductively coupled plasma mass spectrometer (ICP-MS, Argilent Technologies Model 7500 c) [17]. A map of arsenic values from participants' drinking water is shown in Figure 5. The Local Moran analysis was performed on this arsenic dataset resulting in a map of significant clusters (identifying areas as high-high clusters, low-low clusters, low-high outliers, high-low outliers, and areas not significant from background), and a local moran scatterplot. Figure 6 is the result of the local Moran analysis using spatial weights of five (left) and ten (right) nearest neighbours, with 999 randomizations, at the alpha level of 0.05. Generally the two maps look similar, and this is corroborated by similar Global Moran's I values of 0.126279 for five nearest neighbours and 0.129596 for ten nearest neighbours. However, there are differences that arise from analysing spatial pattern at two different local spatial scales. For example, in the northern region of the ten nearest neighbour map we find high-high values indicating high arsenic values surrounded by other high arsenic values. We also see an area of low-low values in the western part of the map, around Lansing. Households in these low-low locations are generally on community water supplies where arsenic values are kept below 50 μg/L to comply with Environmental Protection Agency standards. Conducting the Local Moran analysis at different neighbourhood sizes allows one to evaluate the sensitivity of clustering to different spatial scales.
Figure 5 Arsenic in drinking water (2003/2004) Each point represents an arsenic value taken from the kitchen tap at the present residence of each participant.
Figure 6 Local Moran analysis at two spatial scales Local Moran analysis with five nearest neighbours is on the left, and with ten nearest neighbours is on the right. Notice the appearance of the high-high cluster to the north, and the increase in size of the low-low cluster to the west as the size of the local neighbourhood is increased
Conclusions
In this paper we presented a novel application of a space time information system to analyze some preliminary data in an ongoing case-control bladder cancer study. This approach is significant in that it not only visualizes the movement and attribute changes of spatial objects (including cases, controls, arsenic producing industries, and municipal water supplies) but also allows the user to compare values of these objects over time by time-linking windows. This ability to handle high temporal resolution data is enabling new approaches to exposure assessment. In the near future the STIS will be able to integrate exposure assessment models using an Application Programmers Interface (API). Users will have the flexibility to program specific models outside the software and then visualize their outcome in the STIS using the API. For less technically sophisticated users, a methods toolbar will be included, where common modelling algorithms will be made available using a simple calculator-type interface. Other plans for the software include importing and supporting raster files, exporting animated maps as movies (for presentations), visualizing geospatial lifelines [18,19] in a separate window once objects are selected, and adding spatiotemporal clustering statistics to the methods toolbar.
Competing interests
Some authors are also affiliated with BioMedware a research company that also develops software for the exploratory spatial and temporal analysis of health and environmental data. With funding from the National Cancer Institute, GMJ, AMK, and GAA developed STIS, which is a commercial product of Terraseer.
Acknowledgements
Development of the STIS software was funded by grants R43 ES10220 from the National Institutes of Environmental Health Sciences and R01 CA92669 from the National Cancer Institute. The epidemiologic component was supported by grant R01 CA96002-10, Geographic-Based Research in Cancer Control and Epidemiology, from the National Cancer Institute.
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| 15533253 | PMC529462 | CC BY | 2021-01-04 16:39:01 | no | Int J Health Geogr. 2004 Nov 8; 3:26 | utf-8 | Int J Health Geogr | 2,004 | 10.1186/1476-072X-3-26 | oa_comm |
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Health Qual Life OutcomesHealth and Quality of Life Outcomes1477-7525BioMed Central London 1477-7525-2-571547656110.1186/1477-7525-2-57ResearchThe prevalence and severity of oral impacts on daily performances in Thai primary school children Gherunpong Sudaduang [email protected] Georgios [email protected] Aubrey [email protected] Department of Epidemiology and Public Health, University College London, 1-19 Torrington Place, London WC1E 6BT, United Kingdom2004 12 10 2004 2 57 57 20 7 2004 12 10 2004 Copyright © 2004 Gherunpong et al; licensee BioMed Central Ltd.2004Gherunpong et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Traditional methods of measuring oral health mainly use clinical dental indices and have been complemented by oral health related quality of life (OHRQoL) measures. Most OHRQoL studies have been on adults and elderly populations. There are no systematic OHRQoL studies of a population-based sample of children. The objective of this study was to assess the prevalence, characteristics and severity of oral impacts in primary school children.
Methods
Cross-sectional study of all 1126 children aged 11–12 years in a municipal area of Suphanburi province, Thailand. An OHRQoL measure, Child-Oral Impacts on Daily Performances index (Child-OIDP) was used to assess oral impacts. Children were also clinically examined and completed a self-administered questionnaire about demographic information and oral behaviours.
Results
89.8% of children had one or more oral impacts. The median impact score was 7.6 and mean score was 8.8. Nearly half (47.0%) of the children with impacts had impacts at very little or little levels of intensity. Most (84.8%) of those with impacts had 1–4 daily performances affected (out of 8 performances). Eating was the most common performance affected (72.9%). The severity of impacts was high for eating and smiling and low for study and social contact performances. The main clinical causes of impacts were sensitive tooth (27.9%), oral ulcers (25.8%), toothache (25.1%) and an exfoliating primary tooth (23.4%).
Conclusions
The study reveals that oral health impacts on quality of life in Thai primary school children. Oral impacts were prevalent, but not severe. The impacts mainly related to difficulty eating and smiling. Toothache, oral ulcers and natural processes contributed largely to the incidence of oral impacts.
oral impactsquality of lifechildren
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Background
Contemporary concepts of health suggest that dental health should be defined in physical, psychological and social well-being terms in relation to dental status [1,2]. That is why Cohen and Jago considered that the greatest contribution of dentistry is to the improvement of quality of life because most oral diseases and their consequences interfere with, or have impacts on, daily life performances [3]. Therefore, disruptions in normal physical, psychological and social functioning are important considerations in assessing oral health. Despite these suggestions, traditional methods of measuring oral health use mainly clinical dental indices and focus on the absence or presence of oral diseases. They do not inform us about the oral well-being of people in terms of feelings about their mouths, or, for example, their ability to chew and enjoy their food. The inadequacy of the normative approach in measuring oral health has been recognised and lead to the development of measures of oral health-related quality of life (OHRQoL) [4].
A number of socio-dental or OHRQoL measures have been developed and used for assessing oral well-being and to describe oral impacts on people's quality of life [5]. Generally, they measure the extent to which oral conditions disrupt normal social role functioning and lead to major changes in behaviours, such as changes in ability to work or attend school, or undertake parental or household duties [6,7]. In addition to describing oral impacts on quality of life, some OHRQoL measures were designed specially to assist dental service planning by incorporating them with traditional normative measures in the process of dental needs assessment [8,9].
Most studies using OHRQoL to assess oral impacts of the mouth and teeth have been on adults and elderly populations. Few studies have been done on children possibly because no OHRQoL measures designed for use with children existed until recently. A single measure, dental pain, has been used on children in Malaysia [10] and in South Africa [11]. They found a high prevalence of pain that affected daily living. Similarly, a study in New Zealand found that most school children complained of at least one dental symptom [12]. To date, there are no systematic OHRQoL studies of a large population-based sample of children. In particular, the OHRQoL of primary school children, who are frequently the main target group for dental health services, has not been assessed. Therefore the objective of this study was to use an OHRQoL measure, the Child-OIDP, to assess the prevalence, characteristics and severity of oral impacts in primary school children.
Methods
A cross-sectional survey was carried out in a municipal area of Muang district, Suphanburi province, Thailand. The sample was all 1,126 students aged 11–12 years, in the final year class of all primary schools (grade 6) in the area.
Data were collected through: a) an interview for oral impacts using the Child-OIDP [9], by one interviewer b) a self-administered questionnaire for demographic information such as age, sex and occupation of the father and mother, or male and female guardians [13] and oral health behaviours and c) an oral examination by four calibrated community dentists, mainly based on the WHO guidelines [14]. Orthodontic normative treatment needs were assessed by the Index of Orthodontic Treatment Need (IOTN) [15]. Oral hygiene was also assessed using the Simplified-Oral Hygiene Index (OHI-S) [16]. All documents were translated from English to Thai and the validity was checked by a back-translation method, involving blind re-translation into English. The validity of the translation was verified by experts in the use of questionnaires in both languages. This was also checked after wording modifications, in order to ensure the conceptual and functional equivalences of the questionnaires. A pilot study was carried out to validate all questionnaires before using them in the main data collection. The psychometric properties of the Child-OIDP in terms of face, content and concurrent validity as well as internal and test-retest reliability were excellent. The index was also practical to use with this age group. Full description of the validation process of the Child-OIDP can be found elsewhere [9]. For the main data collection, test-retest reliability of data was tested by ten percent random duplication. Weighted kappa score for the Child-OIDP was 0.91, kappa scores of self-administered questionnaires were 0.7–1.0, and those of intra- and inter-examiner for oral examinations were 0.7–1.0 and 0.6–1.0 respectively indicating good to excellent agreement. The SPSS and Stata programmes were used for statistical analysis.
The protocol of the study was approved by the Ethical Committee of the Ministry of Public Health of Thailand. Primary education and local health authorities as well as all primary schools in the study areas gave permission. Positive consent forms and letters informing parents were sent to parents.
Measuring oral impacts and calculating their severity
Two comprehensive OHRQoL measures specifically for use with pre-adolescent children have recently been developed; the Child Perceptions Questionnaire (CPQ11-14) [17] and the Child Oral Impacts on Daily Performances (Child-OIDP) [9]. Both were validated on a cross-sectional study using a proxy, because no gold standard is available; therefore, at this stage, they should be considered discriminative and not yet evaluative OHRQoL measures. However, they differ mainly in their aims and theoretical frameworks. The Child-OIDP index was developed on a large population-based sample with the aim of being used for dental health service planning. Its theoretical framework is the same as for the original OIDP, namely oral health consequences are categorised into different levels and the index measures only oral impacts on daily performances at the ultimate level [8,9]. The Child-OIDP has the advantage over the CPQ11-14 in that it specifies the different clinical causes of each oral impact and therefore the treatments needed. Although the objective of current study is to assess oral impacts of children, a broader aim of the project was to assess the implications of using measures of oral impacts to estimate dental needs of children. Therefore the Child-OIDP was selected for this study as it is specifically designed to be incorporated into a needs system.
The procedure for using the Child-OIDP began with a self-administered questionnaire carried out with all children as a group in their classroom. The questionnaire contains a list of all oral problems that children are likely to perceive and also include an open answer for any unexpected perceived problem. It was developed during a pilot study, as a modification from the one used in the original OIDP. Children were asked to identify oral problems that they perceived in the last three months. This step aimed to focus children's attention to their oral health problems and to lead to the oral impacts assessment later. Their answers here were used only as a guide to investigate oral impacts on daily performances in the next step and were referred to when they were asked about the causes of oral impacts in individual interviews. Thereafter, children were individually interviewed, irrespective of their answers at the first step, to assess oral impacts on daily life in relation to 8 daily performances. The 8 performances were: a) eating, b) speaking, c) cleaning teeth, d) relaxing, including sleeping, e) smiling, laughing and showing teeth without embarrassment, f) maintaining emotional state, g) study, including going to school and doing homework and h) contact with other people. The individual interviews were aided by 16 pictures (negative and positive pictures for each performance). If children reported an impact on any performance, the frequency of the impact and the severity of its effect on their daily life were scored. Children were also asked to identify oral problems that in their opinion, caused the impact. The oral problems were identified from the list complied in the first step of the assessment.
The oral impact score of each performance is obtained by multiplying severity and frequency scores, 0, 1, 2 or 3 each, in relation to that performance. Therefore scores can range from 0 to 9 per performance. The overall impacts score is the sum of all 8 performances (ranging from 0 to 72) divided by 72 and multiplied by 100. An alternative method of reporting the severity of oral impacts, from the same data set, is to use the 'intensity' and 'extent' of impacts. The intensity refers to the most severe impacts on any of the 8 performances or the highest performance score. It is classified into 6 levels; none, very little, little, moderate, severe and very severe (Table 1). The idea behind this is to differentiate between for example, a child with minor impacts (score of 1) on 6 performances and another child with severe impacts (score of 6) on only 1 performance. In the former case, the child will be in the 'very little', and in the latter one, in the 'severe' category. The extent refers to the number of performances with impacts (PWI) affecting a child's quality of life over the past three months. It ranges from 0 to 8 PWI. The relationships between the impact score and intensity as well as between score and extent were statistically significant (p < 0.001) [18]. Intensity and extent of impacts represent an alternative method of describing or comparing oral impacts on children. They are more straightforward and could give a simpler and clearer picture of impacts than using a single score. Therefore, they provide a more practical aspect to the OHRQoL assessment making it more easily applicable to dental service planning.
Table 1 Classification of the intensity of oral impacts on a performance
The intensity of impacts Severity score Frequency score Performance score
Very severe Severe (3) × Severe (3) 9
Severe Severe (3) × Moderate (2) 6
Moderate (2) Severe (3)
Moderate Moderate (2) × Moderate (2) 4
Severe (3) × Little (1) 3
Little (1) Severe (3)
Little Moderate (2) × Little (1) 2
Little (1) Moderate (2)
Very little Little (1) × Little (1) 1
No impact None (0) × None (0) 0
Results
1101 of the 1126 children returned positive consent forms approved by their parents. 1034 children (91.8% of the total) completed all stages of the survey. 52.4% were male and 47.6% were female. Their mean age was 11.3 years (sd = 0.6). The highest percentage of their fathers were agricultural workers or labourers (34.5%), 30.5% worked in business/private, 27.5% in governmental sectors, 2.1% did not work and 5.4% of children did not have a male guardian. The highest percentage of mothers worked in business/private (38.6%), 24.5% in agriculture, 21.1% in governmental organisations. 14.9% did not work and 1.0% did not have a female guardian.
This population had a low level of dental caries: 43.1% were caries free and the DMFT scores ranged from 0 to 12 with a median score of 1.0 and a mean of 1.5 (±1.8). Almost all children (97.0%) had a Community Periodontal Index (CPI) score of 1 or more; 84.2% had calculus. In terms of oral hygiene status, 5.4% had good, 69.1% had moderate and 25.5% had poor oral hygiene. OHI-S scores ranged from 0.5–5.5 with a median of 2.5 and mean score of 2.5 (±0.9), indicating a moderate level of oral hygiene.
The prevalence of oral impacts was high; 89.8% of children had experienced some kind of oral impact on their daily life during the past three months. There was no difference between the prevalence of impacts in girls and boys (Chi-square test). Impacts on Eating were the most prevalent (72.9%). The prevalence of impacts on Emotion (58.1%), Cleaning teeth (48.5%) and Smiling (40.1%) were also relatively high. The remaining prevalences of impacts were lower, namely Study (15.4%), Relaxing (14.7%), Contact with people (12.2%) and Speaking (9.9%) (Table 2).
Table 2 Prevalence, intensity and score of oral impacts in Thai school children
Performances
Oral impacts on daily performances Overall impacts Eating Speaking Cleaning teeth Relaxing Emotion Smiling Study Contact
Prevalence (%) 89.8 72.9 9.9 48.5 14.7 58.1 40.1 15.4 12.2
Impact intensity (% of children with impacts)
- Very little 15.9 27.9 37.4 33.2 37.4 43.7 25.5 57.8 49.2
- Little 31.1 39.0 33.3 38.8 44.2 37.2 28.2 31.2 38.5
- Moderate 31.7 21.8 19.2 20.8 14.3 13.9 27.4 9.7 10.7
- Severe 18.7 10.8 9.1 6.6 3.4 4.7 15.7 1.3 1.6
- Very severe 2.6 5.5 1.0 0.6 0.7 0.5 3.2 0.0 0.0
Impact score
- Range 0–59.7 0–9 0–9 0–9 0–9 0–9 0–9 0–6 0–6
- Mean (sd) 8.85 (7.4) 1.87 (1.8) 0.23 (0.9) 1.13 (1.6) 0.30 (0.7) 1.17 (1.4) 1.21 (2.0) 0.25 (0.7) 0.21 (0.7)
- Percentiles (25,50,75) 2.8,7.6,12.5 0,2,2 0,0,2 0,0,0 0,0,0 0,1,2 0,0,2 0,0,0 0,0,0
Extent and Severity of impacts
Among the children with impacts, the extent of impacts varied from 1 to 8 performances with impacts (PWI); 16.2% had 1 PWI, 23.3% had 2, 26.9% had 3 and 18.4% had 4 PWIs. Few children had 5 or more PWIs. About 1 in 5 children had severe or very severe intensity of impacts; 18.7% had severe and 2.6% had very severe intensity of impacts.15.9% had very little, 31.1% had little and 31.7% had moderate intensity of impacts (Table 2). The intensity of impacts on each performance showed that Eating and Smiling were the most severely affected while Study and Contact were the least. 16.3% of children with impacts on Eating and 18.9% of those on Smiling had severe or very severe impacts, while the same intensity was reported by 1.3–10.1% of children having impacts on other performances. 57.8% of children with impacts on Study and 49.2% of those on Contact had a very little or little level of impact intensity, whereas none had a very severe intensity of impacts on those two performances.
The distribution of overall impact scores was skewed (Table 2). They ranged from 0.0 to 59.7 with a median of 7.6 and a mean score of 8.8 (sd = 7.4). No difference in overall impact scores were identified between different sexes (Mann-Whitney U test). Mean scores of impacts on each of the 8 performances ranged from 0.21 to 1.87 (maximum possible score is 9). Mean impact score for Eating (1.87) and Smiling (1.21) were the highest while those for Study (0.25) and Contact (0.21) were the lowest (Table 2).
'Causes' of the impacts
There were various oral and dental problems that children perceived as the causes of their overall oral impacts (Table 3). The more prevalent problems leading to impacts were a sensitive tooth (27.9%), oral ulcers (25.8%), toothache (25.1%) and an exfoliating primary tooth (23.4%). Furthermore, oral conditions that related to appearance frequently affected children; position of teeth (20.0%) and colour of teeth (16.2%) were quite frequently cited. In addition, swollen or inflamed gums were related to overall impacts in 13.8% of children.
Table 3 Frequency of oral conditions perceived as causing overall oral impacts
Oral conditions causing overall impacts Frequency (%)
Toothache (t-ache) 25.1
Sensitive tooth (t-sensitive) 27.9
Tooth decay, hole in tooth 5.0
Fractured permanent tooth 4.6
Colour of teeth (colour) 16.2
Shape or size of teeth 2.7
Position of teeth (position) 20.0
Bleeding gum (bleed) 7.4
Swollen or inflamed gum (swollen) 13.8
Calculus 0.9
Bad breath 7.2
Oral ulcer (ulcer) 25.8
Exfoliating primary tooth (exfoliat) 23.4
Tooth space (due to unerupted permanent tooth) (space) 5.3
Erupting permanent tooth 4.9
Deformity of mouth or face 0.4
Missing permanent tooth 0.7
The main perceived causes of impacts on each of the 8 performances are shown in Figure 1. Toothache and oral ulcers were among the main perceived causes of impacts on 6 performances. The majority of impacts on Eating were caused by toothache (64.5%) and on Speaking by oral ulcers (57.8%). An exfoliating primary tooth was one of the main perceived causes of impacts on the following 5 performances; Eating (17.9%), Cleaning (29.5%), Relaxing (11.2%), Emotion (17.5%) and Study (17.6%). Position of teeth was among the main perceived causes of impacts on 3 performances; Smiling (40.7%), Contact (19.8%) and Emotion (10.0%). Space due to a non-erupted permanent tooth (after exfoliation) was one of the main reasons for impacts on Smiling (11.1%). Bad breath was the most frequent perceived cause of impacts on social Contact (27.0%).
Figure 1 Main oral conditions causing impacts on each of the eight performances. Abbreviations refer to Table 3.
Discussion
The prevalence of oral impacts experienced during the past three months by the study population was very high (89.8%). This is surprising in that this was a low caries population in an area with a free accessible school dental service. Although there is no study using OHRQoL index with a population-based sample of 12 year olds to compare with, findings of previous OHRQoL studies suggest that oral impacts are very common in children of this age. In Brazilian adolescent populations, the prevalence of impacts was 32% [19,20] and 62% in Uganda [21]. In child populations, a 88% prevalence of dental pain was reported in South African 8–10-year-old school children [11] and 73% of New Zealand children with good oral status had at least one dental symptom in the past year [12]. That was higher than the 60.1% reported in Malaysian children who also had good oral status and received successful school dental services [10]. A study using the CPQ11-14 index with paedodontic patients found that all the children had oral impacts in the past three months [17]. These findings indicate that oral impacts may be higher in children than in adults. For example, compared to studies using the original OIDP index [8] with other older age groups, the prevalence of oral impacts in a Thai adult population was 73.6% [22] and 52.8% for a Thai elderly population [23]. In a UK national survey of elderly people the prevalence of OIDP impacts was 17% for edentate and 14% for dentate participants [24].
Despite the fact that oral impacts were prevalent in this Thai child population, they were not severe. For example, half of this population had Child-OIDP score less than 7.6 and half of those with impacts had very little or little intensity of impacts (Table 2). Moreover, many clinical causes that contributed to the prevalent impacts do not last long; that is oral ulcers, exfoliating teeth and spaces due to a non-erupted permanent tooth.
This study found that eating was the most important aspect of OHRQoL of children. Difficulty with eating due to oral problems was the most common impact (72.9%), and led to more severe oral impacts on children's quality of life than impacts on other performances. Oral ulcers and exfoliating primary teeth contributed to eating difficulties in nearly half of those with impacts. The finding that eating was the most common performance affected is similar to all studies using the OIDP in all age groups [19,21-24]. They are also similar to a study using the CPQ11-14 with paedodontic patients where impacts on functional limitations were more common than impacts on emotional and social well-being [17].
Difficulty with smiling was another important aspect of children's OHRQoL. It affected 40% of children. The most prevalent cause was position of teeth. Dissatisfaction with position of teeth, moreover, accounted for oral impacts in 1 in 5 of all children (Table 3). Although there is no study documenting the extent of pre-adolescent children's concern about their oral appearance, it is evident that the concern is important when they reach adolescence [25]. For example, de Oliveira and Sheiham found that adolescents with untreated malocclusions were significantly more likely to report oral impacts on their daily lives than those who had completed orthodontic treatment [26]. Chen and Hunter found that psychological impacts of oral health, such as avoiding laughing and being teased about teeth, were more prevalent in children than in adults and elderly [12].
Gum problems were the other important oral conditions affecting children's OHRQoL. More than one fifth of children perceived that bleeding and swollen gums caused oral impacts on their life, particularly in relation to difficulty cleaning, a problem experienced by nearly half of all children (Table 3, Figure 1). Children with difficulty cleaning their teeth because of gum inflammation are unlikely to achieve good levels of oral hygiene because brushing may lead to bleeding, and their gum problems would undoubtedly remain or even get worse. This problem would not be solved by the traditional dental treatment without understanding the affects of oral impacts on behaviour.
An interesting finding was that impacts relating to social dimensions, such as study being affected and contact with people, were less common and least severe. Schor suggested that children's social performances rely more on their physical and psychological performances than adults [27].
It is apparent that an important reason for the high prevalence of oral impacts in children is natural processes such as exfoliating primary teeth or spaces due to a non-erupted permanent tooth. They contributed largely to the high incidence of impacts in these pre-adolescent children. On the other hand, these conditions were not reported as important causes of oral impacts in other age groups [19,22]. The findings on the other clinical causes of oral impacts in this study was consistent with what Jaafar found in Malaysian children, namely, toothache and oral ulcers [10]. Moreover, it is noteworthy that despite the fact that this was a low caries population having access to free dental service, sensitive teeth and toothache were frequently reported causes across the various impacts, particularly so with respect to the more common impact of difficulties with eating.
Although children could often not specify precisely which impairments led to impacts, the question of perceived clinical causes should exclude impacts from some conditions which are definitely not related to actual impairments as well as to treatment needs. For example, toothache, ulcers and conditions relating to appearance definitely require different treatment and could be easily differentiated. However, the accuracy of detecting perceived impairment is limited in a population-based study, while it can be improved at the individual level of investigation.
The specific age group under investigation, particularly in relation to their stage of development, may have influenced the high prevalence of oral impacts. Developmental changes unavoidably affect HRQoL between childhood and adolescence [28]. Maturity and an increase in age generate a more sophisticated understanding and perceptions about health and illness [29]. Therefore, perceptions about health and quality of life of children change as they mature [28,30]. This might make younger children more sensitive to oral symptoms than older age groups. Because of those considerations the modification of the Child-OIDP addressed the main possible problems that might arise when employing adult measures with children [30,31]. They include the adjustment of the 8 items of daily performances, simplification of rating scales, decrease of the time frame and rearrangement and clarification of the complex questions that were beyond the capability of children under 12 years according to Piaget's cognitive development theory [32]. Moreover, the use of pictures as aids is considered of value when interviewing children [33,34]. In addition to the modification, another advantage of the Child-OIDP lies in its conceptual framework where oral health consequences are divided into three levels; the first level represents oral problems (such as tooth decay), the second or intermediate level represents symptoms (such as pain) and the third or "ultimate level" represents difficulty in daily performances. The index measures impact at the ultimate level only, which could reduce double scoring, by not measuring twice the same impacts experienced at different levels. For example, pain is not scored whereas difficulty with eating due to pain is scored. In addition, this approach could reduce the uncertainty of children's perception and interpretation and therefore make the index more applicable for children [35]. Fink explained that HRQoL can be measured through different types of information. Measuring impacts on daily functioning is more objective and reliable than measuring reported health problems or symptoms which are more influenced by individuals' perception and interpretation [36]. Thus, HRQoL measures for children that involve subjective reported problems or symptoms such as pain are frequently problematic, because children's interpretation and perception about health differ from adults [30]. On the other hand, HRQoL measures that focus on information about functioning, such as the Sickness Impact Profile, may readily be applied to children as well as adults [37]. Therefore, to reduce a problem with children's interpretation about their health or symptoms, the technique of assessing HRQoL based on activities of daily living is appropriate [35].
Conclusions
The prevalence of oral impacts on daily performances in this child population was very high. Oral impacts affected children's quality of life mainly through difficulty eating and smiling. There are various oral conditions that contributed significantly to the incidence of impacts, namely, sensitive teeth, toothache, oral ulcers and exfoliating primary teeth. Although the prevalence of impacts was high, the severity was not; many children had their quality of life affected at low levels. This reveals a need for further longitudinal studies to better understand and interpret OHRQoL measures in children.
Authors' contributions
SG carried out all work including data collection, data analysis and writing the paper. GT supervised the project and assisted writing. AS initiated the idea of, and supervised the project and edited writing.
Acknowledgements
The authors acknowledge the help and contribution of Public Health Office, community hospitals and primary schools of Suphanburi province.
* Readers are welcome to request the Child-OIDP questionnaire including pictures of daily performances from the authors.
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| 15476561 | PMC529463 | CC BY | 2021-01-04 16:38:12 | no | Health Qual Life Outcomes. 2004 Oct 12; 2:57 | utf-8 | Health Qual Life Outcomes | 2,004 | 10.1186/1477-7525-2-57 | oa_comm |
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Int Semin Surg OncolInternational seminars in surgical oncology : ISSO1477-7800BioMed Central London 1477-7800-1-81552750410.1186/1477-7800-1-8Case ReportTreatment outcomes in locally advanced colorectal carcinoma Harish K [email protected] YV [email protected] S [email protected] Department of Surgical Oncology, M. S. Ramaiah Medical College & Hospital, Bangalore – 560054, India2 Department of General Surgery, M. S. Ramaiah Medical College & Hospital, Bangalore – 560054, India3 Department of Radiation Oncology, M. S. Ramaiah Medical College & Hospital, Bangalore – 560054, India2004 4 11 2004 1 8 8 8 7 2004 4 11 2004 Copyright © 2004 Harish et al; licensee BioMed Central Ltd.2004Harish et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Locally advanced colorectal cancers form a distinct subgroup where contiguous organs could be involved without distant metastases and so may be amenable to curative surgical resection. It was our objective to report our experience in treating six such patients with operable locally advanced colorectal carcinomas.
Methods
We retrospectively reviewed the case notes of 47 patients who were diagnosed with colorectal cancers at M S Ramaiah Medical Teaching Hospital between the years 1996 – 2001. Six patients were identified with T4 lesions, adjacent organ involvement and with no nodal involvement. The treatments and outcomes for these patients were then reviewed.
Results
Two of three patients with rectal malignancies who underwent pelvic exenteration succumbed to disease recurrence within the first 18 months. One of the three patients with colonic cancers died of non malignant causes. The other two are disease free till date.
Conclusions
Aggressive multivisceral resections for locally advanced colonic cancers might be appropriate. Rectal cancers when locally advanced may be considered for pelvic exenteration, but a more guarded prognosis may apply.
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Background
Locally advanced colorectal tumors constitutes to about 5 – 22% of all colorectal cancers at the time of presentation [1]. This type of tumor forms a distinct sub class of colorectal tumors characterized by aggressive local behavior in the form of invasion of adjacent organs or structures with somewhat surprisingly no distant metastasis at presentation. The survivals of such cases that undergo multivisceral resections are 58% and 43% for UICC stage II and III respectively. These results are similar to those undergoing conventional resections [1]. In addition, there is a suggestion of elevated stage related late results to the same level as that associated with tumors where there is no direct invasion of contiguous organs [1]. Addressing the local disease adequately with multi-visceral resections when necessary could result in favorable out come. However en-bloc surgical resection of the tumor forms a surgical challenge and the risks of complications and death must be weighed against probable survival benefits. The post operative complication rate of multivisceral resection is about 11.5%; 30 day operative mortality is 3.6% and compares favorably with non-multivisceral resections [1]. It was our objective to report our experience in treating six such patients with operable locally advanced colorectal carcinomas.
Methods
During the years 1996–2001, 47 patients with colorectal cancers were treated at our institute. All patients underwent a rectal exam, punch biopsy if it were an accessible rectal lesion or colonoscopy and biopsy if it were a colonic lesion. Screening colonoscopy for a synchronous lesion was however done for all patients. Patients in whom a biopsy confirmed a malignant lesion underwent an ultrasound (US) study of the abdomen, chest X-ray and Carcino-embryonic antigen (CEA) estimation. Patients with doubtful involvement of adjacent organs or structures underwent CT scan. Cystoscopy was performed when urinary bladder involvement was suspected on CT scan. Patients were diagnosed as locally advanced when the staging evaluation showed involvement of adjacent organ or structure. Though nodal evaluation is suboptimal with US or CT scan, all patients with 'N0' status on these investigations were included as locally advanced while those with 'N+' on evaluation were excluded. Patients with metastasis to any organ were excluded. The preoperative staging of the tumor was T4, N0, M0. Elective surgery was performed on all cases. No pre-operative therapy was administered. All patients were administered adjuvant radiation and chemotherapy which was instituted immediately after wound healing (between 16 and 29 days post surgery). A total dose of 5000 centi-Gray of external beam radiation in combination with chemotherapy consisting of 5-Fluoro-Uracil (5-FU) and leucovorin was administered. The dose of 5-FU was 425 mg/m2 and that of leucovorin 20 mg/m2. This combination was administered as infusion on day 1 to 5, and six such cycles every 28 days were instituted as part of chemotherapy regimen. The therapy in colonic carcinoma was sequenced as 1 cycle of chemotherapy followed by radiation and the remaining 5 cycles after completion of radiation. In case of rectal cancers, the chemotherapy was administered concurrently with radiation. The administration of leucovorin was with-held during radiation and instead, the dose of 5-FU was increased to 500 mg/m2. Follow up was by clinical examination every 3 months for first two years, and six monthly until 5 years. The evaluation included alternate year CT scan, annual US abdomen, chest X-ray, colonoscopy and CEA levels every six months.
The two live patients have given consent for publication. Consents have been obtained from the legal heirs of the remaining four patients.
Results
The study group consisted of six patients (29 to 70 years), three of whom had locally advanced colonic disease and three of whom had locally advanced rectal disease. Pre operative CEA levels were surprisingly within normal limits in all six patients. The pre operative colonoscopic or rectal biopsy had determined adenocarcinoma in all six patients (Grade as shown in Table 1). In two of the patients with rectal disease where involvement of the bladder wall was suspected, cystoscopic findings were normal. All the patients were offered radical surgery on elective basis. The radicality included removal of adjacent involved organs which could increase the complication rate of extensive surgery. In addition such extended resections for rectal cancer would result in exenterative pelvic surgery resulting in abdominal stomas. Surgical resection margins were negative in all cases.
Table 1 Salient Clinico – pathologic features of locally advanced colorectal carcinomas
Site of malignancy Age Sex Pathologic 'T' Status Grade M/SR * ca Adjacent
Org † Nodal Status PNS ‡ PN/PV § spread
Colon 56 Male T 4 II - + (abdominal wall) N0 - -
58 Male T4 II - + (abdominal wall) N0 - +
67 Female T3 II - - (bladder dome) N0 - -
Rectum 45 Male T4 I + + (bladder / prostate) N1 + +
27 Female T4 I + + (uterus and vagina) N2 + +
69 Male T4 II + + (bladder / prostate) N2 + +
* Mucinous or signet ring carcinoma
† Pathologic involvement of adjacent organ
‡ Perinodal spread
§ Perineural / perivascular spread
Case 1
Patient presented with a 2 month history of abdominal mass. There was no history of weight loss or altered bowel habits. The mass was firm with irregular borders and restricted mobility. CT scan of abdomen revealed a large mass lesion arising from the caecum and ascending colon apparently infiltrating the anterior abdominal wall (Fig 1). Patient underwent a radical right hemicolectomy with en-bloc resection of involved abdominal wall (Fig 2). The right branch of the middle colic, the right colic and ileocolic vessels were ligated at the origin. Retroperitoneum was not involved. A two layer hand sewn ileocolic anastomosis was performed. The abdominal wall muscles were resected with a 2.5 cm margin and the resultant defect was repaired with a polypropylene mesh. The patient received adjuvant radiation and chemotherapy as described above and is disease free 3 1/2 years after surgery.
Figure 1 CT scan of the abdomen showing the involvement of abdominal wall muscles and adhesions to neighboring intestines.
Figure 2 Specimen of radical right hemicolectomy with en-bloc resection of abdominal wall muscle (indicated by small arrow) and neighboring small intestines (indicated by dotted arrows).
Case 2
Patient presented with a 6 month history of bleeding per rectum. There was no history of weight loss. Patient's hemoglobin was 10.2 gms / dl. Patient had ignored the complaint for 3 months but even at a later date was unfortunately not advised to undergo sigmoidoscopy where he was evaluated. Patient underwent colonoscopic evaluation at our institute after evaluation. CT scan showed possible involvement of abdominal wall with the growth arising from sigmoid colon. Intra-operatively, the mass was found to involve the posterior rectus sheath. Sigmoid colectomy was performed along with resection of the involved posterior rectus sheath and the rectus muscle en-bloc. Splenic flexure was mobilized to obtain colorectal hand sewn anastomosis. Primary closure of the rectus defect was achieved and patient received adjuvant radiation and chemotherapy. The patient is disease free 3 years post surgery.
Case 3
Patient was diabetic, asymptomatic; ultrasound performed for evaluation of kidneys for diabetic nephropathy incidentally detected the mass lesion in sigmoid colon. Biopsy specimen was obtained at colonoscopy; no synchronous lesion was detected in the rest of the colon. CT scan revealed close relation of the sigmoid mass lesion to the bladder dome but was unable to comment categorically on wall infiltration. Urinary bladder mucosa was intact and normal on pre-operative cystoscopy. Intra-operatively, the lesion was found adherent to bladder dome. Sigmoid colectomy and part of the bladder wall was resected en-bloc. There was no pathologic evidence of bladder involvement. Patient had ishcaemic heart disease and diabetic nephropathy; she later developed myocardial infarction and renal failure while on adjuvant therapy 7 months post surgery and succumbed to the same.
Case 4
Patient had bleeding per rectum and constipation for 18 months. Patient was evaluated and treated as hemorrhoids initially (no surgical intervention). About 6 months prior to presentation at our institute, he was diagnosed as rectal cancer and was advised abdomino-perineal resection. Patient was scared of surgery and waited without any therapy. Rest of the large bowel was normal on colonoscopy. A CT scan showed involvement of the urinary bladder and prostate by a rectal mass while bony pelvis appeared free of tumor involvement. The patient underwent a total pelvic exenteration with formation of a urinary diversion by ileal conduit. After a lower midline laparotomy, abdomen was evaluated for any ascites or liver metastasis. Para-aortic area was palpated and rest of peritoneal cavity was evaluated for any metastatic deposit. Inferior mesenteric artery was ligated beyond the origin of the left colic artery. Bilateral pelvic nodal dissection was completed. Sigmoid colon and rectum was mobilized as in abdominoperineal resection over the sacral hollow. Anteriorly the dissection was carried out in the retropubic space to access the urethra beyond the prostate. Lateral dissection was carried out which included the ligation of the superior vesical vessels. Both the ureters were ligated below the pelvic brim in the true pelvis at least 3 cm proximal to palpable disease. Isolated loop of ileum was mobilized based on two vessels. One end of the loop was closed and both ureters were anastomosed separately to the loop. Intestinal continuity was obtained with ileo-ileal end to end anastomosis. This ileal loop was brought out as a urinary stoma on the right side and the cut colon was brought out as colostomy on the left side. The patient's postoperative recovery was unremarkable except for a uereteric leak which settled by 10th post operative day. Patient received adjuvant therapy but was irregular on follow-up and refused to be investigated. Patient developed hepatic metastasis and succumbed to the disease after 18 months.
Case 5
Patient had bleeding per rectum and constipation for 4 months. Patient had significant weight loss over previous 2 months. Patient had circumferential rectal carcinoma involving uterus and upper part of vagina. Rectal carcinoma was proved by biopsy. Rest of the colon was normal on colonoscopy. Upper vaginal mucosa was intact but indurated underneath. CT scan showed infiltration of lower part of uterus and upper vagina. Patient underwent posterior pelvic exenteration. Uterus, including both the fallopian tubes and ovaries were removed en-bloc along with vagina and rectum. Surgery was followed by adjuvant radiation and chemotherapy. Patient succumbed to the disease 1 year later with multiple metastases in liver, brain and malignant ascites.
Case 6
Patient had bleeding per rectum and constipation for 3 months. A preoperative CT scan with rectal and intravenous contrast revealed involvement of prostate and possibly bladder. Colonoscopy did not reveal any other lesion. He underwent a total pelvic exenteration (Fig 3) with ileal conduit similar to case 4. Patient developed fever 48 hours after surgery. Evaluation for infective pathology including cultures from catheters and venous access tips did not reveal any source of infection. There was no pocket of collection on repeated abdominal ultrasound. There was raised leukocyte count. A possibility of sepsis from an occult focus was considered and treated. However, on the 5th post operative day, patient developed vomiting and had raised serum creatinine values. Values of fibrin degradation products were also raised and a diagnosis of disseminated intravascular coagulation and renal failure was entertained. Patient had prolonged prothrombin time but did not have any clinical bleed. He died on 30th day post operative while on recovery from the same. The post surgical pathology reports are summarized in Table 1.
Figure 3 Specimen of pelvic exenteration with cut open rectum showing the rectal adenocarcinoma infiltrating the prostate. Foley's catheter is also seen.
Discussion
Surgical resection remains the primary and most effective treatment for advanced colorectal cancers [2]. The 5-year survival rate of the overall group of patients undergoing multivisceral resection is 42%, that of the subgroup undergoing curative surgery is 51%, and that of the subgroup receiving only palliative resection is 0% [1]. In the presence of local tumor extension the distinction between inflammatory adherence and malignant invasion is impossible to make intra operatively. Adherence of the neoplasm to surrounding structures demonstrates pathological tumor invasion in approximately 50% of cases [1,2]. The operative intent of the surgeon should be to achieve complete extirpation with adequate margins in the involved structures. Dissection between malignancy and adherent structures, or biopsy and frozen section is not recommended as these procedures may promote tumor dissemination [1,2], which may have a detrimental impact on prognosis [3-5]. The concept of en-bloc resection has significantly reduced the local recurrence rate from 77% to 36% and significantly improved the 5 year over all survival of 43% [1,4-6]. Other studies have also shown improved survival of similar staged colorectal cancers with en-bloc resection [7,8]. The survivals reported in various studies are shown in Table 2[1,2,4,9-11]. The 5-year survivals are comparable ranging from 38% to 52%. Although colon and rectal cancers which required multivisceral resections are shown separately, the survivals are not indicated differently. For similar 'T' status, the corresponding 'N+' status of colon versus rectum has not been studied. Our present study is small but the overall survival is 33% and compares with larger studies. In addition, since a very high percentage of even large T4 tumors have not yet metastasized to the regional lymph nodes, a multivisceral resection offers the chance to radically remove the local disease and affect a cure [1,12]. All patients in our study group underwent en-bloc resection. No attempt was made to separate the adherent structure to confirm or negate the involvement of adjacent organ per operatively. Since colon spans the entire periphery of the abdomen, almost all the abdominal organs have been reported to be involved [2]. Multivisceral resections have been recommended whenever necessary as it could offer cure or at least significant palliation [13-15]. En-bloc resections have been recommended even when it warrants a pancreatico-duodencetomy [16,17]. In the present report, the en-bloc resections included abdominal wall in two cases and urinary bladder in one.
Table 2 Summary of survival data from a few large studies
Author Multivisceral Resections Colon Rectum Death Rate 5-year Survival Rate
Reiner (1987) 158 53 105 12% 38%
Heslov (1988) 58 26 32 5% 38%
Montesani (1991) 33 - - - 30%
Hermanek (1992) 197 119 78 3% 52%
Gebhardt (1999) 173 122 51 4% 51%
Lehnert (2002) 201 139 62 7.5% 51%
Rectum cancers involve the pelvic genital organs and or urinary bladder anteriorly. Posteriorly it could involve the sacrum. Exenterative pelvic surgeries are warranted for locally advanced rectal cancers [18]. Pelvic exenteration and sacral resection for primary or recurrent rectal cancer are tolerable procedures with a low mortality rate [19]. Although they provide a survival benefit if curative resection is possible, the associated morbidity remains high [19-21]. The complications described include sepsis, intra-abdominal abscess, pelvic abscess, enteric fistula, enteric anastomotic leak, wound infection, ileoureteral anastomotic leak, ileoureteral anastomotic stenosis, bowel obstruction, vascular injury, bleed, liver dysfunction and pneumonia. In the present study, both the male patients underwent total pelvic exenteration with ileal conduit for urinary diversion. The female patient underwent posterior pelvic exenteration. The male patient who lived for 18 months did not have any major problem to take care of both the stomas. Similar thoughts that a small reduction in patient's quality of life due to urinary diversion should not be a major contraindication when surgery with urinary diversion is warranted to obtain curative resection have been echoed [22].
Pre-operative nodal evaluation by abdominal CT scan, MRI or endoscopic ultrasound may be inadequate. Though endosonography can detect perirectal nodes, its inability to reliably predict pathologic involvement is a constraint [23]. Nodal status assessment is considered adequate when at least 14 nodes are examined [24]. In some studies, nodal metastasis has insignificantly altered survival [4,6,25,26]. In contrast, other studies have shown that presence of lymph node involvement is associated with poor prognosis [21]. Studies have shown that 5 year survival in patients with nodal metastasis was 0% to 11%, significantly lower than the 37% to 76% survival rate in their patients without nodal metastases [11,27,28]. These studies have even cast doubts over usefulness of pelvic exenteration in patients with nodal disease though some would still recommend it [29]. None of the patients in the study were evaluated by endosonography but were evaluated by CT scan. Accepting that it is a poor tool for nodal evaluation, pre-operative involvement was not detected in any patient in the study group. Moreover, lymph node status can be accurately determined by pathologic examination only. Hence the investigations might not contribute to decision making though cases with obvious metastasis could be excluded from major resections. In our series of six cases, all the colonic patients were node negative and had no vascular invasion. With multi visceral resections, T4 colonic cancers had acceptable morbidity and better treatment outcome. However, rectal cancer patients had poor prognostic factors like vascular invasion, lymph nodal involvement and histological type of mucinous / signet ring variety. Another striking feature was that though colonic cancers were grade II tumors, they were pathologically N0. But rectal cancers were pathologically N1, N2 and had perinodal spread despite being grade I tumors.
Down staging of locally advanced rectal cancers have been achieved with pre-operative radiation or chemotherapy or both resulting in decreased recurrence and improved disease free survival [30-35]. The results of IORT hold some promise [36,37]. It would be fair to say that there is still no agreement as to the optimal sequencing of chemotherapy, radiation, and definitive surgery in immediately operable patients, and both preoperative and postoperative approaches have vocal proponents [38]. In the present study, pre-operative chemotherapy or radiation was not administered to any patient. There have been no studies to suggest that pre-operative chemoradiation would decrease the magnitude of surgical resection and hence a patient suitable for exenteration would still require the same after such a therapy. In addition, administration of radiation pre-operatively increases the morbidity after exenteration [39]. It must also be noted that studies on pre-operative chemoradiation have shown improvement is DFS and not in OS. Till such time there are conclusive results in these areas for pre-operative therapy with chemoradiation, such a therapy would continue to be debated.
Although it could be inappropriate to draw conclusions from a small number of cases, certain features require further deliberation. T4 colonic cancers have had better outcomes compared to T4 rectal cancers. Many of the earlier reports have combined results of colonic and rectal cancers. In addition, the reports have combined locally advanced and recurrent cancers. As the tumor biology of these areas is different, the results of colon and rectum have to be viewed separately. In addition, the morbidity and mortality associated with multivisceral resections are different of these areas. Since nodal metastasis were seen in all T4 rectal cancers and in none of the colonic T4 cancers, it would be interesting to evaluate percentage of nodal metastasis separately for colonic and rectal cancers with similar T stages. Another indicator of aggressive biology of rectal cancers compared to colonic cancers is the fact that lower grade rectal cancers had more nodal metastasis, perineural and perivascular invasion compared to higher grade colon cancers.
Conclusions
Locally advanced colonic cancers are to be evaluated and treated aggressively with multivisceral en-bloc resections. The outcomes are likely to be favorable and hence the results gratifying. Locally advanced rectal cancers require exenterative pelvic surgery which carries higher morbidity and mortality. In addition, rectal cancers are biologically more aggressive. Hence exenterative pelvic surgeries are to be done more selectively with guarded disease out come for T4 rectal tumors. Future improvements in chemotherapeutic agents and radiation techniques could make down staging of these malignancies a reality not only in terms of operability but in improving overall survival.
List of abbreviations used
US: Ultrasound
CEA: Carcinoembryonic antigen
CT: Computerized tomogram
5FU: 5-Fluoro-uracil
MRI: Magnetic Resonance Imaging
IORT: Intraoperative Radiotherapy
DFS: Disease free survival
OS: Overall survival
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
KH: Was the principal treating surgeon apart from designing and conceptualizing the article.
YVN: Was the assistant in the procedures and made the preliminary draft of the article.
SN: Planned and administered adjuvant radiation.
Acknowledgements
Dr. Nalini Kilara, Professor of medical oncology, MS Ramaiah Hospital, planned and administered adjuvant chemotherapy. We thank her for the inputs regarding chemotherapy
The authors wish to thank the faculty of The Department of Pathology, MS Ramaiah Medical College for reviewing all the pathology specimens to specifically address the issues of adjacent organ involvement and nodal involvement microscopically.
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| 15527504 | PMC529464 | CC BY | 2021-01-04 16:38:35 | no | Int Semin Surg Oncol. 2004 Nov 4; 1:8 | utf-8 | Int Semin Surg Oncol | 2,004 | 10.1186/1477-7800-1-8 | oa_comm |
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Int Semin Surg OncolInternational seminars in surgical oncology : ISSO1477-7800BioMed Central London 1477-7800-1-91553325210.1186/1477-7800-1-9ResearchResults of a planned interim toxicity analysis with trimodality therapy, including carboplatin AUC = 4, paclitaxel, 5-fluorouracil, amifostine, and radiation for locally advanced esophageal cancer: preliminary analyses and treatment recommendations from the North Central Cancer Treatment Group Jatoi Aminah [email protected] James [email protected] Michelle R [email protected] Bradley S [email protected] Jeffrey S [email protected] Frank [email protected] Normand [email protected] Kendrith [email protected] Loren [email protected] Steven [email protected] Mayo Clinic, Rochester, Minnesota, USA2 Iowa Oncology Research Association Cancer Cooperative Oncology Program, Des Moines, Iowa, USA3 Altru Health Systems, Grand Forks, North Dakota, USA4 Toledo Community Hospital Cancer Cooperative Oncology Program, Toledo, Ohio, USA5 Sioux Community Cancer Consortium, Sioux Falls, South Dakota, USA2004 8 11 2004 1 9 9 22 6 2004 8 11 2004 Copyright © 2004 Jatoi et al; licensee BioMed Central Ltd.2004Jatoi et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Purpose
An aggressive trimodality approach from the Minnie Pearl Cancer Research Network [carboplatin AUC = 6, days 1 and 22; 5-fluorouracil 225 mg/m2 continuous infusion, days 1–42, paclitaxel 200 mg/m2, days 1 and 22; 45 Gy] has resulted in remarkable pathologic response rates but notable toxicity. This trial was designed to mitigate this toxicity by starting with a lower carboplatin dose, AUC = 4, and by adding subcutaneous amifostine.
Methods
This phase II trial included patients with locally advanced, potentially resectable esophageal cancer. All were to receive the above regimen with modifications of carboplatin AUC = 4 and amifostine 500 mg subcutaneously before radiation. All were then to undergo an esophagectomy. A planned interim toxicity analysis after the first 10 patients was to determine whether the carboplatin dose should escalate to AUC = 6.
Results
Ten patients were enrolled, and all required dose reductions/omissions during neoadjuvant therapy. One patient died from paclitaxel anaphylaxis. Six patients manifested a complete pathologic response.
Conclusion
With this regimen, carboplatin AUC = 4 for patients with locally advanced esophageal cancer is appropriate.
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Combined modality treatment is often administered for locally advanced esophageal cancer [1,2]. Particularly noteworthy are data from Meluch and others from the Minnie Pearl Cancer Research Network [3]. These investigators published an innovative trial that examined neoadjuvant therapy that consisted of the following: carboplatin area under the curve (AUC) of 6 on days 1 and 22, paclitaxel 200 mg/m2 days 1 and 22, 5-fluorouracil 225 mg/m2 per day by continuous infusion days 1–42 along with radiation 45 Gy in 25 fractions. Yielding some of the most remarkable response rates observed in the treatment of esophageal cancer, this regimen resulted in a pathologic complete response rate of 46% within an initial cohort of 37 patients. Although long-term survival had not been reported, these data appear promising when one acknowledges that pathologic complete response predicts a longer survival and better prospect for cure for patients with this malignancy [4]. Moreover, recent preliminary data suggest that these response rates are reproducible [5,6].
However, this high complete response rate occurred at the expense of substantial treatment-related toxicity. During neoadjuvant therapy, grade 3 or worse leukopenia occurred in 65% of patients. Twenty-two percent of patients required hospitalization for neutropenic fevers. Grade 3 or worse esophagitis occurred in 31%. Over half of patients suffered weight loss of 10 pounds or more during neoadjuvant therapy. Finally, although there were no deaths during the administration of neoadjuvant therapy, the postoperative death rate was 9%.
These toxicity data, coupled with these high response rates, underscore the need to test such novel, aggressive treatments in conjunction with methods to mitigate toxicity. This report describes the planned interim toxicity analysis from an ongoing North Central Cancer Treatment Group (NCCTG) trial that was undertaken with this goal in mind. The NCCTG trial employed two modifications to the Minnie Pearl Regimen in an attempt to mitigate toxicity: 1) carboplatin dosing was reduced from AUC = 6 to AUC = 4 in the first 10 patients with the possibility of dose escalation thereafter; 2) amifostine 500 mg subcutaneously to be given before radiation, as prompted by data from Koukourakis and others [7] as well as by other subsequent reports [8]. An interim analysis of toxicity in this NCCTG trial was planned after enrollment of the first 10 patients to decide whether to continue this trial and, if so, whether to escalate the carboplatin to AUC = 6. The results of this interim toxicity analysis are presented below.
Methods
Overview
This trial was initiated and conducted within the NCCTG. The Institutional Review Boards at each site approved the protocol before patient enrollment, and all patients provided signed informed consent at the time of enrollment.
Eligibility and Exclusion Criteria
Eligibility criteria consisted of the following: 1) age ≥ 18 years; 2) histologic or cytologic evidence of squamous cell carcinoma or adenocarcinoma of the esophagus; 3) surgically resectable tumor, as deemed by a surgeon; 4) Eastern Cooperative Oncology Group (ECOG) performance score of 0–2; 5) anticipated life expectancy of ≥ 12 weeks; 7) the laboratory parameters < 14 days prior to registration of absolute neutrophil count ≥ 1.5 × 109/L, platelet count ≥ 100 × 109/L, total bilirubin ≤ 1.5 times the upper limit of normal, serum creatinine ≤ 1.5 times the upper limit of normal, asparatate aminotransferase ≤ three times the upper limit of normal.
Exclusion criteria consisted of the following: 1) uncontrolled infection; 2) prior chemotherapy for esophageal cancer; 3) pregnancy or unwillingness to utilize contraception if pregnancy was a possibility; 4) New York Heart Association classification III or IV; 5) other severe underlying illness that, in the opinion of the treating oncologist, would make the patient inappropriate for study entry; 6) prior radiation that would overlap anticipated radiation fields; 7) antihypertensive or diuretic medications that could not be safely discontinued, if necessary, for several days during study treatment.
Treatment Regimen
The neoadjuvant treatment regimen consisted of both chemotherapy and radiation. Chemotherapy consisted of carboplatin with an area under the curve (AUC) of 4 to be given intravenously on days 1 and 22, paclitaxel 200 mg/m2 to be given intravenously on days 1 and 22, and 5-fluorouracil 225 mg/m2/day to be given by continuous intravenous infusion on days 1 through 42. In addition, amifostine was to be administered as a 500 mg flat dose subcutaneously immediately before each radiation treatment.
For evaluation purposes, a treatment "cycle" is defined by the administration of carboplatin and paclitaxel, where the initiation of these agents heralded the start of a new chemotherapy "cycle." In effect, cycle 1 occurred during days 1–21 of the treatment, and cycle 2 between days 22–42.
The protocol was written to allow for a dose increase of carboplatin to an AUC of 6 in the event that a planned interim toxicity analysis after the first 10 patients deemed this increase could be undertaken safely.
Radiation was prescribed at a total dose of 4500 centigray. Each fraction size was 180 centigray, and a total of 25 fractions were to be given.
Chemotherapy dose reductions were initiated based on toxicity. As determined by the National Cancer Institute's Common Toxicity Criteria (NCI CTC), version 2, Grade 3 or worse stomatitis, esophagitis, or diarrhea prompted holding 5-fluorouracil until symptoms were grade 2 or better. If treatment was held for diarrhea, the protocol called for resuming the continuous infusion 5-fluorouracil at 80% of the prior dose. 5-Fluorouracil that was held was not to be made up. Severe myelosuppression on weekly blood counts (absolute neutrophil count ≥ 0.5 × 109/L for greater than 2 days and/or platelet count ≤ 25 × 109/L) prompted a 25% dose reduction of both paclitaxel and carboplatin on day 22. Similarly, on day 22, the protocol mandated that paclitaxel and carboplatin be held until the absolute neutrophil count was ≥ 1.5 × 109/L and the platelet count ≥ 100 × 109/L. For grade 3 or worse esophagitis, the paclitaxel and carboplatin were held until the esophagitis resolved to grade 1 or less. Paclitaxel was also held for grade 3 or worse neuropathy. Re-treatment was permitted with a 30% dose reduction in the event the neuropathy resolved to grade 2 or better. For any grade 3 or 4 event attributable to amifostine, the amifostine was to be decreased to 300 mg and subsequently discontinued if the event recurred at the lower dose. Finally, radiation was to be held for myelosuppression (absolute neutrophil count < 1.0 × 109/L and/or the platelet count < 50 × 109/L) or for grade 4 esophagitis.
Aggressive supportive care measures were recommended throughout the neoadjuvant portion of the regimen. These measures included, but were not limited to, premedication with corticosteroids prior to paclitaxel, use of antiemetics before and during chemotherapy and before amifostine, and nutrition and hydration support, as needed.
An esophagectomy was to be performed within 4–8 weeks after completion of radiation for all patients still deemed to be operative candidates.
The protocol also included an optional, additional two cycles of post-operative chemotherapy with paclitaxel and carboplatin with dosing, for the most part, left to the discretion of the treating oncologist.
Pretreatment and Follow Up Evaluations
All patients underwent a history and physical examination within 21 days of trial registration. Other testing was performed within this time frame as well and included a complete hemogram, chemistry profile, computed tomography scan or magnetic resonance imaging of the chest and abdomen, chest radiograph, electrocardiogram, and an esophagoscopy. A bone scan was required if the alkaline phosphatase was two times the institution's upper limit of normal, and a bronchoscopy was required if the primary tumor was adjacent to the trachea or left main stem bronchus or if the patient was experiencing respiratory symptoms. Other testing was optional and included endoscopic ultrasound of the upper gastrointestinal tract and positron emission tomography scanning.
All patients were monitored throughout the period of radiation and chemotherapy administration with a weekly history and physical examination and weekly hemograms. History, physical examination, hemogram and chemistry profiles were mandated before days 1 and 22 of chemotherapy.
Tumor assessments were performed two weeks prior to surgery, and RECIST criteria, as recommended by the NCI , were used to determine tumor response [9]. Additionally pathological response was assessed post-operatively.
Other testing, such as quality of life assessment and genotyping, were also obtained but will be reported at a later date.
Statistical Analyses
The purpose of this study was to assess the safety of this treatment regimen for patients with locally advanced esophageal cancer. The protocol called for a three-stage phase II study design with a safety analysis in the first 10 patients. The first 10 patients received treatment, as described above, with a carboplatin AUC = 4. It was decided a priori that if more than two of the initial patients developed grade 4 or 5 non-hematologic adverse events during neoadjuvant therapy or died within 15 days of surgery, then the study would not permit a carboplatin dose increase to an AUC = 6.
All data on toxicity and response are presented descriptively in this initial 10-patient report on the first phase of this trial. Summary statistics, including means and median values, frequency tables, and graphical methods were used to describe the distributions of drug administration, clinical characteristics, and adverse event rates. Unless otherwise specified, all adverse event data are reported regardless of relationship to study treatment.
Results
Demographics
Ten patients were recruited from late April 2001 to early March 2002. Patient characteristics are listed in Table 1, which shows that the median age of the cohort was 63 years (range 49–81) and that nine patients had an Eastern Cooperative Performance Score of 0 with one having a score of 1.
Table 1 Characteristic N = 10
Age Median, in years (range) 63 (49–81)
Male/Female 9/1
Performance Score (ECOG)
0 9
1 1
Drug Administration
During administration of neoadjuvant therapy, all 10 patients required a dose reduction or an omission of drug administration because of treatment-related toxicity (Table 2). Overall, nine patients received both cycles of neoadjuvant chemotherapy.
Table 2 Patient Amifostine dose received (%)* Neoadjuvant chemotherapy dose received (%) 1stcycle/2ndcycle* Disease Status
1 31 81/75 CR**
2 7 71/57 CR
3 16 64/11 PR***
4 27 100/26 PR
5 73 100/100 CR
6 24 52/3 CR
7 63 62**** PR
8 87 86**** dead
9 7 91/28 CR
10 67 72/79 CR
*denotes the lowest percentage of administered drug for a cycle
** complete response (CR)
***partial response (PR)
****second cycle not give
Toxicity
Severe adverse events during neoadjuvant therapy consisted of 1 death (paclitaxel-related allergic reaction); 15 grade 4 events (myelosuppression (11); mucositis (1); dyspnea (1); neutropenia (1); and cerebral vascular accident (1); and 55 grade 3 events of various types (Table 3). Six of eight patients experienced grade 3 or greater adverse events within 15 days of surgery, and four of these were grade 4 events. Some patients experienced more than one event. Specifically, two patients suffered acute respiratory distress syndrome; one patient thrombophlebitis; two patients dyspnea; one patient non-specific constitutional symptoms; one patient neutropenia; and one patient a dysrhythmia. Of the two patients with acute respiratory distress syndrome, one remains alive 6 months after surgery as of this report, and the other died roughly 3 months after surgery.
Table 3 Salient Grade 3 or Worse Adverse Events Attributed to Neoadjuvant Study Treatment
ADVERSE EVENT ABSOLUTE INCIDENCE NUMBER OF PATIENTS WITH EVENT
death (paclitaxel reaction) 1 1
neutropenia 11 9
leukopenia 12 10
febrile neutropenia 5 5
hypotension 4 3
vomiting 4 3
nausea 2 2
dehydration 2 2
diarrhea 2 2
dysphagia 6 6
electrolyte abnormalities 6 4
dyspnea 1 1
pain (non-specific) 1 1
rash 1 1
mucositis 1 1
fatigue 2 2
syncope 1 1
hypersensitivity 1 1
neuropathy 1 1
infection 1 1
melena 1 1
Preliminary Response Data
A total of 9 patients underwent an esophagectomy, demonstrating 6 complete pathologic responses.
Discussion
This report describes a planned interim toxicity analysis of the first 10 patients who were enrolled on a multi-institutional trial for patients with locally advanced esophageal cancer. This NCCTG trial tested a version of the Minnie Pearl Regimen with two main modifications: a drop in the carboplatin AUC dose from 4 to 6 and an addition of amifostine 500 mg subcutaneously (flat dose) prior to each dose of radiation. Preliminary analyses show that this modified regimen resulted in a substantial rate of severe toxicity: one death from a paclitaxel allergic reaction, several episodes of grade 4 neutropenia/leukopenia, one episode of grade 4 mucositis as well as several grade 3 events – all of which were attributable to the study regimen. At the same time, however, this regimen resulted in 6 of 10 patients manifesting a complete pathologic response. The results of this planned analysis led to the NCCTG's decision to reopen this trial with a carboplatin dose of AUC = 4. In effect, the results of this interim analysis suggest that oncologists who might choose to treat patients with the Minnie Pearl Regimen should consider treating with this lower carboplatin dose, especially should they choose to include amifostine for its purported cytoprotective effects.
In fact, a major modification of the Minnie Pearl Regimen in the NCCTG trial described here is the addition of subcutaneous amifostine. It remains unclear whether the addition of amifostine compounded or mitigated the toxicity observed in this trial. Certainly, the inclusion of amifostine into this regimen did not permit a dose escalation of carboplatin to an AUC of 6, as tested in the original Minnie Pearl Regimen. Although amifostine is considered cytoprotective, it also carries toxicity in its own right including nausea, vomiting, and hypotension. Preliminary data suggest that subcutaneous administration of amifostine might be better tolerated than intravenous administration, but these conclusions are not based on large, comparative studies. The fact that the amifostine, carboplatin, 5-fluorouracil, paclitaxel, and radiation were all given in combination as part of the phase II study presented here makes it impossible to sort out attribution as it pertains to an individual study agent, such as amifostine. Should the results of this trial continue to appear promising, a larger phase III trial would be necessary to provide a definitive answer on the contribution of subcutaneous amifostine to the efficacy and toxicity profile of this regimen.
In short, the preliminary toxicity assessment of this trial suggests that the Minnie Pearl Regimen should include a carboplatin dose with an AUC of 4 rather than 6, especially if subcutaneous amifostine is included as part of the regimen.
==== Refs
Walsh TN Noonan N Hollywood D A comparison of multimodality therapy and surgery for esophageal adenocarcinoma N Engl J Med 1996 335 462 7 8672151 10.1056/NEJM199608153350702
Urba S Combined modality therapy of esophageal cancer – standard of care? Surg Oncol Clin N Am 2002 11 377 86 12424857
Meluch AA Hainsworth JD Gray JR Preoperative combined modality therapy with paclitaxel, carboplatin, prolonged infusion 5-fluorouracil, and radiation therapy in localized esophageal cancer: preliminary results of a Minnie Pearl Cancer Research Network phase II trial Cancer J Sci Am 1999 5 84 91 10198730
Kleinberg L Knisely JPS Heitmiller R Mature survival results with preoperativ cisplain, protracted infusional 5-fluorouracil, and 44 Gy radiotherapy for esophageal cancer Int J Radiation Oncol Biol Phys 2003 56 328 334 10.1016/S0360-3016(02)04598-4
Meluch AA Greco FA Burris JR Preoperative paclitaxel, carboplatin, 5-FU and radiation therapy for localized esophageal cancer: final results of a Minnie Pearl Cancer Research Network phase II trial Proceedings of the American Society of Clinical Oncology 2001 636A
Zemanova M Pazdro A Novak F Neoadjuvant chemotherapy with paclitaxel, carboplatin, 5-fluorouracil with consecutive surgery in esophageal cancer Annals of Oncology 2002 13 188
Koukourakis MI Kyrias G Kakolyris S Subcutaneous administration of amifostine during fractionated radiotherapy: a randomized phase II study J Clin Oncol 2000 18 2226 2233 10829042
Anne PR Curran WJ A phase II trial of subcutaneous amifostine and radiation therapy in patients with head and neck cancer Semin Radiat Oncol 2002 12 18 19 11917279
Therasse P Arbuck SG Eisenhauer EA New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada J Natl Cancer Inst 2000 92 205 16 10655437 10.1093/jnci/92.3.205
| 15533252 | PMC529465 | CC BY | 2021-01-04 16:38:34 | no | Int Semin Surg Oncol. 2004 Nov 8; 1:9 | utf-8 | Int Semin Surg Oncol | 2,004 | 10.1186/1477-7800-1-9 | oa_comm |
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World J Surg OncolWorld Journal of Surgical Oncology1477-7819BioMed Central London 1477-7819-2-361549407510.1186/1477-7819-2-36Case ReportRupture of totally implantable central venous access devices (Intraports) in patients with cancer: report of four cases Filippou Dimitrios K [email protected] Christoforos [email protected] Georgios K [email protected] Athanasios [email protected] Spiros [email protected] Dept. of Surgical Oncology, Agii Anargiri Kifissias Anticancer Hospital, Greece2 1st Surgical Department, GPHP "Tzanion" Hospital, Piraeus, Athens, Greece3 3rd Surgical Department, Piraeus Anticancer Hospital "Metaxa", Piraeus, Greece2004 19 10 2004 2 36 36 1 7 2004 19 10 2004 Copyright © 2004 Filippou et al; licensee BioMed Central Ltd.2004Filippou et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Totally implantable central venous access devices (intraports) are commonly used in cancer patients to administer chemotherapy or parenteral nutrition. Rupture of intraport is a rare complication.
Patients and methods
During 3 years period, a total of 245 intraports were placed in cancer patients for chemotherapy. Four of these cases (two colon cancer and one each of pancreas and breast cancer) had rupture of the intraport catheter, these forms the basis of present report.
Results
Mean time insitu for intraports was 164∀35 days. Median follow-up time was 290 days and total port time in situ was 40180 days. The incidence of port rupture was 1 per 10,000 port days.
Three of the 4 cases were managed by successful removal of catheters. In two of these the catheter was removed under fluoroscopic control using femoral route, while in the third patient the catheter (partial rupture) was removed surgically. One of the catheters could not be removed and migrated to right ventricle on manipulations.
Conclusion
Port catheter rupture is a rare but dreaded complication associated with subcutaneous port catheter device placement for chemotherapy. In case of such an event the patient should be managed by an experienced vascular surgeon and interventional radiologist, as in most cases the ruptured catheter can be retrieved by non operative interventional measures.
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Background
Totally implantable central venous access devices (intraports) are commonly used in patients with cancer to administer chemotherapy, blood and blood products, antibiotics, parenteral nutrition and to obtain blood samples for laboratory analysis. The catheter is usually placed in the subclavian vein under local anesthesia. There are many complications associated with this technique like hemothorax, pneumothorax, pocket infection, infection of the tunnel or the port, bleeding, hematoma and thrombosis of the catheter or the vein. A very rare complication of the intraport catheter is rupture inside the subclavian vein [1].
The literature regarding common complications is abound, however there is very little information on port ruptures. Some attempts has been made to correlate these with type of port used, site of placement, type of chemotherapy, duration of catheter use etc [2,3]. However, what is ideal is still debatable. We report here 4 cases of intraport ruptures encountered in our practice.
Patients and methods
Between June 1995 and May 1998, 245 patients underwent intraports insertions at the "Agii Anargiri" Anticancer Hospital of Kifissia, Athens, Greece, and were followed up for possible device-related morbidity. Baseline demographic information and the indication for the placement were obtained from the retrospective review of patients' medical and operative reports. Follow-up continued till the device was removed or the patient died. Follow-up time ranged from 1 month to over 3 years. Port-A-Cath® with titanium portal and detachable silicon rubber catheter (Arrow International™, USA) were used in all cases. All patients requiring removal or replacement of the device, prior to completion of the intended treatment were identified from the operative room and hospital records.
All intraports were inserted by percutaneous access technique to the subclavian vein. The tip of the catheter was positioned in the superior vena cava or the right atrium. The ports were then placed within a subcutaneous pocket created on the anterior chest wall. The position was documented by immediate intraoperative chest roentgenogram. Four of these patients had port rupture of which three were complete and one partial, one of these ruptures was accidental.
Routine removal of the subcutanous intraport was carried out in the operating room under local anesthesia with xylocaine 1% (10 cc). An incision was placed on the skin in the area over the drum of the device. Then the drum was prepared by cutting off the tissues that surround the port. The port and the catheter were caught with Kocher forceps. The tissues around the catheter were dissected and the catheter was slowly pulled out. The catheter was cut into three pieces and was sent for culture. Patients were discharged after two hours of observation.
Results
Over a three year period between 1995 and 1998 a total of 245 port devices were fitted in cancer patients (139 females and 106 males). Mean age of the patients was 58 ± 6.3 years. Total port time in situ was 40180 days, while mean (SD) port time in situ was 164 (35) days.
After a median follow-up of 290 days (range 30–690 days) four ports ruptured, thus bringing the number of fractures per 1000 port days to 0.01% or one rupture every 10,000 port days. Two patients had cancer of the colon and one each had cancer of breast and pancreas. Both patients with colonic cancer received 5 flurouracil and leukovorin, patient with breast cancer received epirubicin and paclitaxal and patient with pancreatic cancer received gemcitabine, paclitaxal and cisplatinum. No correlation was observed with type of chemotherapy or site of disease. Three of the port ruptures were full and one partial one of the ruptures was accidental (case 1). The detail of the cases has been provided in next section. Three of the patients survived for two years after the catheter removal, while the fourth patient with accidental port rupture and whose catheter was left behind in the right ventricle died of progressive disease two month later.
Case 1
A 65-years-old female with colonic cancer, received intraport for chemotherapy administration. After 6 months due to the poor response to chemotherapy it was decided to remove the catheter. During the manipulations for removal, the catheter, accidentally ruptured at the point of its entrance in to subclavian vein. The peripheral part of the catheter remained in the vein, while only the central part could be removed. Another attempt to uncover the subclavian vein till superior vena cava failed. Patient underwent thoracotomy for removal of remaining catheter two days later. Superior vena cava was opened and catheter removal was attempted, however, during this process the catheter slipped into right atrium and further attempts to retrieve it were abandoned. Patient was started on anticoagulant treatment using enoxaparin sodium, 1 mg/kg/12 h for 5 days and 40 mg/day for further 14 days in order to prevent thromboembolic event. The patient died two months later due to progressive disease without obvious complications related to the retained catheter.
Case 2
A 68-year-old female with colonic cancer received intraport for administration of chemotherapy. After one and a half year of treatment it was decided to remove the catheter as the catheter has thrombosed due to non heparinization. At the time of its removal the catheter ruptured at the point of its entry to subclavian vein. The peripheral part of the catheter remained in the vein. An unsuccessful attempt was made to expose subclavian vein till superior vena cava. Later this catheter migrated to right ventricle. The catheter was removed using the technique of Yedlicka et al [5], through the left femoral vein by advancing a vessel catheter to right ventricle under fluoroscopic control. The broken catheter was caught with endovessel forceps and was removed through femoral vein.
Case 3
A 74-years-old female suffering from breast cancer underwent intraport insertion for administration of chemotherapy. After fourteen months of treatment it was decided to remove the catheter as the catheter had thrombosed. On attempted removal the catheter was found to be ruptured at its entry to subclavian vein (Figure 1). Next day, the broken part of the catheter was removed successfully under fluoroscopic control using the technique described above for case 2 (Figure 2). No complications were observed. The biomechanical analysis of removed catheter showed a significant decrease in the elasticity of the material (Figure 3).
Figure 1 Chest X-ray showing the port in correct position while the catheter is not shown here. The catheter had moved in the right atrium, from where it was removed fluoroscopically.
Figure 2 X-ray showing the intravascular catheter being fluoroscopically removed by means of a special endo-vessel grasper.
Figure 3 (a) The ruptured catheter had been sent for biomechanical analysis that identified alteration of its elastic properties. (b) The port removed by means of the open technique
Case 4
In a 56-year-old female patient with pancreatic carcinoma underwent an intraport placement for chemotherapy. Eight months later, the patient complained of pain in the back during the administration of chemotherapy. A fluoroscopic examination showed partially broken catheter in the vein while the other part was lying in the subcutaneous tissue. The catheter was removed from the subclavian vein carefully to avoid complete rupture of the catheter. Similar to case 3 above, the biomedical examination showed a significant reduction in the elasticity of the catheter material.
Discussion
Since Aubaniac first described the percutaneous central venous catheterization in 1952, insertion of central venous access devices for fluid administration has increased rapidly. The total complication rate associated with central venous catheter devices ranges from 0.4 to 29% [3,4,6].
Spontaneous rupture of the port catheters appears to be a very rare and dreaded event. Biffi et al, in 1997 [1], reported three cases of port catheter rupture out of 178 ports inserted by them. The incidence of port rupture was estimated to be 1.68 % (0.09/1000 port days). In their series the rupture occurred 66 days after the placement, during a pause between subsequent chemotherapy cycles. The symptoms consisted of palpitations and chest discomfort in two while the third patient remained asymptomatic. All the catheters in their series were removed by interventional technique without any complications [1,5].
The biomechanical analysis, of ruptured catheters in our series showed a significant decrease in the elasticity of the material. No correlation between changes of mechanical properties of the material and specific chemotherapy administrated through the port has been established so far [7,8]. Even in our series all patients received differing chemotherapy and no correlation was observed between the agent and property of the material, this is also due to smaller number of such events.
In case 1 the port catheter rupture was due to wrong manipulations during catheter removal and accidental cutting of peripheral part without first holding it in clamp. The further catheter movement was induced by negative intra-thoracic pressure during respiration [9]. Other potential cause of catheter fracture could be incorrect fixation of the catheter to the locking steel ring, repeated high pressure injections to resolve clot formation, alteration of the catheter mechanical properties etc. In two of our cases decreased elasticity of the material was found however one case could not be attributed to any know cause. There was no correlation of port rupture with type of chemotherapy or site of cancer probably due to only 4 events.
Ballarini et al [10], suggested that catheter thrombosis mainly occurs due to incorrect fixation of the locking steel ring to the port where it is associated with rupture. The estimated incidence of port catheter rupture and embolization varies from 0.9 to 1.7% of the cases [1,10-12]. It was 1.65% in our series.
There is no controversy that the foreign bodies inserted in the systematic circulation must be removed. This is best achieved under fluoroscopy with specific catheters and snare loops. Removal of intravascular material by means of minimally invasive techniques presents excellent results, while at the same time it minimizes morbidity and mortality [13,14]. If such an attempts fail, open surgery should be considered.
Conclusions
Intravascular rupture of subcutaneous port catheters is a rare complication. Etiology still remains elusive, however wrong placement has been advocated as the most important cause. Other causes include catheter material faults and alterations of the material's mechanical properties, probably due to the administered substances; however, there is no data to support the effect of administered substances. Ruptured catheters are best removed by minimally invasive radiological techniques.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
DF participated in the operations, and drafted the manuscript.
CT participated in the operations, patients follow-up search of literature and preparation of draft.
GF conducted the follow-up and helped to draft the manuscript.
AN and SR participated in the operations, design of the study and preparation of manuscript for publication.
All authors read and approved the final manuscript.
Acknowledgments
Written informed consent was obtained from all the four patients reported here.
We also thank Mrs Argiro Triga for her help in the revision of the manuscript.
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Biffi R Corrado F de Braud F de Lucia F Scarpa D Testori A Orsi F Bellomi M Mauri S Aapro M Andreoni B Long-term, totally implantable central venous access ports connected to a Groshong catheter for chemotherapy of solid tumors. Experience of 178 cases using a single type of device Eur J Cancer 1997 33 1190 1194 9301441 10.1016/S0959-8049(97)00039-7
Kurul S Saip P Aydin T Totally implantable venous-access ports: local problems and extravasation injury Lancet Oncol 2002 3 684 692 12424071 10.1016/S1470-2045(02)00905-1
Talfer S Conessa C Herve S Rogouet E de Rotalier P Poncet JL [Complications with totally implantable venous access device. A retrospective of 116 cases] Presse Med 2003 32 1263 1268 French 14506448
Aubaniac R L' injection intraveineuse sous-claviculaire: Avantages et technique Presse Med 1952 60 1456 1458 13027062
Yedlicka JW JrCarlson JE Hunter DW Castaneda-Zuniga WR Amplatz K Nitinol goose-neck snare for removal of foreign bodies. Experimental study and clinical evaluation Radiology 1991 178 691 693 1994404
Vasquez RM Brodski EG Primary and secondary malposition of silicon central venous catheters Acta Anaesthesiol Scand 1985 81 22 25
Koulas S Karahalios N Kotzias N Filippou D Efthimiadou S Nissiotis A Infectious and other complications in totally implantable venous access port catheters In the Proceedings of the Sixth Annual Congress of Intensive Care Medicine, 9–10 October 1997; Athens, Greece Produced by the Hellenic Society of Intensive Medicine 1997 41
Brothers TE Von Moll LK Niederhuber JE Roberts JA Walker-Andrews S Ensminger WD Experience with subcutaneous infusion ports in three hundred patients Surg Gynecol Obstet 1988 166 295 301 3127896
Marie O Leverger G Douard MC Mourey F Roche A Eurin B Migration intravsculaire de fragments de catheters veineux centraux Presse Med 1986 15 1270 1272 2945181
Ballarini C Intra M Pisani Ceretti A Cordovana A Pagani M Farina G Perrone S Tomirotti M Scanni A Spina GP Complications of subcutaneous infusion port in the general oncology population Oncology 1999 56 97 102 9949293 10.1159/000011947
Tazzioli G Roncaglia G Impiego dei sistemi venosi totalmente impiantabili nelle terapie infusive di lunga durata Chirurgia 1995 8 93 95
Barrios CH Zuke JE Blaes B Hirsch JD Lyss AP Evaluation of an implantable venous access system in a general oncology population Oncology 1992 49 474 478 1465287
Biffi R de Braud F Orsi F Pozzi S Mauri S Goldhirsch A Nole F Andreoni B Totally implantable central venous access ports for long-term chemotherapy. A prospective study analyzing complications and costs of 333 devices with a minimum follow-up of 180 days Ann Oncol 1998 9 767 773 9739444 10.1023/A:1008392423469
Biffi R Pozzi S Agazzi A Pace U Floridi A Cenciarelli S Peveri V Cocquio A Andreoni B Martinelli G Use of totally implantable central venous access ports for high-dose chemotherapy and peripheral blood stem cell transplantation: results of a monocentre series of 376 patients Ann Oncol 2004 15 296 300 14760125 10.1093/annonc/mdh049
| 15494075 | PMC529466 | CC BY | 2021-01-04 16:38:38 | no | World J Surg Oncol. 2004 Oct 19; 2:36 | utf-8 | World J Surg Oncol | 2,004 | 10.1186/1477-7819-2-36 | oa_comm |
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Hum Resour HealthHuman Resources for Health1478-4491BioMed Central London 1478-4491-2-141550930510.1186/1478-4491-2-14ReviewDual practice in the health sector: review of the evidence Ferrinho Paulo [email protected] Lerberghe Wim [email protected] Inês [email protected]ólito Fátima [email protected] André [email protected] Associação para o Desenvolvimento e Cooperação Garcia de Orta, Lisbon, Portugal2 World Health Organization, Geneva, Switzerland2004 27 10 2004 2 14 14 5 5 2004 27 10 2004 Copyright © 2004 Ferrinho et al; licensee BioMed Central Ltd.2004Ferrinho et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
This paper reports on income generation practices among civil servants in the health sector, with a particular emphasis on dual practice. It first approaches the subject of public–private overlap. Thereafter it focuses on coping strategies in general and then on dual practice in particular.
To compensate for unrealistically low salaries, health workers rely on individual coping strategies. Many clinicians combine salaried, public-sector clinical work with a fee-for-service private clientele. This dual practice is often a means by which health workers try to meet their survival needs, reflecting the inability of health ministries to ensure adequate salaries and working conditions.
Dual practice may be considered present in most countries, if not all. Nevertheless, there is surprisingly little hard evidence about the extent to which health workers resort to dual practice, about the balance of economic and other motives for doing so, or about the consequences for the proper use of the scarce public resources dedicated to health.
In this paper dual practice is approached from six different perspectives: (1) conceptual, regarding what is meant by dual practice; (2) descriptive, trying to develop a typology of dual practices; (3) quantitative, trying to determine its prevalence; (4) impact on personal income, the health care system and health status; (5) qualitative, looking at the reasons why practitioners so frequently remain in public practice while also working in the private sector and at contextual, personal life, institutional and professional factors that make it easier or more difficult to have dual practices; and (6) possible interventions to deal with dual practice.
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Introduction
Doctors and nurses in government employment are labelled "unproductive", "poorly motivated", "inefficient", "client-unfriendly", "absent" or even "corrupt". These labels are often associated with coping strategies associated with widespread "demotivation", due partly to "unfair public salaries". These are presented as the de facto justification of "inevitable" predatory behaviour and public-to-private brain drain [1-6]. In many countries, developed and developing alike, this has eroded the implicit psychological and social contracts that underlie the civil service values of well-functioning public organizations [7]. As a result, public servants often resort to dual or multiple employment.
This paper reports on income generation practices among civil servants in the health sector, with a particular emphasis on dual practice. It first approaches the subject of public–private overlap. Thereafter it focuses on coping strategies in general and on dual practice in particular.
Public–private overlap
Private providers capture a significant and growing share of the service delivery market for health care, and ensure an important part of the uptake of services. For a sample of 40 developing countries, an average of 55% of physicians worked in the private sector and an average of 28% of health care beds were private beds (21% private, for profit) [8].
Asia has more than 60% of private sector contributions to health care financing (excluding China and India) and is the part of the world where the private sector normally plays the dominant role. In Malaysia, for example, the proportion of physicians in private practice increased from 43% in 1975 to 70% in 1990. In Indonesia half of the hospitals are privately run. In Thailand the share of beds in private hospitals grew from 5% in 1970 to 14% in 1989.
The contribution of the private sector to health care financing in Africa is 50%, with a significant preponderance of private, not-for-profit, nongovernmental (PNFP-NGO) entities, particularly church organizations. In Zimbabwe church missions provide nearly 70% of all beds in rural hospitals and in Tanzania they run 40% of the hospitals. In Kenya, about one third of the total health services and 40%–50% of the family planning services are provided by PNFP-NGOs. In Latin America the private sector finances 40% to 60% of the health sector [9].
For many populations, especially in rural areas, these PNFP-NGOs are the main – if not the only – providers of health care. For example, in a mail survey of 88 nongovernmental hospitals in sub-Saharan Africa at the end of the 1980s, 39 were the only service provider in their district [10,11]. It is also the case, for example, of rural Congo in the 1990s, where only NGO-supported districts continued working [12]. Following the reforms of the early 1990s in Mali, most ambulatory health care – nearly all in rural areas – is now provided by community-owned PNFP health centres organized in a nongovernmental federation [13]. But these are mainly rural situations.
In urban areas PNFP-NGOs usually share the work with private, for-profit (PFP) providers and government services. In Alexandra, South Africa – a poor periurban township – for example, a PNFP university clinic, a municipal clinic and PFP "general dispensing cash practices" worked side by side [14,15]. But in general, the market share of private providers is more limited.
PFP health providers are no doubt an important source of ambulatory care throughout the developing world, but tend to concentrate on the more profitable niches of the market. The development of private practice in most developing countries is notoriously unregulated. Private practices are not easily forthcoming with information, at times for fear of tax implications, at times because existing regulations are not respected, often because of a lack of respect for discredited ministries of public health and not infrequently because of the non-existence of information systems. In transitional countries, such as Tunisia, the growth of the entrepreneurial sector is well documented.
Although hard data are hard to come by for the poorer countries, it is sufficient to walk around Luanda or Dar es Salaam or to look at the advertising sections of any newspaper in Maputo to see that private health care provision is a thriving growth industry. If there are ever more private providers on the market, not all provide the whole range of health services. They tend to select niches according to demand and competition rather than providing comprehensive care.
There are huge differences between and within countries. This (patchy and scarce) evidence confirms that both PFP and PNFP providers have a significant and growing share of the market of health care, but this statement needs qualification. There are wide differences between and within countries. Overall, the market share of the private sector is smaller for inpatient than for ambulatory care, and limited for preventive and public health services [8].
Coping strategies
Individual coping strategies represent the health professionals' ways of dealing with unsatisfactory living and working conditions. In many countries their prevalence has increased over recent years. The notion of the full-time civil servant exclusively dedicated to his/her public sector job is disappearing. Were this without consequences for the performance of the public health sector, it would not be much of a problem. Not all coping strategies can be classified as predatory behaviour or corruption, and their effects on the health care system can be positive as well as negative. They cannot, however, be ignored as, in many countries – particularly the poorest – the situation has gotten out of hand.
Most would agree that public sector salaries are most often "unfair". For example, in 1999 a Mozambican nurse's salary was only 10% to 15% of what it had been 15 years before [16]. In Sierra Leone most health professionals, physicians included, are employed by the public sector at salaries under USD 100 per month [17]. In many other countries health staff is going through similar experiences. In Russia, state doctors earn between USD 15 and USD 50 per month [18]. In the Dominican Republic, in 1996, physicians with 20 years of experience earned the same as new medical graduates, rewards for good performance were impossible and personnel were paid regardless of whether they performed their duties [19]. In such a context, demotivation, overall lack of commitment and low productivity are to be expected.
To compensate for unrealistically low salaries, health workers rely on individual coping strategies [1-6,18,20-54]. Many clinicians combine salaried public sector clinical work with a fee-for-service private clientele [3-6,25-29,32,38-41,44,45,53]. Others resort to absenteeism [24,44,45], or predatory behaviour, asking under-the-counter payments for access to services intended to be free of charge [33,34,46] or goods and/or misappropriating drugs or other supplies [20,21,27,43,49] and referral of public sector patients to private practices [23].
In Thailand 37% of patients pay their public sector obstetricians "gratitude money" [55]. In France, mainly for surgical interventions, under-the-table remuneration is very common in hospital or ambulatory settings [56]. In Greece, doctors and nurses have been criticized for receiving gifts and bribes [57].
Another example is fee splitting, whereby a specialist shares a fee with the referring physician [23]. In 1998, for example, a group of Italian general practitioners was suspended for accepting payments to send their patients to a particular private centre for radiology examinations [58]. It is common practice in the United Kingdom for consultants to spend time in private clinics when they should be attending to their public duties [23].
Patients and practitioners may collude to deceive a third agent: in Kazakhstan, for example, it is reported that doctors regularly provide false health reports in return for a fee, so that patients can obtain driving licenses [23]. A further form of fraud through misinformation is exemplified by the case of the English GP who forged consent forms for patient participation in medical trials in order to boost income [53]. The problems these coping strategies create are increasingly recognized [6,38-40]., although the subject remains taboo for many ministries of health and development agencies.
Drugs are – in the current context of scarce resources, health care reform, promotion of generics, the HIV epidemic and growing demand for health care – a sensitive issue, as in many low-income countries, pharmaceuticals make up 50% or more of health care costs [59]. With health sector reforms, private sector pharmacies are increasingly becoming the first and sometimes the only outlet for the delivery of health services [60-63]. In this environment, and for several reasons – including the "business profession dilemma" in private pharmacy practice – irrational prescription can become a major problem [64-67]. Antibiotics are often sold without a prescription [68,69].
In other settings, such as hospitals and health centres, misappropriation is a widespread practice by all categories of professionals. This is infrequently acknowledged explicitly or documented, even in studies that have looked into the coping strategies of health professionals [3-5,28,29,41,44,45]. It has nevertheless been documented in a number of African [20,21,27,43,49] and Latin American countries [22]. Where documented it is perceived as common practice.
In Uganda, for example, misuse of pharmaceuticals was reported by facility health workers as well as by the District Health Teams and the Health Unit Management Committees; resale of drugs represented the greatest single source of income for health workers in most units [27,43]. In many other developing countries the situation is supposed to be similar if not worse.
Dual practice
We have stated that many clinicians combine salaried public-sector clinical work with a fee-for-service private clientele [3-6,25-29,32,38-41,44,45,53]. Dual practice may be considered present in most countries, if not all. Nevertheless, there is surprisingly little hard evidence about the extent to which health care workers resort to dual practice, about the balance of economic and other motives for doing so, or about the consequences for the proper use of the scarce public resources dedicated to health. Dual practice is often a means by which health workers try to meet their survival needs, reflecting the inability of health ministries to ensure adequate salaries and working conditions.
In this paper dual practice is approached from six different perspectives: (1) conceptual, regarding what is meant by dual practice; (2) descriptive, trying to develop a typology of dual practices; (3) quantitative, trying to determine its prevalence; (4) impact on personal income, the health care system and health status; (5) qualitative, looking at reasons for its prevalence and for staying in public practice while working in the private sector and at contextual, personal life, institutional and professional factors that make it easier or more difficult to have dual practices; and (6) possible interventions to deal with dual practice.
What is meant by dual practice
Dual practice is approached in the literature with great diversity. It can mean health professionals with multiple specializations – for example, among Egyptian physicians the most popular areas of multiple specializations are "cardiology and internal medicine, internal medicine and fever and cardiology and chest" [70].
It can also mean health professionals working within different paradigms of health (allopathic medicine combined with traditional medicine – Chinese, African or otherwise – or with other paradigms such as osteopathy, homeopathy and others). It may involve combining different forms of health-related practice – clinical with research, with teaching or with management. It can also mean health professionals combining their professional health practice with an economic activity not related to health (such as agriculture).
In this paper, dual practice will be used to describe multiple health-related practices in the same or different sites.
An operational typology of dual practices
Therefore, in terms of sector location, dual practice may be public on public, public on private or private on private (Table 1).
Table 1 Typologies of dual practice
Public Private, for-profit Private, not-for-profit
Public + + +
Private, for-profit + +
Private, not-for-profit +
Overtime may be considered a form of dual employment. It is increasing because of cost-containment measures or shortages of staff. In 1992, the German Union of Salaried Employees estimated that the overtime worked by health workers was equivalent to 20 000 extra full-time staff posts. Today, trade unions in Canada and the United Kingdom express particular concern about the use of overtime to substitute for recruitment and about the increase in unpaid overtime [71].
The extent of dual practice
As mentioned and supported by the limited literature available (Table 2), dual practice is probably present in all countries regardless of income, even in settings – such as China – where there are major regulatory restrictions [29,55,70,72-82]. In Latin America physicians usually hold jobs in both public/social security and private systems [83].
Table 2 The extent of dual practice in several countries
Country Type and frequency of dual practice
Angola Dual (public and private) practice is ubiquitous and unregulated [72].
Cambodia Dual (public and private) practice is ubiquitous [89].
Egypt Rural-based Egyptian physicians in private practice are more likely to have a second job (85%) than urban-based physicians (71%). However, there is not much difference between urban and rural physicians in the likelihood of having a third or fourth job: 15% of urban and 11% of rural physicians have a third job and 2% of urban and 1% of rural physicians have a fourth job. Twenty percent of 113 single private practice dentists work only in their private clinic; 73% have 2 jobs, 6% have 3 jobs and 1% have 4 jobs. Among 261 pharmacists 91% have only one job, 8% have two jobs and 1% have 3 jobs. Among 80 other health service providers, mainly unlicensed, who are officially not allowed to operate, but yet provide a significant amount of health care, 66% of the sample have a second job and 1% has a third job [70].
Indonesia Most doctors have dual practices in the public and private sectors [77].
Malawi The government allows serving medical personnel in its facilities to set up private surgeries where they can practice after official duty hours; it further allows those without professional qualifications (e.g. "paramedics") to set up a health care business for minor health complaints [78].
Mozambique Common among urban, but not rural, health professionals [29].
Papua New Guinea Semi-private wards in public facilities are well patronized in the larger hospitals but tend to be underutilised in the smaller provincial centres [79].
Peru Almost all physicians have both public and private practices [77].
Portugal 23% of public sector health centre workers have a second job, the highest rate being for doctors – 43%; 58% of public sector hospital workers have a second job, the highest rate being for doctors – 50% [75, 76,75, 76].
South Africa Half of general practitioners in private practice have other employment. While 36% worked in the public sector, this was more common in rural (62%) compared with urban (21%) areas [80].
Syria Most physicians have dual practice [77].
Thailand An estimate suggested that in Bangkok alone there were over 2000 private clinics, many of these run by government doctors [81]. Private practice by public sector obstetricians is very frequent [55].
Viet Nam Most doctors complement public sector work with private practice [77]. Full-time government employees are supplementing their incomes through part-time private practice. One village-based health survey found that 70% of the drug sellers were moonlighting government workers [82].
The impact of dual practice
Predatory behaviour
Dual practice may lead to predatory behaviour (behaviour in which self-gain is pursued to the detriment of the legitimate interests of colleagues, services and/or patients)[84] by health workers. This is particularly strong in situations where market conditions – usually high physician supply, as is the case of capital cities in Africa and other urban more than rural areas – would otherwise reduce their incomes. In these situations clinicians use their authority to prescribe treatment for their patients to generate additional demand for their own services.
This hypothesis, controversial for some practices such as caesarean sections, seems consensual regarding other surgical practices [85]. In Thailand, for example, it is well demonstrated that antenatal care is sought in private ambulatory facilities, while caesarean sections are offered in public hospital facilities. Caesarean-section rates among private patients (46%) are three times higher than among non-private patients (16%), which indicates that private practice by public obstetricians is a strong determinant of caesarean sections [55].
The predatory behaviour of individual clinicians constitutes, in many cases, a de facto financial barrier to access to health care [21]. More important, in the long run, is that it delegitimizes public sector health service delivery and jeopardizes the necessary relation of trust between user and provider [20].
Conflicts of interest
A more insidious problem is that of conflicts of interest. Effects on the system can best be looked at separately for each type of side activity. When health officials set up in dual practice to improve their living conditions – or merely to make ends meet – this may not interfere with their work as civil servants (although it is likely to compete for time and to reinforce rural-to-urban migration). When they take up teaching as an extra job, usually in public sector institutions, that may actually be beneficial to the public agenda, as it reinforces the contact of trainees with the realities of the health services.
For doctors who are basically managers, moonlighting in private practice presents less of a conflict of interest than for clinicians. The latter must compete for patients with themselves, and thus they have an incentive (and the opportunities) to lower the quality of the care they provide in the public services. This is not the case for managers: involvement in NGO projects or work for donors can foster better coordination in the provision of services, but may constitute a conflict of interest when NGO or project policies are not necessarily congruent with national health policies or the agenda of the public service [4,44,52,55].
Other business activities, such as agriculture, are neutral towards health services, although they may constitute a de facto internal brain drain [3].
Brain drain
Dual practice is to a large extent a question of creating and making use of opportunities. The pursuit of such opportunities contributes also to brain drain.
Brain drain of health professionals is often thought of only in terms of intercountry migration [86]. But failure to post and retain the right person at the right place is not merely a question of a Congolese doctor's deciding to move to South Africa or a Philippine nurse's moving to the United States. It is also a question of internal – including public to private sector – "migration".
This public-to-private migration compounds the rural-to-urban migration because cities also offer more opportunities to diversify income generation [28,29]. The need to make up for inadequate salaries – and to be in a setting where there are opportunities to do so – thus fuels rural-to-urban migration and resistance against redeployment to rural areas [4,28,29]. Professionals who have successfully taken advantage of these urban opportunities increase their market value over time, until they are ready to leave public service. Rural-to-urban brain drain thus is later compounded by public-to-private brain drain.
Competition for time and limits to access
Coping strategies, including dual practice, also affect access, but through competition for time. In many countries, civil service medical staff is available only nominally full-time to fulfil its assigned tasks. This has been well described regarding Colombia, Costa Rica and Venezuela. In Venezuela doctors and head nurses missed about one third of their contracted service hours, 37% and 30% respectively, while resident doctors and nurses were absent about 7% and 13% of the time, respectively [47]. In Peru 32% of doctors and nurses considered absenteeism common or very common [24]. In Costa Rica 65% of doctors and 87% of nurses felt that physicians were unjustifiably absent from work or, even if present at work, often saw private patients on public time in public facilities [22]. Absenteeism in the hospitals of Bogotá, Colombia, may cost over USD 1 million a year [35].
If public sector medical staff is moonlighting in private practice, this evidently limits access by public patients. This corresponds to a net flow of resources out of the public sector. Competition for time is also something that concerns managers, whose coping strategies are often more oriented towards collaboration with development agencies [44,45].
Competition for time is a nagging problem for many development agencies and ministries of health. At times it is blatant. In Mali, for example, regional health staff was found to spend 34% of its total working time in (income-generating) workshops and supervision missions supported by international agencies; for chief medical officers it was 48% (El Abassi & Van Lerberghe, unpublished data, 1995). The 73% of working time spent on official duties that was self-reported by the respondents to one of the surveys reviewed may well be an overly optimistic estimate [44,45].
In Egypt the number of hours worked in the private clinic falls as the number of a physician's jobs increases, indicating that the physician replaces hours away from the private clinic with other employment. Secondly, as the number of jobs increases, the amount of time spent in the second job, usually a government job, decreases. This reduces the access of low-income people to medical care, as they cannot afford to seek care in the private sector.
Econometric analysis of the data also found that physicians replace hours they should be working at the government job by hours in the private clinic. This has important policy implications, as multiple employment is not increasing the access of the population to medical care. On average, physicians see one patient per hour in their private clinic. This increases to 3.2 patients per hour in the second job, 4.4 in the third job and 2.9 in the fourth job. An extreme example is that of general practitioners who see one patient per hour in their private clinic, approximately four in the second job and 12 in their third job. In general, the number of patients seen per hour increases with the job number. This is true for physicians as well as dentists [70].
Competition for time automatically results in a transfer of salary resources out of the public sector through reduced availability – at least the equivalent of 27% of the salary mass [44,45].
Outflow of resources
Besides competition for time, in many cases the use of the public sector's means of transportation, office infrastructure and personnel represent additional hidden outflows of resources, often in association with dual practice. The overall impact of this outflow of resources is hard to quantify in any country. Reports from Moscow suggest that up to 30% of the federal budget is not accounted for [18,34]; in the United Kingdom, estimates of GBP 115 million are given for prescription fraud alone [23].
The loss to the public sector associated with redirection of diagnostic and therapeutic resources, such as pharmaceuticals, to private practice or into the black market is obviously difficult to assess. In Uganda, for example, it results in a significant loss to the public health facilities: the median drug leakage in health facilities was estimated at 78% [27,43]. In the Dominican Republic, almost one third of total hospital expenditure remains unaccounted for, representing some combination of theft of materials and supplies, diversion of funds and gross mismanagement [37]. In Panama, high-value medications were stolen on a daily basis, with significant losses to the hospital [87]. In Venezuela, between 10% and 13% of all medical supplies and medications were stolen [47].
In Costa Rica, 71% of doctors and 83% of the nurses reported that equipment and materials had been stolen in their hospital [22]. In the United Kingdom it is estimated that pilfering – of bandages, medications and stationery, for example – adds up to more than GBP 15 million annually. In an Andalusian hospital in Spain, it was estimated that pilfering of food supplies led to per capita catering costs that were higher than those of a good restaurant [23]. The ultimate purpose of this stealing is not studied, but it is possible to speculate that many stolen resources find their way into the dual practices of public servants.
Competition for time and transfer of resources are compounded by the fact that the best-trained and most competent officials are also the most likely to divert their time to other activities outside the health sector (a de facto brain drain). This in turn reinforces the attraction of what starts out as a job-on-the-side, and quickly becomes not only more rewarding financially but also professionally and in terms of social prestige.
The impact of these coping strategies is seen as negative. These strategies weaken the public sector health structure and damage people's health [20,88].
Impact on income
Individual income topping-up strategies allow professionals a standard of living that is closer to what they expect. In one study, these strategies more than doubled the median income of public sector health managers, and brought it from 20% to 42% of that of a full-time private practice [44,45] (Tables 3 and 4. Fig. 1: The box-plot chart represents for each variable the maximum, 75th percentile, 25th percentile and the minimum [44,45].)
Table 3 Median and interquartile range of take-home salaries of civil servant health service managers
Low-income countries (61 respondents) Middle-income countries (39 respondents)
In USD at official exchange rate 3802 (2137–5249) 11 253 (6704–18 900)
In USD corrected for purchasing power parity 13 890 (9411–20 956) 26 376 (18 416–38 931)
As % of the income of a private practice serving 15 patients per day 14% (10%–33%) 29% (22%–41%)
As % of the income of full-time consultancy work (250 days / year) 31% (23%–44%) 81% (45%–108%)
Source: [44, 45]
Table 4 Median and interquartile range of total income (salary plus extra activities) of civil servant health service managers
Low-income countries (61 respondents) Middle-income countries (39 respondents)
In USD at official exchange rate 5899 (2712–8137) 11 372 (6000–23 040)
In USD corrected for purchasing power parity 21 438 (4081–84 640) 39 377 (26 149–64 338)
As % of the income of a private practice serving 15 patients a day 26% (17%–52%) 42% (29%–64%)
As % of the income of full-time consultancy work (250 days/year) 49% (30%–96%) 115% (74%–172%)
Source: [44, 45]
Figure 1 Distribution of income in USD purchasing power parity, with the increase from extra jobs, compared to distribution of potential income through consultancies or private practice
In Phnom Penh, Cambodia, 90% of a physician's income in dual practice is derived from the private sector activity [89]. In Thailand earnings from private practice among physicians constitute 55% of their total income [55]. The upside is that income topping-up helps to retain valuable expertise in public service [90-96].
Corruption in the health sector
Corruption has been a long-standing concern in development circles. The literature is rich on theories ranging from macrosociological analyses of sociocultural processes to dyadic game theory modelling. Although impressive, this theorizing has not resulted in useful, empirically validated tools to redress this problem [97].
For these reasons, we prefer to understand this issue from the perspective of the context that expects health professionals to have a standard of living that cannot be met by existing social systems, sometimes to a level where they cannot even satisfy their basic needs through their public sector salary.
In Mozambique, for example, medical students seem to know they will be needed in the public sector, and that this would represent an opportunity to contribute to the public's welfare. Nevertheless, in order to improve their earnings their expectations are to combine their public sector practice with private medical work. One third of the medical students expect an income of between USD 715 and USD 1071 a month, and another third expects over USD 1429, at a time when the salary of a newly graduated doctor is about USD 357 a month. This sets the scene for the reality, often unregulated, of dual practice that plagues many countries [98]. It is not surprising that once graduated they resort to dual practice (often already initiated as medical students).
One must distinguish between individual coping strategies and orchestrated activities, acknowledging, nevertheless, that they may be closely interrelated. For example, hospitals with limited budgets in Mozambique, Portugal, Russia and other countries see dual practice as a means of retaining the most senior personnel.
There is a fine line separating coping strategies, including dual practice, from corruption. The difficulty in differentiating between the two starts with the definition of corruption. Widely accepted definitions often have ideological connotations. The definition of corruption as the "private use of public goods" is frequently associated with authors who ultimately defend a greater role for the private sector in the provision of public services such as education and health (as proposed by Van der Geest) [21], but without sufficient evidence of its effectiveness. They fail to acknowledge that corruption in the private sector may be a significant problem [99] and that liberalization and transition from state-controlled systems to systems in which the market plays a greater role have often resulted in more corruption, not less [100].
Another problem with current research is its treatment of corruption as something that would be the same everywhere, with essentially the same causes and implications wherever it occurs [101].
A third problem is that of the moralistic and criminal connotations of the word "corruption". It should be kept in mind that not all that is illegal is corrupt and not all that is corrupt is illegal, and that also a distinction should be maintained between corrupt transactions and those that are immoral [99].
The literature tends to focus on the "corrupt", failing to acknowledge that contexts that generate so-called corrupt behaviours generate them across the whole spectrum of society, to the extent that they become an ingrained and acceptable part of society, a necessary evil to survive in a very harsh environment. This shift from focusing on the persons involved to the system in which the professionals are integrated has taken place in other areas, such as in quality management [102] and in the prevention and control of industrial accidents [103] or medical errors [104]. Sooner or later this is likely to happen with the corruption literature as well.
Corruption has a negative impact on development in general. It hits the poor the hardest, directly and indirectly by, inter alia, reducing their access to public services such as education and health care [105,106].
This negative impact is also felt in indicators of health status. There is an inverse relationship between indices of corruption and ratio of public health spending to GDP and child mortality rates [107]. As such, it cannot be ignored by health sector managers, but labelling is not only misleading and counterproductive: it also does not help mobilize the coalitions necessary to address the problem.
When compared with other sectors, health is frequently classified as being of median to high levels of corruption, with sectors such as public works contracts and construction, arms and defence, energy and industry appearing as more corrupt [108].
The professional literature hardly touches on corruption in the health sector. It is anecdotal (Yudkin reports, from two Kenyan newspaper articles, that two ministry of health officials had been bribed to purchase sufficient quantities of two medicines made by one company to last the nation for more than 10 years, whereas at least one of the medicines would expire in two to three years) [109] (see also Baxter, 1998) [110], biased, peripheral to the core issues [22] or lacking in empirical data. Empirical data are, nevertheless, available from a number of studies and reports [16,18,21-23,105,110-113], most of which were reviewed in previous sections.
One of the earlier papers on this was by Van Der Geest [21]. It attempted to explain why health services in southern Cameroon functioned so inefficiently, with special attention to the distribution of medicines. It calculated that the "elementary health centres received on the average about 65% of the medicines they should have received", this proportion increasing for the "more developed health centres" and even more for the hospitals. Once in the institutions, "many of the medicines which finally arrive...and which should be distributed freely among patients is taken by health personnel for private use or distribution among friends and relatives. Medicines are also sold to petty traders or directly to patients visiting health workers in their private homes", resulting in a further loss of medicines of 30% for health centres and of 40% for hospitals.
The main consequences of these practices were: underutilization of health services known to be without medicines; a very limited stock of medicines, which forced health professionals to treat patients with inadequate doses of medicines; referral of patients with prescriptions to expensive private pharmacies; and an increase in inequity, as rural populations were clearly at a disadvantage compared to urban populations. The author came to the reluctant conclusion that the root cause of the observed inefficiency was corruption, deeply embedded in socially accepted practices of "gift-giving, with the preponderance of traditional loyalties over obligations to the state, and with a proprietary view of public offices".
The most important single factor encouraging corruption, was, however, "the position of the state as the main source of goods, services and employment and the relative underdevelopment of the private commercial sector". In the end Van Der Geest reluctantly acknowledged that "suggestions to ameliorate the situation are hard to make".
Reasons for dual practice
With current salary levels in many countries, it is surprising that many people remain in public service, when they could earn much more in private practice. Dual practice allows a standard of living that is closer to what clinical doctors – still a rare resource in many situations – expect, and thus helps retain valuable elements in public service.
Many spend comparatively little time, or none at all, on private practice. It is unlikely that this is only for lack of opportunities, such as a saturated private health care market, or too much competition from the "real" clinicians.
There must be other sources of motivation to keep working in public services. The involvement in (relatively unrewarding) teaching, or in unpaid NGO work shows that other factors – social responsibility, self-realization, professional satisfaction, working conditions and prestige – still play a significant role [3,4,44,45]. A study from Phnom Penh, Cambodia, suggests that the links with the public sector are highly valued, as they give physicians access to information, opinions of influential doctors, recruitment of patients, privileges for treating and referring patients and an opportunity to make a contribution to the community [89].
The gap between income and expectations makes it unavoidable that managers, like other health care workers, will seize opportunities that are rewarding, professionally and financially. Some are of the opinion that dual practice can at times be justified. Health workers in Mozambique and Cape Verde rationalize it: "... to help a sick neighbour" or to help patients "because there are patients that, on account of the long waiting times, do not go to the hospital, they will rather go to these persons in order to avoid wasting their time in the hospitals" [20].
Most, however, implicitly or explicitly condemn such practices while still attempting to explain and/or justify them in various ways. An obvious explanation is that of "serious lack of motivation" and insufficient salaries: "economic reasons, and low salaries ... those are the reasons ... it is a means of surviving" [20].
The reasons for dual practice are not well studied. A number of Portuguese case studies [72,74-76] suggest that these reasons are contextual and vary between professional groups and site of employment (hospital versus health centres). The extent of dual practice seems to vary according to urban or rural residence, according to professional group (it is more common among health workers with university degrees and, for these, more common for doctors than for other professional groups) and within a professional group, according to specialty or occupation. For example, health system managers have fewer opportunities for dual practice than clinicians.
This limited evidence suggests that being a migrant worker, being on temporary contracts and doing shift work are important determinants. This evidence is not conclusive or generalizable, but is welcome as it suggests that dual practice depends not so much on the personal (age and sex), social (marital status) and professional characteristics of health workers – although these are not insignificant – but on factors that are manageable.
Sometimes dual practice may be the unexpected result of health care reform. In Canada, within the public system, designating sites for different levels of surgical acuity during the early stages of regionalization has resulted in a 3.5-fold increase in the number of surgeons working in more than one setting after this restructuring compared to before, as most surgeons do both high- and low-acuity surgeries. This resulted in interference with continuity of care, increased commuting time for both surgeons and medical residents and increased reliance on house staff (with whom surgeons spent less time and are thus less familiar with the limits of their skills) [114].
Another area where provider strain was reported as high was among paid home care staff: low wages, irregular hours, inadequate training and high turnover resulted in lack of continuity of care, staff shortages, waiting lists, health risks to both workers and recipients and impoverishment. Some home workers reported working several jobs to make ends meet [114].
Reforms frequently result in the increase of staff employed on fixed term and temporary contracts [71]. This trend seems to induce dual employment [72,74,75].
Interventions to deal with dual practice
At the core of the reliance on individual coping strategies, including dual practice, is a very strong motor: the gap between the professional's financial (but also social and professional) expectations and what public service can offer.
Most public responses to individual coping strategies, including dual practice, fail to acknowledge the obvious: that individual employees are reacting individually to the failures of the organizations in which they work, and that these de facto choices and decisions become part of what the organization is.
Adequate responses also imply that the main underlying reason for the observed dual practice can be identified. They call for an understanding of how endemic are the practices observed: Are they isolated, individual cases? Are they specific to the health sector? Or are they widespread in other sectors of society? It is equally important to identify the impact of these practices, particularly the impact in terms of reduced access, inequity and other dangers for the health of the public.
Dual practices have, in some countries, become so prevalent that it has been widely assumed that the very notion of a civil service ethos has completely – and possibly irreversibly – disappeared. But some of the literature reviewed reflects, from the health workers themselves, a conflict between what it means to be an honest civil servant who wants to do a decent job, and the brute facts of life that make them betray that image. The manifest unease that this provokes is an important observation as such. It suggests that even in the difficult circumstances observed in many countries, behaviours that depart from traditional civil servant duty and ethics have not been interiorized as a norm. This ambiguity suggests that interventions to mitigate the erosion of proper conduct would be welcome.
The most relevant conclusion is that there is no single recipe to address the reality of dual practice. Its cause and logic vary, and the resulting differences among situations need to be taken into account in the design of corrective measures [115].
What does not work
Pretending that the problem does not exist or that it is a question merely of individual ethics, or approaching it as a problem merely of corruption, does not do justice to the complex nature of the problem and will not make it go away.
Prohibition is equally unlikely to meet with success, certainly if the salary scales remain blatantly insufficient. In situations where it is difficult to keep staff performing adequately for want of decent salaries and working conditions, those who are supposed to enforce such a prohibition are usually in the same situation as those who have to be disciplined. As an isolated measure, restrictive legislation, when not blatantly ignored, only drives dual practice underground and makes it difficult to avoid or correct negative effects [6]. Despite this, governments still resort to prohibition as the main means of controlling dual practice [116].
Closing the salary gap by raising public sector salaries to "fair" levels may not be enough to break the vicious circle. This was attempted by Louro, in Greece, in his restructuring of the health sector in 1945. When public-sector doctors were prohibited by law from pursuing private practice, their average remuneration was raised to take account of their lost income. But there was great resistance by doctors not prepared to give up private practice and professional autonomy [57]. The 1983 Greek NHS Act again required doctors to have a heavily disputed exclusive full-time status in their public sector employment; correspondingly the hospital doctors' salaries were raised so that seniority was favoured – at an average of 112%, or at a range of 11% for junior doctors to almost 211% for directors. Categories such as university and military doctors escaped from the restrictions introduced.
The 1990–1994 reform again allowed doctors to become part-timers if they wished to practice simultaneously in private surgeries or to treat on a per-case basis, but in this case their salaries would be reduced. Notably, though the Ministry of Health expected that around 2500 doctors would become part-timers, only 150 opted for it.
In 1994 an international committee chaired by Abel-Smith reopened this issue, recommending that either rights to private practice would be denied to all doctors working for the NHS or they would be confined to senior doctors for a limited number of sessions. Higher salaries were to be paid to professors to compensate them for their loss of unlimited private practice rights.
In 1997 this still had not been implemented [57]. And it is not a realistic option in many poor countries. In the average low-income country, salaries would have to be multiplied by at least a factor of five to bring them to the level of the income from a small private practice [44,45]. Doing this for all civil servants is unimaginable; doing it only for selected groups would be politically difficult.
Downsizing central bureaucracies and delinking health service delivery from civil service [117] would make it possible to divide the salary mass among a smaller workforce, leaving a better individual income for those who remain. However, experience shows that such initiatives often generate so much resistance among civil servants that they never reach implementation [118]. Where retrenchment becomes a reality it is rarely followed by substantial salary increases, so that the problem remains and the public sector is even less capable of assuming its mission.
Lastly, a mere increase in salary would not automatically reinstate the sense of purpose that is required to make public services function: as such it would not be enough to make moonlighting disappear spontaneously [119,120]. That does not mean that nothing can be done. Improvement is likely to come from a combination of small piecemeal measures that rebuild a proper working environment.
Addressing the problem of dual practice openly
A prerequisite is to address the problem of dual practice openly. Where it is not realistic to expect health care workers to dedicate 100% of their time to their public service job, this should be acknowledged. That is the only way to create the possibility of containing and discouraging income-generating activities that present conflicts of interest, in favour of safety valves with less potential for negative impact on the functioning of the health services.
Besides minimizing conflicts of interest, open discussion can diminish the feeling of unfairness among colleagues. It then becomes possible to organize things in a more transparent and predictable way.
There are indications that the newer generations of professionals have more modest expectations and are realistic enough to see that the market for dual practice is finite and to a large extent occupied by their elders. This gives scope for the introduction of systems of incentives that are coherent with the organization's social goals [121].
Incentives
Where, for example, financial compensation for work in deprived areas is introduced in a context that provides a clear sense of purpose and the necessary recognition, this may help to reinstate lost civil service values [122]. The same goes for the introduction of performance-linked financial incentives [121]. These can, in principle, address the problem of competition for working time, one of the major drawbacks of dual practice. However, such approaches require well-functioning and transparent bureaucracies, making the countries most in need also those where they are a priori most difficult to implement on a large scale [117,123].
Improving working conditions
It makes no sense to expect health workers to perform well in circumstances where the equipment and resources are patently deficient. But improving working conditions involves more than providing an adequate salary and the right equipment. It also means developing career prospects and providing perspectives for training [119,120]. Perhaps most important, it requires a social environment that reinforces professional behaviour free from the favouritism and arbitrariness prevalent in the public sector of many countries.
Professional value systems
However ill-defined they may be, the value systems of the professionals are a major determinant in making the difference between good service to the public and bad. It would be naïve to think this could be achieved through mere bureaucratic regulation by governments or donor agencies. With the building up of pressure from donors and from peers as well as from users, civil servant health professionals will be more likely to invest in patterns of behaviours and practices that visibly uphold the professional value system [119,120].
Peer pressure
The social and professional culture within a profession may have a major impact on the practice [64,120,121]. Peer influence, building on the concept of group responsibility for self-education and monitoring, as well as multi-component interventions, have been shown to be effective in improving professional practice in the public sector of high-income countries [124,125].
The effect of peer pressure may be positive or negative. Pressure from local practice styles is particularly relevant in situations where there is the most uncertainty concerning the most appropriate treatment protocol [85]. This reflects the practice-styles hypothesis of Wenberg and colleagues in 1982 [126].
Practice styles can be changed through "peer influence meetings", particularly if the change is seen as building up public reputation and status, once more showing that simple income topping-up is not the principal driving force of professional behaviour [127]. This points to the importance, in the absence of effective regulatory mechanisms, of the role of professional societies in ensuring peer-pressure mechanisms to reduce undesirable coping strategies associated with dual practice.
A further possibility is workers' forming peer pressure groups to reduce undesirable coping strategies associated with dual practice. These groups could function to support members to maintain their personal stance as well as to inform the public of their rights. Making public the membership of such a group could be a way of identifying the non-members, an indirect way of increasing pressure [23].
A significant problem with individual coping strategies associated with dual practice is the difficulty of assigning individual responsibilities in situations where these are endemic. In these circumstances it might be relevant to introduce legislation that makes the head of an organization or department legally responsible for the actions of that body. This would be a further means of increasing peer pressure and accountability [23].
Pressure from users
Civil society has a particularly important role, specifically in linking reform measures to the experiences and expectations of real people. But civil society must not be seen as a neutral body, particularly in developing countries where patron–client networks or kinship networks have a strong influence on the state and on the patterns of corruption and/or of coping strategies observed. In these situations the reform of civil society itself should be an objective of the interventions to correct such strategies.
In many countries, users/clients/patients are not protected against the consequences of the asymmetry of information they face – with health and financial consequences. From the history of the workers' movement in Europe and as the recent evolution in a number of middle-income countries – such as Thailand's National Forum on Health Care Reform [128] – points out, perhaps the most effective way to help the State regulate professional practice is to increase pressure from civil society. (Fear of malpractice may have a paradoxical effect in that may result in excessive and inappropriate recourse to caesarean sections, for example [85,129].)
Creating opportunities for users to voice their discontent effectively implies that patient's rights must be clear, channels for complaints must be simple, regulatory agencies must be strong and trusted by the public, processes must be explicit and transparent and the judiciary system must be strengthened [23].
Recruitment practices
International development agencies, even when they do not have formal, explicit policies regarding dual practice, have become more sensitized to the problem over recent years. This has resulted in a number of recommendations to help minimize the problem. To limit the brain drain due to their own employment policies, organizations such as the World Bank, Norwegian Agency for Development Cooperation (NORAD), German Technical Cooperation (GTZ) or the World Health Organization in principle implement human resources recruitment policies that emphasize the employment of task-specific and short-term consultants, with a commitment of national institutions to retain such staff [90-92,130].
Regulating the private sector
The anti-corruption literature, without the necessary empirical evidence to support such claims, actually blames government monopoly of service provision as one of the key determinants of the emergence of some of the coping strategies reviewed above [105]. It has also been argued that the presence of a significant quasi-private system operating within the public sector, i.e. the form of dual practice most common in transitional economies and in developing countries, is detrimental to the development of a strong private sector [23].
The claims for a greater role for the private sector in the provision of health care are based on a number of assumptions that are not all based on empirical evidence and ignore that private practice, in most developing countries, is notoriously unregulated. The fragmentary evidence shows that blanket recommendations regarding the role of the private sector are inappropriate [131].
There is a case for public sector support of the private sector where this serves the public's interest and allows redirection of scarce resources. If that is not the case, support has no rationale. Support, but also mere control, carries costs for the public sector administrative machinery. The costs of the "new" state responsibilities must be compensated for by savings resulting from gains in efficiency and from complementarity [124,131,132].
A key policy question is whether doctors should be allowed to work in both the public and private sectors. As discussed before, prohibition is unlikely to be effective. The real issue is what types of private practice should be allowed in order to minimize conflicts of interest, and what forms of regulatory mechanisms can be introduced to isolate coping strategies that are associated mostly with lack of regulation rather than just with low income [23]. It seems that efforts should be undertaken to ensure multiple and independent channels of accountability, by means of penalties for not satisfying contractual obligations, through channels of accountability to professional councils and associations and to the public.
Regulation is one important factor influencing the coping strategies that result from the interface with the private sector [62,124]. Even when regulations exist, effective enforcement mechanisms are often absent in low- and middle-income countries [125,133]. Therefore, good legislation is not enough. The state must have the means to enforce it. In India, for example, private clinics and mobile teams promote prenatal sex determination by advertising in local newspapers, in spite of government prohibition of the practice [134].
Pressure on donors
International collaboration is seen as particularly important regarding the support of international development agencies for actions such as: good-governance interventions in specific domains; supporting methods to curb corruption, including policy dialogue, capacity building, documentation and analysis of best practices and support to national programmes; and making reformers aware of the importance of country conditions in programme development [115].
Anti-corruption strategies have also been approached by donors with different objectives: to reduce poverty, to improve the functioning of democratic institutions, to sustain economic development, political stability and social justice. The lesson for the management of coping strategies and dual practice is that international collaboration cannot be neglected, as donors may be important inducers of coping strategies and dual practice as well as essential partners in the search for solutions.
One way to increase donors' and governments' commitment to deal with the causes of individual coping strategies as well as dual practice might be to include a formal "human resources impact assessment" as a condition for the approval of health projects or components of sector-wide approaches. This could force governments and their partners to face the problems caused by dual practice before it becomes part of the public organization's culture. This would not be a guarantee that it would be effectively dealt with, but might limit the damage [135].
Conclusions
In terms of sector location, dual practice may be public-on-public, public-on-private or private-on-private. Dual practice is probably present in all countries regardless of income, even in settings where there are major regulatory restrictions, such as China.
Dual practice may lead to predatory behaviour by health workers. This constitutes, in many cases, a de facto financial barrier to access to health care. It delegitimizes public sector health service delivery and jeopardizes the necessary relation of trust between user and provider. Clinicians in dual practice have to compete for patients with themselves, which is an incentive to lower the quality of the care they provide in the public services.
Dual practice contributes also to brain drain, specifically public-to-private brain drain. If public sector medical staff is moonlighting in private practice, this limits access. Besides competition for time, in many cases, the use of the public sector's means of transportation, office infrastructure and personnel represent additional hidden outflows of resources, often in association with dual practice.
This is not the case for managers: involvement in NGO projects or work for donors can foster better coordination in the provision of services, but may constitute a conflict of interest when NGO or project policies are not necessarily congruent with national health policies or the agenda of the public service.
On the other hand, dual practice allows professionals a standard of living that is closer to what they expect, as well as a standard of practice closer to their own perceptions of good professional practice, resulting in higher professional satisfaction.
There is no evidence that dual practice by public sector health professionals complements public practice or promotes greater equity of health care distribution.
The reasons for dual practice are contextual. The extent of dual practice seems to vary according to urban or rural residence, according to professional group (more common among health workers with university degrees and, for these, more common for doctors than for other professional groups) and even within a professional group, according to specialty or occupation. The limited evidence suggests that being a migrant worker, being on temporary contracts and doing shift work are important determinants. This evidence suggests that dual practice depends not so much on the personal (age and sex), social (marital status) and professional characteristics of health workers, although these are not insignificant, but on factors that are manageable.
Sometimes dual practice may be the unexpected result of health care reform. Reforms frequently result in the increase of staff employed on fixed-term and temporary contracts. This trend seems to encourage dual employment.
Therefore, at the core of the reliance on dual practice is the gap between the professional's expectations and what public service can offer. Adequate responses imply the identification of the main underlying reason for the observed dual practice. The most relevant conclusion is that there is no single recipe to address the reality of dual practice.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
AB, IF, FH were responsible for the Portuguese case studies. PF and WVLconceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.
Acknowledgements
We acknowledge comments, suggestions on further contacts or literature from the following colleagues: Alan Leather (Public Services International), Christiane Wiskow (International Labour Office), Gilles Dussault (World Bank Institute), Göran Tomson (Division of International Health, Karolinska Institutet (IHCAR)), James A. Rice (Governance Institute, International Health Summit, Cambridge International Health Leadership Programme), Jan Boyes (Institute of Development Studies, University of Sussex), Mike Waghorne (Public Services International), Mireille Kingma (International Council of Nurses), Piya Hanvoravongchai (International Health Policy Program, Thailand), Rolf Wahlstrom (IHCAR). We also acknowledge support from Margarida Carrolo. This paper was produced in the context of the Joint Learning Initiative on Human Resources for Health, supported by the Rockefeller Foundation, the World Bank and the World Health Organization.
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| 15509305 | PMC529467 | CC BY | 2021-01-04 16:37:43 | no | Hum Resour Health. 2004 Oct 27; 2:14 | utf-8 | Hum Resour Health | 2,004 | 10.1186/1478-4491-2-14 | oa_comm |
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Malar JMalaria Journal1475-2875BioMed Central London 1475-2875-3-361549622510.1186/1475-2875-3-36ResearchCommunity effectiveness of chloroquine and traditional remedies in the treatment of young children with falciparum malaria in rural Burkina Faso Mueller Olaf [email protected] Oliver [email protected] Corneille [email protected] Bocar [email protected] Department of Tropical Hygiene and Public Health of the Ruprecht-Karls-University Heidelberg, Germany2 Centre de Recherche en Santé de Nouna, Burkina Faso2004 20 10 2004 3 36 36 27 7 2004 20 10 2004 Copyright © 2004 Mueller et al; licensee BioMed Central Ltd.2004Mueller et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
There is little information on the effectiveness of modern compared to traditional malaria treatment from the rural areas of Africa.
Methods
Follow-up of 402 episodes of clinical malaria among pre-school children in Nouna Health District, northwestern Burkina Faso. The exposure of interest was the type of treatment (chloroquine versus traditional); the outcome was clinical response to treatment.
Results
Out of the 402 observed malaria episodes, 87% were treated with chloroquine and 13% with traditional remedies. Overall, community effectiveness was 67% with chloroquine and 54% with traditional treatment. Chloroquine effectiveness was associated with age and ethnicity. An additional interview survey demonstrated wide variations in the dosages of chloroquine given to young children in this community.
Conclusions
The effectiveness of chloroquine, when used within the community, was significantly lower in this study than its known efficacy in the study area. This concerns, in particular, the very young children. These findings demonstrate the need for better education of parents about correct dosage of first-line malaria drugs, and for particular attention in the treatment of very young children.
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Introduction
It has been estimated that at least one million annual malaria deaths occur among young children in rural sub-Saharan Africa (SSA). Most of these deaths are in areas with little access to health services [1-4]. Under such circumstances, home treatment with chloroquine, antipyretics and traditional remedies is the most frequent response of caretakers to fever episodes in children [5]. There is also increasing evidence that improved home management of malaria in young children of SSA can be effective [6,7].
The situation is now complicated by the increasing resistance of Plasmodium falciparum to chloroquine in most SSA countries [8]. Moreover, compared to the efficacy under trial conditions, the community effectiveness (the drug efficacy under real life conditions) of malaria treatment is significantly lower [9]. Chloroquine has been used in Burkina Faso for decades and reported clinical failure rates in children with uncomplicated malaria were around 5% in the early 1990s [10]. However, data from a more recent study on the efficacy of chloroquine in a representative sample of villages in the Nouna Health District in north-western Burkina Faso demonstrated a clinical failure rate of 10% in young children [11]. Nevertheless, chloroquine has remained the official first-line treatment for uncomplicated malaria in Burkina Faso until today.
Traditional treatment for malaria is very common, particularly in the rural areas of SSA, but only few data exist regarding the efficacy of such treatments [5,12]. In rural north-western Burkina Faso, treatment of uncomplicated malaria usually comprises a combination of modern and traditional methods. Chloroquine and antipyretics, often combined with traditional treatments, are the most common community-based treatment regimens for young children during fever episodes [4]. Traditional treatments usually comprise oral and/or skin applications of extracts from eucalyptus plants, acacia, citronella, papaya, guava and the neem tree [13].
This paper reports on the community effectiveness of chloroquine compared to traditional remedies in the treatment of uncomplicated falciparum malaria in young children of rural Burkina Faso.
Methods
Study area
The study was conducted in the rural part of the research zone of the Centre de Recherche en Santé de Nouna (CRSN), in Nouna Health District, north-western Burkina Faso. The CRSN study zone comprises 41 villages and Nouna town, with a total population of 55,000. The Nouna area is a dry orchard savanna, populated by subsistance farmers of different ethnic groups. There is a short rainy season which usually lasts from June until October. Malaria is holoendemic but highly seasonal in the study area [14]. Formal health services in the CRSN study zone consist of four village-based health centres and the district hospital in Nouna town [4].
Study design
This is an observational study at the community level. Data for this study were extracted from the database of a trial on the effects of zinc supplementation on malaria morbidity conducted in the Nouna area in 1999 [14]. During this trial, 709 children aged 6–31 months at enrollment in June 1999 were recruited from 18 villages of Nouna Health District. Study children were followed up until December 1999.
Field methods for data collection have already been described [14]. In brief, the children in the study were regularly visited by village-based field staff for temperature measurement and malaria slide preparation from finger-prick blood in case of fever over the main malaria transmission period (June to December). If fever persisted or recurred, another blood slide was prepared at least one week after the start of the febrile episode. If children were found to have fever or other obvious illness, their caretakers were advised by the field staff to use chloroquine for febrile episodes or to seek diagnosis and treatment at the next governmental health centre. Children found ill during four cross-sectional surveys were treated appropriately by the study physician or referred to Nouna hospital, where they were treated free of charge.
All relevant signs and symptoms manifested by the children were recorded on a standard questionnaire during regular visits of field staff. Moreover, a comprehensive questionnaire was filled in for each disease episode which included information on specific signs and symptoms as well as place, time and type of treatment received. No specific information was recorded on the dosage of chloroquine and on the type of traditional remedies given to the study group.
For this study, only children with a diagnosis of falciparum malaria, defined as an axillary temperature of ≥37.5°C together with a density of ≥5,000 P. falciparum parasites per μl of blood were considered. Inclusion criteria were falciparum malaria treated with chloroquine (with or without antipyretics) but without traditional remedies (group CQ), or with traditional remedies only (group TRAD). Exclusion criteria were a history of febrile illness during the previous four weeks, malaria treatment during the previous four weeks, any treatment with another antimalarial drug during follow up, and missing temperature measurements during the two-weeks follow up period. Multiple episodes of malaria in individual children were considered to be independent with regard to treatment choice [4].
To evaluate treatment outcome, a modified definition of the WHO protocol for assessment of therapeutic efficacy of antimalarial drugs in areas with intense transmission was used [15]. Treatment failure (TF) was defined as development of severe malaria on day 1–14 or fever recurrence (axillary temperature ≥37.5°C) on day 3–14. Adequate clinical response (AR) was defined as the absence of fever on day 3–14, without meeting any of the criteria of TF.
Chloroquine dosage survey
To retrospectively estimate the chloroquine dosages usually given to febrile young children in the study area, 55 individual interviews with randomly selected mothers of young children from three villages in the study area were conducted during a malaria survey at the end of the 2001 rainy season. The villages were purposely selected to represent the rural study population in its socio-cultural, demographic and geographical diversity. Questions concerned the kind of treatment (western and/or traditional) and the dosages of western drugs used during treatment of the last febrile episode of survey children, the current availability of chloroquine in the household and the origin of chloroquine drugs. The weight of children was measured with a Salter hanging spring scale.
Laboratory procedures
Blood films were kept in closed slide boxes until they were transported to Nouna (two to three times per week). They were Giemsa-stained at the Nouna hospital laboratory and transported afterwards to the Centre National de Recherche et de Formation sur le Paludisme (CNRFP) in Ouagadougou for reading. All films were examined by two experienced laboratory technicians using a ×100 oil immersion lens and ×10 eyepieces. In case of significant discrepancy between the results of the two technicians, blood slides were read by a third investigator. Blood films were analysed for the species-specific parasite density per μl by counting against 500 white blood cells and multiplying by sixteen (assuming 8,000 white blood cells per μl of blood). Slides were declared negative if no parasites were seen in 400 fields on the thick film.
Statistical analysis
Data were entered at the data management department of the CRSN into a data bank (Microsoft Access, version 97). All questionnaires were checked by supervisors before computer entry. Parasitological data were entered into EpiInfo (version 6.0) at the CNRFP, and the data were transferred to the CRSN. All data were checked for range and consistency before data entry.
The outcome of malaria episodes (AR vs. TF) was assessed dependent on the treatment given by the caretakers. Episodes in which the child had received at least one dose of chloroquine were grouped in the treatment group CQ, and episodes in which caretakers had given only traditional treatment in the treatment group TRAD. The hypothesis was tested that the samples in the CQ and the TRAD treatment groups were from populations with the same distribution of baseline variables using the Wilcoxon ranksum test for continuous variables and the chi-squared test for categorical variables. Community effectiveness of treatment with chloroquine was assessed both by intention to treat (i.e. adherence to advice by the field workers to give chloroquine) and considering only those cases who actually complied. A stratified analysis by age bands was started, assuming that age would be an important determinant of treatment outcome. Logistic regression modelling was then used to control for differences in the distribution of baseline characteristics such as age, sex and parasite density in the two treatment groups. Stata 7.0 statistical software was used for all calculations.
Ethical aspects
Approval for the RCT from which the data used were drawn was granted by the Ethical Committee of the Heidelberg University Medical School and the Ministry of Health in Burkina Faso.
Results
Follow-up study
A total of 330 children (175 boys and 155 girls), who contributed 402 episodes of falciparum malaria, were followed up. Most of the episodes (65.7%) occurred between August and October (Table 1).
Table 1 Distribution of febrile episodes by treatment type over the main malaria transmission period in young children of rural Burkina Faso
Month
7/99 8/99 9/99 10/99 11/99 total
Group CQ 25 126 108 75 16 350
Group TRAD 1 15 15 19 2 52
Total 26 141 123 94 18 402
In 350 (87.1%) of the episodes, the child had received at least one dose of chloroquine (treatment group CQ); in 52 episodes (12.9%), the caretakers had given only traditional treatment (treatment group TRAD). Table 2 shows the distribution of baseline characteristics in the two treatment groups. The median parasite density was significantly lower in the TRAD group (p = 0.001), but diarrhoea was observed in a larger proportion of episodes in this group than in the CQ group (p = 0.04). In the Bwaba ethnic group, 33.3% of malaria episodes had been treated with traditional treatment, compared to 8.7% in the other ethnic groups (p < 0.0001).
Table 2 Baseline characteristics of malaria episodes in study children in rural Burkina Faso
Group CQ (n = 350) Group TRAD (n = 52) p-value *
Male/female 183/167 30/22 0.47
Median age, range (months) 20 (8 – 35) 19 (9 – 33) 0.36
Median parasite density/μl, D0 Range 26 220
5000 – 1 140 000 16 352
5000 – 86 656 0.001
Median temperature D0 (°C) 38.2 38.2 0.72
Diarrhoea D0 (%) 64/350 (18.3) 16/52 (30.8) 0.04
Vomiting D0 (%) 39/350 (11.1) 3/52 (5.8) 0.24
Ethnicity <0.0001
Marka 180 16
Mossi 72 8
Bwaba 46 23
Peulh 44 5
Others 8 0
* Chi-squared test for categorical variables, ranksum test for continuous variables
Group CQ: received at least one dose of chloroquine
Group TRAD: received only traditional treatment
D0: Day of onset of malaria episode
The caretakers had been advised to give chloroquine to all children participating in this study. Compliance with this advice, stratified by age group, is shown in Table 3. Compliance was independent of sex (data not shown), except in the age group 15–21 months, in which 98.2% of episodes in girls, but only 86.8% of episodes in boys, were treated with chloroquine (p = 0.02). Community effectiveness as a result of following the advice to give chloroquine, measured by the proportion of episodes in which the child had an AR was 64.9% (95% CI: 60.0–69.6%). This proportion did not increase appreciably when we restricted the analysis to episodes in which the child had actually received chloroquine (Table 3). In the TRAD group, children had an AR in 53.9% of the episodes. Our study did not have sufficient power to show a difference between the TRAD and the CQ groups as small as the one observed (-11%) at the 5% significance level. In the CQ group, outcome was strongly and positively associated with age, as demonstrated by the chi-squared test for linear trend (p = 0.014). A comparable result was not shown in the TRAD group, possibly because of small case numbers.
Table 3 Compliance with treatment and outcome of treatment, stratified by age group
Compliance Outcome of treatment
Age group CQ given n (%) AR, total n (%) AR, CQ group n (%) AR, TRAD group n (%)
8 – 14 months 87 (81.3) 58 (54.2) 49 (56.3) 9 (45.0)
15 – 21 months 121 (91.7) 89 (67.4) 83 (68.6) 6 (54.6)
22 – 28 months 86 (87.8) 63 (64.3) 57 (66.3) 6 (50.0)
29 – 35 months 56 (86.2) 51 (78.5) 44 (78.6) 7 (77.7)
Total 350 (87.1) 261 (64.9) 233 (66.6) 28 (53.9)
CI a - 60.0 – 69.6% 61.4 – 71.5% 39.5 – 67.8%
p-value 0.13 b 0.004 c 0.014 c 0.17 c
a CI: Binomial exact 95% confidence interval
b p-value: Chi-squared test for heterogeneity (within each column)
c p-value trend: Chi-squared test for linear trend (within each column)
CQ group: Received at least one dose of chloroquine
AR: Adeaquate clinical response
The crude odds ratio for TF in the CQ group vs. the TRAD group was 0.59 with a 95% confidence interval that included unity (Model 1 in Table 4). Logistic regression modelling was used to assess the degree to which the observed advantage of chloroquine treatment over traditional treatment was due to a different distribution of baseline characteristics in the two treatment groups. Inclusion of the variables age group (with four seven-month age bands) and sex (Model 2 in Table 4), as well as parasite density (log-transformed and grouped in three bands) and diarrhoea on day 0 (Model 3 in Table 4) did not change the odds ratio much; if there was any advantage of chloroquine over traditional treatment it was slightly attenuated. However, removing all episodes among children of the Bwaba ethnic group from the analysis decreased the odds ratio, and the corresponding confidence interval no longer included unity (Model 4 in Table 4). This means that in the other ethnic groups, chloroquine treatment carried only 0.43 (95% CI: 0.19–0.91) times the odds of treatment failure of traditional treatment and thus conveyed a significant advantage.
Table 4 Regression modelling of treatment failure, CQ vs. TRAD group
Model Variables Odds Ratio (95% CI) p-value
1 crude 0.59 (0.33 – 1.06) 0.08
2 age group, sex 0.61 (0.34 – 1.11) 0.10
3 age group, sex, parasite density, diarrhoea 0.64 (0.35 – 1.17) 0.15
4 age group, sex, parasite density, diarrhoea excluding Bwaba ethnicity 0.43 (0.19 – 0.97) 0.04
CQ group: received at least one dose of chloroquine
TRA group: received only traditional treatment
Table 2 already showed evidence of an interaction between treatment pattern and ethnic group. Therefore, a subgroup analysis was done in which additional differences between the Bwaba and the other ethnic groups were found. First, among the other ethnic groups, children who received chloroquine had a significantly higher log-transformed mean parasite density than those who received traditional treatment (p = 0.002). The difference in parasite density was smaller and not significant among the Bwaba (p = 0.26). Secondly, treatment outcome was not significantly associated with age group among the Bwaba, while it was so among the other ethnic groups. In Model 4, an increase in age by one age band (equivalent to seven months) was associated with an almost 30% reduction in the odds of treatment failure (p = 0.004) after control for treatment type, sex, parasite density and diarrhoea on day 0 (also see Table 3). With a number of important factors determining treatment type and/or outcome varying by ethnic group, the stratified analyses were warranted.
Chloroquine dosage survey
A total of 55 mothers participated in the survey with 28 girls and 27 boys aged 3–27 months (median: 14 months). During the most recent fever episodes of respective children, home treatment with chloroquine was reported in 32/55 (58%) (8/32 in combination with paracetamol and 1/32 in combination with traditional treatment), treatment at the local health centre in 15/55 (27%), traditional treatment in 6/55 (11%), and only paracetamol treatment in 2/55 (4%). The mean weight of chloroquine-treated children was 8.5 kg (range 3.2 – 12.0 kg). The mean (median) total dose of chloroquine used was 34 mg/kg and 25 mg/kg respectively (range 5–110 mg/kg) (figure 1). Only 9/55 (13%) mothers reported current availability of chloroquine tablets in their household (median 3 tablets, range 1–10). Most chloroquine drugs were purchased from local shops and drug sellers.
Discussion
The main finding of this study is a rather low community effectiveness of chloroquine in the treatment of malaria episodes among young children in rural Burkina Faso. Overall, a failure rate of 35% was observed which declined only marginally to 33% when malaria episodes in which caretakers did not comply with the advice to give chloroquine were excluded. This is considerably higher than the 10% treatment failure rate observed in a study on chloroquine efficacy in young children of the area [11]. These findings support the hypothesis that treatment efficacy declines drastically under real life conditions in the community and demonstrate that efficacy trials need to be complemented with observational studies to assess the actual health benefits conferred by an intervention [9,16].
The effectiveness of treatment with chloroquine increased significantly and almost linearly with age, even after controlling for parasite density. The most likely explanation is a rapid development of immunity within the age range of our study children, as has been observed in other studies [1,17,18]. This implies that particular care is needed in the treatment of the youngest children in whom immunity will be weakest. Given the fact that the children who participated in the study on chloroquine efficacy in the Nouna area were in the most susceptible age group for developing clinical malaria (mean age 10 months, range 6–15), the loss of efficacy under community conditions observed in this study becomes even more pronounced [11].
Only a small number of episodes were treated solely with traditional treatment, possibly a consequence of the view of the local population that western malaria treatment is more effective than traditional treatment [13]. This may not apply to all strata of the population, as there was an interaction between type of treatment and ethnicity. Moreover, while children receiving traditional treatment had a higher risk of treatment failure than children treated with chloroquine after control for age, sex and parasite density, this association did not reach statistical significance until episodes of children belonging to the Bwaba ethnic group were removed from the analysis. Interestingly, crude failure rates were not higher among cases from the Bwaba group compared to those belonging to other ethnic groups. Also these results support the findings from another study in Burkina Faso which has also shown a lower effectiveness of traditional compared to modern malaria treatment, traditional treatments are not identical in different populations [17]. Thus, it may well be that the traditional treatment used in the Bwaba ethnic group is more effective against malaria compared to other traditional treatments in the area. Finally, the observed significant association of traditional treatment with diarrhoea on day 0 may well be a chance finding, given the lack of a difference between treatment groups in all other clinical and parasitological parameters.
This study has two limitations. First, information on the dosage of chloroquine and on the nature of traditional remedies given to children in the study was lacking. However, data from the interview survey provide a good estimate of chloroquine dosages used. Although these data demonstrate a wide variation of chloroquine dosages, it is reassuring that the reported median dosage complies with the national recommendations of a total dose of 25 mg/kg chloroquine for uncomplicated malaria. So why is there such a difference between efficacy and effectiveness? The reason may well be a combination of partly chloroquine underdosing, poor quality of drugs purchased from local shops and drug sellers and high frequency of vomiting in febrile young children [19-23]. Moreover, the possibility of a significant reporting bias during interview surveys also has to be taken into consideration. Secondly, a purely clinical definition of treatment failure was used. Intercurrent febrile illnesses may thus have been misclassified as persisting malaria, thereby overestimating failure rates. However, this is likely to have only a small effect, since the fraction of febrile episodes attributable to malaria (using a highly specific definition) in this cohort of children was found to be above 50 percent [24].
In conclusion, this study provides clear evidence for a rather low and age-dependant community effectiveness of chloroquine in the treatment of P. falciparum malaria in an area of rural Burkina Faso where the efficacy of chloroquine is still sufficiently high in use as first-line treatment. The observed discrepancy between efficacy and community effectiveness implies that many children received insufficient doses of chloroquine. Our findings thus point to the importance of (1) better education of parents on correct dosage of first-line malaria drugs, as well as on danger signs requiring a visit at a health centre, (2) quality-control of all malaria drugs used in the community, and (3) particular care in the treatment of immunologically naïve very young children. Finally, more studies on the types and effects of traditional treatments used in different population groups are needed.
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| 15496225 | PMC529468 | CC BY | 2021-01-04 16:37:27 | no | Malar J. 2004 Oct 20; 3:36 | utf-8 | Malar J | 2,004 | 10.1186/1475-2875-3-36 | oa_comm |
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BMC Fam PractBMC Family Practice1471-2296BioMed Central London 1471-2296-5-231549810610.1186/1471-2296-5-23Research ArticleSymptomatic hypogammaglobulinemia in infancy and childhood – clinical outcome and in vitro immune responses Kidon Mona Iancovici [email protected] Zeev T [email protected] Rivka [email protected] Irit [email protected] Michael [email protected] Israel [email protected] Department of Pediatrics, Kaplan Medical Center, Rehovot, Israel2 Allergy and Clinical Immunology Unit, Kaplan Medical Center, Rehovot, Israel3 Department Human Microbiology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel2004 21 10 2004 5 23 23 15 3 2004 21 10 2004 Copyright © 2004 Kidon et al; licensee BioMed Central Ltd.2004Kidon et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Symptomatic hypogammaglobulinemia in infancy and childhood (SHIC), may be an early manifestation of a primary immunodeficiency or a maturational delay in the normal production of immunoglobulins (Ig). We aimed to evaluate the natural course of SHIC and correlate in vitro lymphoproliferative and secretory responses with recovery of immunoglobulin values and clinical resolution.
Methods
Children, older than 1 year of age, referred to our specialist clinic because of recurrent infections and serum immunoglobulin (Ig) levels 2 SD below the mean for age, were followed for a period of 8 years. Patient with any known familial, clinical or laboratory evidence of cellular immunodeficiency or other immunodeficiency syndromes were excluded from this cohort. Evaluation at 6- to 12-months intervals continued up to 1 year after resolution of symptoms. In a subgroup of patients, in vitro lymphocyte proliferation and Ig secretion in response to mitogens was performed.
Results
32 children, 24 (75%) males, 8 (25%) females, mean age 3.4 years fulfilled the inclusion criteria. Clinical presentation: ENT infections 69%, respiratory 81%, diarrhea 12.5%. During follow-up, 17 (53%) normalized serum Ig levels and were diagnosed as transient hypogammaglobulinemia of infancy (THGI). THGI patients did not differ clinically or demographically from non-transient patients, both having a benign clinical outcome. In vitro Ig secretory responses, were lower in hypogammaglobulinemic, compared to normal children and did not normalize concomitantly with serum Ig's in THGI patients.
Conclusions
The majority of children with SHIC in the first decade of life have THGI. Resolution of symptoms as well as normalization of Ig values may be delayed, but overall the clinical outcome is good and the clinical course benign.
Humoral immunodeficiencyTransient hypogammaglobulinemiaMitogens
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Background
Pediatric patients with "recurrent infections" within our area are referred to the pediatric immunology clinic in the Kaplan Medical Center. Few fulfill the clinical criteria of the immune deficiency "red flags", Table 1, and only in a small minority, quantitative or qualitative defects in immunological function are documented. As expected, most such defects, involve the humoral immune system, the most common of the primary immune deficiencies [1,2]. Classically, the clinical presentation, includes a neonatal "grace" period, during which the baby is protected from infection by the presence of passively acquired maternal antibodies. As the level of these antibodies decline, the babies present at the end of the first year of life or the beginning of the second with recurrent respiratory, ENT and GI infections. The pathogens involved are mostly the "usual" bacteria, Streptococcus pneumonia, Haemophilus influenza and Staphilococcus aureus, but the infections may be of unusual severity, persistence, or frequency.
Table 1 Clinical "Red Flags" for Immunodeficiency
1 Eight or more new ear infections within 1 year
2 Two or more serious sinus infections in 1 year
3 Two or more months on antibiotics with no effect
4 Two or more pneumonias within one year
5 Failure of an infant to gain weight or grow well
6 Recurrent, deep skin or organ abscesses
7 Persistent thrush in the mouth or elsewhere on the skin, after age 1
8 Need for intravenous antibiotics to clear infection
9 One or more deep-seated infections such as sepsis, meningitis or cellulitis
10 A family history of primary immune deficiency or early infant death from infection, recurrent infection, malignancy, or autoimmune disease
Adapted from: Primary Immunodeficiency Diseases: A molecular and genetic approach New York, Oxford University Press, 1999.
In the last 10 years, tremendous advances in the fields of molecular medicine and genetics, have made possible the definitive diagnosis of most combined immunodeficiency patients, agammaglobulinemia patients and clinical syndromes associated immunodeficiency patients, on the basis of a recognized genetic aberration leading to a protein product dysfunction [3,4]. Nevertheless, the diagnosis of some of the most common forms of primary immune deficiency, IgA deficiency [5], common variable immunodeficiency [6] and transient hypogammaglobulinemia of infancy (THGI), are still based on clinical criteria and the exclusion of other specific diagnoses [7,8]. THGI is thought to be caused by a poorly understood maturation delay in the normal production of Ig, extending the physiologic hypogammaglobulinemia of the new born beyond the first year of life [1,9]. Currently, there are no diagnostic tests that differentiate, on initial presentation of a young child with recurrent infections and low Ig levels, those that will spontaneously correct on follow-up from those where a primary and permanent immune deficiency will develop, except for B cell numbers below 2%, which point towards X-linked agammaglobulinemia (XLA)[10].
In this study we aimed to evaluate the natural course of disease in symptomatic hypogammaglobulinemia of infancy and correlate in vitro lymphoproliferative and secretory responses to mitogens in this population with recovery of immunoglobulin values and clinical resolution.
Methods
Patients
Children more than 1 year of age, with recurrent infections, defined as more than three episodes of acute otitis media and/or more than one episode of acute sinusitis and/or more than one episode of pneumonia or the presence of a severe deep seated infection (meningitis, septicemia, etc.) within the last 6 months, or fulfillment of one of the "red flags" of immunodeficiency, see Table 1, and hypogammaglobulinemia, defined as serum Ig values 2 SD below the age defined norms on two or more measurements [11], have been prospectively recruited from a cohort of children referred to our clinic because of recurrent or severe infections. Patients were seen at presentation and reevaluated periodically at 6 to 12 month intervals, up to 1 year after resolution of symptoms.
All procedures were performed according to accepted ethical standards of the Institutional Review Board of Kaplan Medical Center. The parents of all the children were informed accordingly and gave their permission for participating in the study and blood sampling.
Ig assessment
Total serum Ig levels were measured by nephelometry (Beckman Immunochemistry Systems, IgM, IgG and IGA test, Beckman Instruments, Galway, Ireland) and serum IgG subclasses have been assayed with an immunodiffusion commercial kit (Human IgG Subclasses single dilution BINARID, Birgminham, UK). Specific antibody production was not evaluated.
In vitro cell proliferation and Ig secretion
Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood of patients and age-matched donors (children hospitalized or attending the outpatient clinic for unrelated conditions) on ficoll isopaque gradients (Sigma, St. Louis, MO, USA). Patient and normal donor cells were cultured in microtiter plates with culture medium: RPMI medium supplemented with 10% FCS (Bio-Lab, Jerusalem, Israel), 10 mM Hepes buffer, 100 U/ml penicilin 100 μg/ml streptomycin, 2 mM L-glutamine, and 100 μg/ml kanamycine (Sigma Israel), and were grown at 37°C with 7.5% CO2 in air. 5 × 105 cells were stimulated for four days with 0.01% w/v SAC (Calbiochem, La Jolla, CA, USA), 2.5 μg/ml PWM, 20 μg/ml E. coli: O55:B5 LPS or with 20 μg/ml PHA (Sigma, St. Louis, MO, USA). The cells were then pulsed with 1 μCi/well of [3H]-Thymidine (Nuclear Research Center, Negev, Israel) and incorporated radioactivity was measured by a β scintillation counter. Proliferation was expressed as stimulation index (SI).
In parallel, cell culture supernatant aliquots were harvested and Ig isotype concentrations were measured in the culture supernatants, by a solid-phase immunoassay, in Nunc- Immunoplate Maxisorp 96 wells (Nunc, Denmark). The plates were coated with goat anti-human IgM, IgG or IgA antibodies (Jackson Immunoresearch Laboratories, West Grove PA, USA). Biotinilated goat anti-human IgM, IgG or IgA (Jackson, West Grove, PA, USA) and streptavidin-alkaline phosphatase (Amersham, Buckinghamshire, England) were used for measurement of IgM, IgG and IgA respectively. Resulting yellow dye intensity was read by an ELISA reader (Microplate Auto-Reader Bio-Tek Instruments, VT, USA). Dye units were converted to immunoglobulin concentrations by extrapolation from standard curves determined by using purified myeloma proteins of known concentration in every assay.
Statistical analysis
We used non-parametric tests to compare the means (Kruskal Wallis test for k independent samples) and a standard analysis of variance to compare between groups. Logistic regression analysis was used for comparison of distribution of dichotomous values between the groups. Analysis was performed using SPSS for windows ver 9.0.
Results
32 patients were included in the study, 24 males (75%) and 8 females (25%) with a mean age at diagnosis of 3.4 years (range 1.2 – 7.0). Clinical presentations included severe and recurrent Ear-Nose-Throat (ENT) infections – 22 patients (69%), pneumonia, bronchopneumonia or severe, recurrent upper respiratory infections – 26 patients (81%), diarrhea – 4 (12.5%) and atopy related complaints – 20 patients (63%). A positive family history of recurrent, unusual or severe infections was obtained in 7 patients (22%). Demographical and clinical data of patient cohort is summarized in Table 2.
Table 2 Clinical and Demographic Data of Patient Cohort
THGI Non Transient p
Demographics No of Patients 17 (53%) 15 (47%) > 0.05
Males 15 (88%) 9 (60%) > 0.05
Females 2 [30] 6 [31] > 0.05
Average age at Diagnosis 3.6 years 3.1 years > 0.05
Clinical Data ENT 10 (59%) 12 (80%) > 0.05
Respiratory 15 (88%) 11 (73%) > 0.05
GI 2 [32] 2 [33] > 0.05
Atopy 11 (65%) 9 (60%) > 0.05
Family History 4 (24%) 3 (20%) > 0.05
Diagnosis IgAD 9 (53%) 7 (48%) > 0.05
IgGD 12 (71%) 10 (68%) > 0.05
IgMD 2 [34] 3 (20%) > 0.05
Treatment Antibiotics 4 (24%) 8 (53%) > 0.05
IVIg 2 [35] 1 [36] > 0.05
Outcome No Infections 14 (83%) 13 (87%) > 0.05
Follow-up (years) Age at last follow-up 7.1 5.5 > 0.05
Length of follow-up 3.5 2.5 > 0.05
THGI – Transient Hypogammglobulinemia of Childhood
Non Transient – Hypogammaglobulinemic patients who did not correct during follow-up
IgAD – IgA 2SD bellow age specific norms
IgGD – One or more IgG isotype 2SD bellow age specific norms
IgMD – IgM 2SD bellow age specific norms
Out of the initial 32 patients, 17 (53%) spontaneously corrected their Ig abnormalities. This group included 15 boys (88%) and 2 girls[12], mostly in the second or third year of life, average age at diagnosis being 3.6 years (range 1.3–9). The two groups, those with essentially transient hypogammaglobulinemia – THGI and those who did not correct their Ig values during the follow up period did not differ significantly at diagnosis, see Table 2.
All defects were partial, no patient in this group, showing a complete absence of a given Ig isotype. The clinical course was benign, only 9.4% (3/32) patients requiring IVIg (2 in the THGI group, 1 in the non corrected group). 38% (12/32) of patients received prolonged antibiotic prophylaxis (4/17 in the THGI and 8/15 in the non corrected group, p = 0.09) and resolution of clinical symptoms occurred in 84% of patients (14/17 in the THGI and 13/15 in the non corrected group). All calculated p values, non significant for comparison between the 2 groups.
Comparative analysis of serum Ig isotype levels at diagnosis showed no significant difference, between the transient and non-transient group.
In vitro lymphocyte proliferation and Ig secretion were measured in 9 patients (5 patients with THGI and 4 with non-corrected IGD) on one or more occasions. Lymphocytes proliferative responses to SAC, PWM and LPS showed no differences between the groups and no significant differences from childhood norms. The proliferative response to PHA was significantly increased (p < 0.005) after the correction of Ig abnormalities, overshooting normal controls (Fig 1).
Figure 1 Lymphocyte proliferation after in-vitro mitogenic stimuli in Pediatric Hypogammaglobulinemia. SAC – Staph. Aureus Cowan. PWM – Pokeweed Mitogen PHA – Phytohemmaglutinin LPS – Lipopolysacharide Values are given as Stimulation Index (SI) mean ± 95% CI, ** p < 0.005
Quantification of the in-vitro immunoglobulin secretion in response to the various mitogens showed significant isotype and mitogen dependent variation. The IgM secretory response to PWM and LPS, was low in hypogammaglobulinemic patients. After normalization of serum Ig values, the IgM response to LPS stimulation increased, see table 3. However, the improved response to LPS was still lower than age matched controls (p < 0.05).
Table 3 Immunoglobulin Secretion from B-cells after in-vitro mitogenic stimuli in Pediatric Hypogammaglobulinemia
SAC PWM LPS
IgM IgG IgA IgM IgG IgA IgM IgG IgA
Hypogamma 1.6 1.2 * 1.1 ** 1.1 * 1.4 * 1.3 * 1.3 * 2.0 * 1.1 *
(0.6–2.6) (0.9–1.5) (1.0–1.2) (0.9–1.3) (0.9–1.9) (1.0–1.6) (1.1–1.5) (1.1–2.9) (1.0–1.2)
Corrected 2.8 1.0 * 1.3 * 1.0 * 1.1 ** 1.3 * 2.2 * 1.5 * 1.2 *
(1.7–3.9) (0.9–1.1) (1.0–1.6) (0.9–1.1) (1.0–1.2) (1.1–1.5) (1.4–3.0) (1.2–1.8) (1.0–1.4)
Normal 3.0 2.5 2.3 2.6 3.0 2.2 4.4 5.0 3.4
(2.5–3.5) (1.9–3.1) (1.8–2.8) (2.2–3.0) (2.4–3.6) (1.7–2.7) (3.9–4.9) (3.5–6.5) (3.0–3.8)
SAC – Staph. Aureus Cowan. PWM – Pokeweed Mitogen
LPS – Lipopolysacharide
Values are given as Secretion Index Mean and (95% confidence interval)
** p < 0.005 compared to normal controls
* p < 0.05 compared to normal controls
For both IgG and IgA, the response in normal children was significantly better than in either group of patients. The IgA secretion index to all 3 stimulatory mitogens was minimal in hypogammaglobulinemic patients, even after serum Ig correction and differed significantly from age matched controls (p < 0.005 for SAC, p < 0.05 for PWM, p < 0.05 for LPS), table 3. The IgG secretion index in hypogammaglobulinemic patients was slightly better to LPS than to other mitogens (non significant) and differed significantly from age matched controls (p < 0.05 for SAC, p < 0.005 for PWM, p < 0.05 for LPS). No difference was observed on either test between THGI and non corrected patients on initial presentation.
Discussion
Young patients with recurrent infections represent a sizeable portion of the daily practice of all primary care pediatricians and family physicians, with parents clamoring for a solution with the accumulation of lost daycare or school days. The physician is faced with the dilemma when parental assurance will suffice, rather than initiation of a costly immunological investigation. Often, the presenting clinical signs and symptoms are insufficient for an educated diagnosis, as well as Ig levels in infants below the age of one year. The present investigation was initiated in order to try to contribute additional understanding how to differentiate between cases of primary immune deficiency and those who are not.
We prospectively studied the outcome of SHIC in 32 patients during a period of 8 years, mean follow up of 3.2 years. During this time more than half corrected their Ig abnormalities. The mean follow up of the non-corrected IGD group is slightly shorter (though not statistically significant) than that of the THGI group (2.5 years and 3.5 years respectively) which leads us to speculate that some patients in the "non-transient" group may eventually correct. This is consistent with previously published reports from Dalal et al. who, after a follow up of 10 years, found that 70% of patients had complete resolution of their Ig abnormalities [13].
Interestingly, the majority of our patients were males (75%), and a higher proportion of males corrected their serum Ig (15/24 males and 2/8 females). This finding of male preponderance is not uniquely ours. In the reports of Dalal et al. [14], 24/35 (69%) and Walker et al. [15] 29/39 (74%) of patients included in the study were male. We found no readily available explanation for this phenomenon.
It is impossible to estimate the prevalence of THGI in Israel, from our data, since our patients are part of a biased referral clinic population. Still, our impression is that this diagnosis is perhaps the most common of the Ig deficiencies in childhood, an impression supported by a number of publications from different groups [16,17].
In the paragraph on THGI in the most recent Scientific Group Report on the subject [1], the age cutoff of recovery of normal Ig synthesis capability "may be delayed for as long as 36 months". In contrast, in our series of patients, normalization to age adjusted Ig levels, has occurred in some cases at 8 or 9 years of age. These results are consistent with Dalal et al [18], who also describe resolution of THGI over the first decade of life. A more recently published series [19], 7/40 [20] of patients with THGI, still had low levels of antibodies at age 5 years. More over, the benign course of the group of patients who did not correct their Ig values during the course of our follow up, may indicate that a significant portion of these patients may actually still belong to the THGI group and will correct on subsequent follow up. These combined observations may induce a change in the classical definition and diagnostic criteria for THGI [21].
The majority of patients (84%) resolved their tendency for recurrent infections irrespective of their Ig values. Only a minority of patients required any medical intervention, 38% received antibiotic prophylaxis and only 9% intravenous Ig replacement therapy. We observed no difference in the clinical presentation or follow up of the transient group as compared to the non corrected IGD patients and resolution of serum Ig abnormality did not cause a complete clinical remission in all patients. This may be due to a residual inability to mount an adequate antibody response to specific antigen challenge, data that has not been evaluated in our series. The impaired IgG and IgA in vitro secretory responses, seen in hypogammaglobulinemic patients, even after serum Ig normalization, may be an expression of such an inability, which warrants further investigation.
Atopy was a prominent associated complaint in 21/37 (57%) of our patients. Especially so in comparison with the prevalence of asthma in this age group in Israel – 7% [22,23] and the estimated prevalence of other atopy associated diseases, about 20%. This finding was inconsistently reported by other investigators [24,25].
Though no clinical evidence of T-cell functional impairment was observed in our patients (no opportunistic, fungal or chronic viral infections), previous reports [26-28] suggest that the apparent B cell defect may be secondary to a T cell dysfunction such as T cell cytokine disregulation. We have shown that cellular and humoral responses to mitogenic stimuli tend to be lower in SHIC patients as compared to normal children but do not differentiate THGI from patients with more persistent hypogammaglobulinemia. Lymphocyte proliferation in response to PHA is significantly increased after Ig correction in THGI, overshooting normal controls. The molecular basis of this observation is unclear. In vitro IgM secretion is less impaired than other isotypes and in THGI a definite improvement of IgM secretion with SAC and LPS stimulation occurs concomitantly with correction of serum Ig. The IgG and IgA secretion in response to mitogenic stimuli is severely impaired and does not normalize concomitantly with serum Ig, indicating a possible impairment in the isotype switching mechanism. This observation is supported by the previous long term follow-up reported by Dalal [29] where specific IgG antibody responses to polysaccharide antigen were reduced even after the resolution of the serum Ig deficiency in a large subgroup of patients with apparently resolved THGI.
Conclusions
THGI is a relatively common cause of symptomatic hypogammaglubulinemia in infancy in our area. Most children will spontaneously correct their Ig abnormalities during the first decade of life. Though tests of cellular or humoral stimulation index, are not as yet capable of differentiating the transient from the non-transient patients upon their presentation, significant isotype and mitogen specific variability is evident. The relative preservation of the in vitro IgM secretory response and the lack of IgA/IgG response in patients with hypogammaglobulinemia, argues for a delay in isotype switching as the molecular basis underlying the clinical entity of transient hypogammaglobulinemia of infancy.
Competing interests
The author(s) declare that they have no competing interests.
Abbreviations
SHIC – Symptomatic Hypogammaglobulinemia in Infancy and Childhood
THGI – Transient Hypogammaglobulinemia of Childhood
Ig – Immunoglobulin
IGD – Imunoglobulin deficiency
SAC – Staphylococcus aureus cowan I
PWM – Pokeweed mitogen
LPS – Lipopolysaccharide
PHA – Phytohemagglutinin
IVIg – Intravenous Immunoglobulin
ENT – Ear, nose & throat
Authors' contribution
MIK – carried out the patient care and follow-up, was responsible for the database organization, data analysis and for manuscript coordination and writing
ZT – is one of the research initiators, carried out the patient care and follow-up and contributed to the manuscript writing
IA – carried out the in vitro cell proliferation and Ig secretion studies
RS – carried out the in vitro cell proliferation and Ig secretion studies
MS – carried out the patient care and follow-up, was responsible for the database initiation
IZ – is one of the research initiators, carried out of the laboratory evaluation and contributed to the manuscript writing
All authors read and approved the final manuscript
Pre-publication history
The pre-publication history for this paper can be accessed here:
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| 15498106 | PMC529469 | CC BY | 2021-01-04 16:29:00 | no | BMC Fam Pract. 2004 Oct 21; 5:23 | utf-8 | BMC Fam Pract | 2,004 | 10.1186/1471-2296-5-23 | oa_comm |
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BMC UrolBMC Urology1471-2490BioMed Central London 1471-2490-4-121550930210.1186/1471-2490-4-12Research ArticleA comparison of vas occlusion techniques: cautery more effective than ligation and excision with fascial interposition Sokal David [email protected] Belinda [email protected] Mario [email protected] Michel [email protected] Mark A [email protected] Family Health International, PO Box 13950, Research Triangle Park, NC 27709, USA2 Department of Family Medicine, Laval University, Quebec City, Canada3 EngenderHealth, 440 Ninth Avenue, New York, NY 10001, USA2004 27 10 2004 4 12 12 16 5 2004 27 10 2004 Copyright © 2004 Sokal et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Vasectomy techniques have been the subject of relatively few rigorous studies. The objective of this analysis was to compare the effectiveness of two techniques for vas occlusion: intraluminal cautery versus ligation and excision with fascial interposition. More specifically, we aimed to compare early failure rates, sperm concentrations, and time to success between the two techniques.
Methods
We compared semen analysis data from men following vasectomy using two occlusion techniques. Data on intraluminal cautery came from a prospective observational study conducted at four sites. Data on ligation and excision with fascial interposition came from a multicenter randomized controlled trial that evaluated the efficacy of ligation and excision with versus without fascial interposition. The surgical techniques used in the fascial interposition study were standardized. The surgeons in the cautery study used their customary techniques, which varied among sites in terms of type of cautery, use of fascial interposition, excision of a short segment of the vas, and use of an open-ended technique. Men in both studies had semen analyses two weeks after vasectomy and then approximately every four weeks. The two outcome measures for the analyses presented here are (a) time to success, defined as severe oligozoospermia, or <100,000 sperm/mL in two consecutive semen analyses; and (b) early vasectomy failure, defined as >10 million sperm/mL at week 12 or later.
Results
Vasectomy with cautery was associated with a significantly more rapid progression to severe oligozoospermia and with significantly fewer early failures (1% versus 5%).
Conclusion
The use of cautery improves vasectomy outcomes. Limitations of this comparison include (a) the variety of surgical techniques in the cautery study and differences in methods of fascial interposition between the two studies, (b) the uncertain correlation between sperm concentrations after vasectomy and the risk of pregnancy, and (c) the use of historical controls and different study sites.
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Background
Vasectomy techniques have been the subject of relatively few rigorous studies. The Royal College of Obstetricians and Gynecologists [1] noted that more research is needed to compare different methods of vasectomy. Experts have recommended the use of fascial interposition or cautery [1], cautery with clips [2], and cautery with fascial interposition [2,3].
We have shown in a randomized controlled trial that fascial interposition can reduce failure rates by about half when the occlusion method is suture ligation with vas excision [4]. However, the failure rates defined by semen analyses in that study were relatively high, with a failure rate of 5.7% even in the fascial interposition group.
At an experts' meeting organized by Family Health International (FHI) and EngenderHealth in April 2001, experts reviewed data on vas occlusion techniques, including the preliminary pooled results from the randomized controlled trial of fascial interposition mentioned above. Given the apparently high rate of vasectomy failures in that study, the experts recommended that an observational study be done to assess sequential sperm concentrations after vasectomies with cautery to see if there was a qualitative difference between sperm concentrations after vas occlusion by cautery and sperm concentrations after occlusion by ligation and excision. Some participants suggested that if such a study showed a clear difference in the rate of success or the frequency of apparent recanalizations between cautery and ligation and excision with fascial interposition, then this might provide sufficient evidence for service providers to consider switching to the use of a cautery technique [5].
Based on that recommendation, we conducted an observational study of vasectomy by cautery [6] and then conducted this comparative analysis. Even if some researchers decide that additional research is needed, the data from this comparative analysis should provide a strong basis for planning further research. The objectives of the analysis presented here were to compare early failure rates, sperm concentrations, and time to success for vas occlusion by ligation and excision with fascial interposition versus those for vas occlusion by cautery.
Methods
The methods of the fascial interposition and cautery studies have been previously described [4,6]. In brief, the fascial interposition study involved eight sites in seven countries. It was a randomized controlled trial comparing two occlusion techniques: ligation and excision with versus without fascial interposition. All surgeons used the no-scalpel approach to the vas and a standardized occlusion technique. The vas was occluded using two silk sutures, and an approximately 1-cm segment of vas between the ligatures was excised. For the fascial interposition technique, a suture was used to contain the testicular end of the vas inside the fascial sheath; the prostatic end remained outside [4]. The study was halted following a planned interim analysis that demonstrated a clear benefit from the use of fascial interposition [7]. Of 419 men who had fascial interposition in that study, 410 were included in this comparative analysis. Nine men were excluded because of lack of follow-up data.
The cautery study involved four sites in four countries. It was designed to estimate the effectiveness of cautery as currently performed and to describe the trends in sperm counts after vas occlusion by cautery. The surgeons used their customary cautery occlusion techniques, which differed among the sites. At two sites, surgeons used electro-cautery without fascial interposition: one with and one without excision of a short segment of the vas. At the other two sites, they used thermal cautery with fascial interposition: one with and one without an open-ended technique and excision of a short segment of the vas. Three sites used the no-scalpel approach to the vas. Graphic depictions of the four methods used have been published [6]. Of 400 men enrolled, 389 are included in this comparative analysis. Eleven men were excluded because of lack of follow-up data.
Follow-up and semen analysis methods
Both studies included frequent semen analyses, beginning at two weeks after vasectomy. In the fascial interposition study, subsequent semen analyses were conducted every four weeks until a man had provided two consecutive azoospermic specimens, was declared a vasectomy failure, or reached the end of study follow-up at 34 weeks. In the cautery study, after the first sample at two weeks, subsequent semen analyses were conducted at weeks 5, 8, 12, 16, 20, and 24, regardless of semen analysis findings. In both studies, participants were asked to record all ejaculations between semen analyses on a wallet-sized card, which they gave to study personnel at each follow-up visit.
Semen analyses methods for both studies were based on World Health Organization recommendations, but the methods differed somewhat between the two studies. Freshly collected semen was examined in the fascial interposition study, and data were obtained on sperm concentration, motility, and viability. For the cautery study, two of the four sites did not routinely collect fresh specimens, so semen analysis data from those two sites were limited to sperm concentrations. Therefore, for this comparative analysis, we did not consider sperm motility as an outcome measure. In addition, specimens showing azoospermia or very low sperm concentrations were centrifuged in the fascial interposition study but not in the cautery study. During both studies, the laboratories conducted periodic quality-control tests.
Outcome measures
In both studies, we used frequent semen analyses rather than pregnancy as the vasectomy effectiveness outcome measure, to minimize the risk of pregnancy, sample size, and study duration. Vasectomy success is commonly defined as two azoospermic specimens [2]. However, small numbers of nonmotile sperm may persist for many months in some men. Surgeons' experience [8] and guidelines recently published by the British Andrology Society [9] suggest that low concentrations of nonmotile sperm (<100,000 sperm/mL) are of less concern than higher concentrations. We found in the fascial interposition study [4] that severe oligozoospermia (<100,000 sperm/mL) was a more robust measure of success than was azoospermia, at least for research purposes. In both studies, men of different ages tended to reach severe oligozoospermia at about the same time, but older men took longer to reach azoospermia than did younger men.
Consequently, we used two definitions for vasectomy success for this comparative analysis. Our primary definition of success was severe oligozoospermia, defined as <100,000 sperm/mL in two consecutive specimens taken at least two weeks apart. Our alternate definition of success was the occurrence of two consecutive azoospermic specimens taken at least two weeks apart, with no subsequent samples showing sperm concentrations of 100,000 sperm/mL or higher. Motility was not considered, for reasons mentioned earlier. The date of success was the date of the first of the two oligozoospermic or azoospermic semen samples.
For early failure, we used a criterion of >10 million sperm/mL at week 12 or later, regardless of motility. This is an adaptation of Alderman's criteria specifying 5 million motile sperm/mL or more as evidence of "overt failure" [10]. This definition is different from the definitions of failure used by each of the two studies, but it was necessary for a comparative analysis because some sites in the cautery study did not measure sperm motility. Thus, the failure rates reported here may differ slightly from the failure rates reported by each study. In addition, to avoid bias from the two studies' different lengths of follow-up, we included semen analysis data from the fascial interposition study through only 26 weeks of follow-up.
The data collection forms, study monitoring, and laboratory quality-control procedures were similar for both studies, though only one research site was common to both. Both studies were organized and managed by researchers and staff at FHI and EngenderHealth, and both received approval from FHI's institutional review board and from institutional review boards at the study sites.
Statistical methods
Kaplan-Meier product-limit estimates of the probabilities of severe oligozoospermia, at each scheduled week of follow-up through week 24, and their 95% confidence intervals (CIs) were produced overall, by study group (i.e., fascial interposition and cautery), and by study group and age group (i.e., <35 years and 35 years and older). Peto's standard error [11] was used to compute the 95% CIs. The Kaplan-Meier probabilities were compared between the study groups using a two-sided log-rank test with an alpha of 0.05. Given that the participants in the fascial interposition study had a longer follow-up period than the participants in the cautery study (34 versus 24 weeks), the information on the fascial interposition participants was censored at the 26-week visit for the purpose of this comparative study.
The comparison of failure rates between the two study groups was based on a Fisher exact test with a two-sided alternative hypothesis and an alpha of 0.05. In addition, a logistic model was fit to estimate an age-adjusted odds ratio of the failure rates and its 95% CI.
Unlike in the cautery study, participants in the fascial interposition study were discontinued after azoospermia was confirmed or after vasectomy failure was declared. Therefore, for the purpose of comparing the distribution of the participants in the different sperm concentration categories by week of follow-up, we kept azoospermic cases in the azoospermic category for all follow-up weeks after their discontinuation due to confirmed azoospermia. Similarly, we kept participants with a declared vasectomy failure in the sperm concentration category that they were in at the moment of discontinuation, for all subsequent follow-up weeks.
Results
Detailed results for the two studies have been reported [4,6]. We report here the results of the comparison of the semen analysis data from the two studies.
Baseline population data
Among the baseline population characteristics (Table 1), age distribution was somewhat different between the two studies, with a younger population in the fascial interposition study. Marital status, number of children, use of condoms, and years of education were similar between the two studies.
Analysis of early failures
We found significantly fewer early failures in the cautery study than in the fascial interposition group from the randomized controlled trial: 1.0% (4/389) versus 4.9% (20/410) (p = 0.0014 by the Fisher exact test). The adjusted odds ratio was 4.8 (95% CI, 1.6–14.3), indicating nearly a five-fold higher risk of early failure in the fascial interposition study than in the cautery study. No significant age effect was detected (data not shown).
Sperm concentrations
The distribution of sperm concentrations by week is shown for the two studies (Figure 1). The difference in early failures can be appreciated by examining the percentages of men with high sperm concentrations. In the fascial interposition study, the percentage of men with sperm counts of 10 million or more stayed about the same from 6 to 26 weeks. However, in the cautery study, the percentage decreased dramatically from 5 to 8 to 12 weeks. This difference was probably due to recanalizations, which become apparent in the first 6 to 10 weeks after the procedure.
Time to success
Life-table analyses of time to success showed that the participants in the cautery study reached severe oligozoospermia significantly more rapidly than did those in the fascial interposition study (p = 0.0049) (Figure 2). Ninety-seven percent of the men in the cautery study had reached success by 12 weeks, while only 91% in the fascial interposition study had reached success by 14 weeks. The analyses of the data stratified by age group showed similar results (data not shown). Using the time to azoospermia outcome, the difference between the two groups was also significant (p < 0.0001) (data not shown).
Discussion
The difference in the observed failure rates suggests that vas occlusion techniques that include cautery are significantly more effective than ligation and excision plus fascial interposition, at least based on semen analysis. We believe that most of the failures in the fascial interposition group were due to early recanalizations within the first two to three months after vasectomy (data not shown). Possible explanations for the surprisingly high failure rate among the fascial interposition group have been previously presented and discussed [4].
Recent reports suggest that pregnancy rates may be higher in low-resource settings, where semen analysis is usually not available and where most vasectomies are done by ligation and excision [12,13]. The use of cautery devices may have the potential to reduce failure rates in low-resource settings.
Which cautery technique is best?
When a ligation and excision technique is used, we have shown that fascial interposition provides an important improvement in effectiveness [4]. The addition of fascial interposition may be less important when cautery is used as the occlusion method, but this study was not designed to answer that question.
Schmidt was a pioneer in the use of cautery for vasectomy. His preferred technique was thermal cautery of about 5 mm of each end of the vas, combined with fascial interposition and no excision [3]. Two of the sites in the observational cautery study used techniques very similar to Schmidt's technique. The other two sites used electro-cautery without fascial interposition, an approach that has been used for many years at the Elliot-Smith Clinic in Oxford [14] and similar to one used for many years by Marie Stopes clinics with excellent results [15]. Little evidence is available to support one type of cautery over another. Schmidt [16] preferred thermal cautery to electro-cautery based on histological examination of specimens at vaso-vasostomy, and Li [17] found a lower failure rate with thermal cautery than with electro-cautery, but the difference in Li's study was not statistically significant.
The length of the vas segment that is cauterized can vary by the type of cautery. In the United States and Canada, marketed thermal cautery "vasectomy tips" have a functional length of about 0.8 cm, so it would be difficult to cauterize more than 1 cm of each end. The cautery tip of the Sturgeon cautery device used by Schmidt was 0.5 cm long. However, the tips available for electro-cautery devices are much longer, which could permit cauterization of longer segments of the vas and potentially cause more difficulty in the event of a request for reversal.
Since several reports suggest that the combination of thermal cautery plus fascial interposition is one of the most effective methods available [18], this procedure can be recommended with few reservations. However, there is at least one other reason to consider the inclusion of fascial interposition in the vasectomy procedure, especially in low-resource settings. If providers in low-resource settings adopted a cautery technique, cautery instruments could occasionally become unavailable for various reasons. In those cases, a provider might want to be able to perform fascial interposition as part of a ligation and excision procedure.
Schmidt suggested that cautery should not be combined with suture or clip ligation of the vas [3]. He noted that while blood vessels will thrombose after ligation, the vas will remain open. Thus, using a ligature in addition to cautery could reduce the value of cautery by causing necrosis of some or the entire cauterized end, potentially reducing the benefit from cautery. None of the cautery techniques in this study included the use of ligatures or clips on top of a cauterized vas.
Study limitations
Limitations of this comparison include (a) the variety of vas occlusion techniques used in the cautery study and differences in methods of fascial interposition between the two studies, (b) the uncertain correlation between post-vasectomy sperm concentrations and the risk of pregnancy, (c) the lack of sperm motility data and centrifugation for the cautery study, and (d) the use of different study sites and surgeons in the two studies. The difference in time to vasectomy success could in part be related to the lack of centrifugation in the cautery study. However, the difference in centrifugation between the two studies would not have affected the detection of early failures, especially since the cautery study gathered semen samples throughout the 24-week follow-up period (i.e., men continued providing semen samples even after they reached azoospermia). Another potential limitation of this comparison was the difference in follow-up schedules between the two studies.
In addition, other factors could have an unknown impact on the comparability of the data. Even though the results are encouraging for the use of cautery in vasectomy, we have to be cautious about making definitive statements based on this nonrandomized comparison.
Future vasectomy research
Results from this comparative analysis suggest that cautery may be a more robust and less technique-dependent method than is fascial interposition. However, additional research would be useful to directly compare the effectiveness of the following standardized techniques in a randomized controlled trial: cautery without fascial interposition, cautery with fascial interposition, and ligation and excision with fascial interposition. Additional research would also be of interest to compare an open-ended procedure with a closed-end procedure. Several investigators have suggested that leaving the testicular end open reduces post-vasectomy pain, but no randomized controlled trials have examined this issue [1,18].
Conclusions
We compared data from two prospective multicenter studies conducted using similar methodologies. We found that the use of cautery as part of the vasectomy procedure significantly reduced vasectomy failure rates compared with ligation and excision plus fascial interposition as part of the procedure. It is unclear from our results and those of others whether fascial interposition used with cautery improves vasectomy success rates when compared with cautery alone.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
DS participated in the conception, design and analysis of the study, and drafted the manuscript. BI participated in the conception, design, and analysis of the study, and was primarily responsible for managing the study implementation. MC participated in the design of the study and was primarily responsible for the statistical analysis. ML participated in the conception, design and analysis of the study. MB participated in the conception, design, management and analysis. All authors reviewed and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The authors would like to thank the clinical investigators and study coordinators for the two studies whose work formed the basis of this comparison. Partial support for this work was provided by FHI with funds from the U.S. Agency for International Development (USAID) Cooperative Agreement # CCP-A-00-95-00022-02, and by EngenderHealth with funds from the USAID Cooperative Agreement # HRN-A-00-98-00042-00, although the views expressed in this article do not necessarily reflect those of FHI, EngenderHealth, or USAID.
Figures and Tables
Figure 1 Distribution of sperm concentrations by week after vas occlusion by ligation and excision with fascial interposition (left) versus cautery (right).
Figure 2 Cumulative probability of vasectomy success by week: vas occlusion by cautery (solid line) versus fascial interposition (dashed line). Success was defined as severe oligozoospermia (i.e., <100,000 sperm/mL).
Table 1 Baseline characteristics by study/vas occlusion technique
Characteristic Ligation and excision with fascial interposition Intraluminal cautery
N % N %
Total 410 100.0 389 100.0
Age group <30 109 26.6 39 10.0
30–34 125 30.5 115 29.6
35–39 95 23.2 121 31.1
40+ 81 19.8 114 29.3
Marital status Married 344 83.9 276 71.0
Co-habiting 52 12.7 92 23.7
Single 14 3.4 21 5.4
Number of children 0 20 4.9 16 4.1
1 22 5.4 44 11.3
2 210 51.2 196 50.4
3+ 158 38.5 133 34.2
Prior condom use No 213 52.0 178 45.8
Yes 197 48.0 211 54.2
Years of education * Mean (SD) 11.0 (4.3) 13.1 (3.7)
Median 11 13
* One "years of education" value was missing for the fascial interposition group.
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| 15509302 | PMC529470 | CC BY | 2021-01-04 16:03:53 | no | BMC Urol. 2004 Oct 27; 4:12 | utf-8 | BMC Urol | 2,004 | 10.1186/1471-2490-4-12 | oa_comm |
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BMC Cardiovasc DisordBMC Cardiovascular Disorders1471-2261BioMed Central London 1471-2261-4-191550929910.1186/1471-2261-4-19Research ArticleHospitalization for heart disease, stroke, and diabetes mellitus among Indian-born persons: a small area analysis Muennig Peter [email protected] Haomiao [email protected] Kamran [email protected] Department of Health Policy and Management, Mailman School of Public Health, Columbia University, New York, NY 10003, USA2 Department of Community Medicine, Mercer University School of Medicine, Macon, GA, 31207, USA3 Inner City Health Research Unit, St. Michael's Hospital and the University of Toronto, Toronto, ON, M5V2M4, Canada2004 27 10 2004 4 19 19 12 3 2004 27 10 2004 Copyright © 2004 Muennig et al; licensee BioMed Central Ltd.2004Muennig et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
We set out to describe the risk of hospitalization from heart disease, stroke, and diabetes among persons born in India, all foreign-born persons, and U.S.-born persons residing in New York City.
Methods
We examined billing records of 1,083,817 persons hospitalized in New York City during the year 2000. The zip code of each patient's residence was linked to corresponding data from the 2000 U.S. Census to obtain covariates not present in the billing records. Using logistic models, we evaluated the risk of hospitalization for heart disease, stroke and diabetes by country of origin.
Results
After controlling for covariates, Indian-born persons are at similar risk of hospitalization for heart disease (RR = 1.02, 95% confidence interval 1.02, 1.03), stroke (RR = 1.00, 95% confidence interval, 0.99, 1.01), and diabetes mellitus (RR = 0.96 95% confidence interval 0.94, 0.97) as native-born persons. However, Indian-born persons are more likely to be hospitalized for these diseases than other foreign-born persons. For instance, the risk of hospitalization for heart disease among foreign-born persons is 0.70 (95% confidence interval 0.67, 0.72) and the risk of hospitalization for diabetes is 0.39 (95% confidence interval 0.37, 0.42) relative to native-born persons.
Conclusions
South Asians have considerably lower rates of hospitalization in New York than reported in countries with national health systems. Access may play a role. Clinicians working in immigrant settings should nonetheless maintain a higher vigilance for these conditions among Indian-born persons than among other foreign-born populations.
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Background
Immigrant populations in industrialized nations are on average healthier than their native-born counterparts [1]. However, there is considerable debate surrounding the risk of ischemic heart disease and other related vascular and metabolic conditions among South Asian Indians, both within India and in the countries to which they immigrate [2-7]. The relationship between ischemic heart disease and Indian ethnicity is complex. For instance, lower rates of smoking are juxtaposed against higher mean cholesterol levels and unfavourable cholesterol ratios, higher rates of central obesity, and a higher prevalence of diabetes mellitus among Indian immigrants relative to either native-born or other foreign-born populations[3-5], [7-9].
Studies conducted in countries with national health-care systems have noted approximately twice the risk of hospitalization for ischemic heart disease or myocardial infarction among South Asian immigrant men relative to native-born men aged 45–64 [10]. This amounts to nearly four times the risk of Caribbean-born men in the same study. However, India-born women were found to be at similar risk as native-born women. South Asian immigrants may also experience the onset of symptomatic heart disease at a younger age than native born populations [11]. Nonetheless, mortality rates of ischemic heart disease among Indian immigrants to Canada were comparable to Canadians of European ancestry [12]. There is therefore considerable confusion surrounding the actual underlying risk of ischemic heart disease among South Asian persons.
We set out to describe relative differences in the rates of hospitalization for heart disease, stroke, and diabetes among Indian-born persons relative to native-born persons or other foreign-born persons living in New York City. Unfortunately, there are no national datasets that provide individual level data for the risk of hospitalisation among specific immigrant sub-groups. It is, however, possible to compare the risk of hospitalization among immigrant sub-groups on an area-level using established small area analytic techniques via local datasets with a large sample size [13]. Herein, we quantify the risk of hospitalization for heart disease among Indian-born persons relative to other groups using a large hospitalization dataset covering New York City.
Methods
We obtained all hospital admission records for residents of New York City from the Statewide Planning and Research Cooperative System (SPARCS) and population data for New York City from Census 2000 [14]. The SPARCS dataset contains information on 2.45 million hospitalizations for the civilian population of New York State, including basic demographic variables and diagnosis codes [15]. A logistic model was used to calculate rate ratios and risk ratios (RR) rather than odds ratios. All analyses were conducted on SAS version 8 (Cary, NC).
We used this model to calculate hospitalizations for manifestations of atherosclerotic heart disease (myocardial infarction, angina, and congestive heart failure, International Classification for Disease, 9th Revision codes 410, 413, and 428 respectively), stroke (430–438), and diabetes (250) as the dependent variables in each regression model. The following categorical variables were entered as independent predictors: age (0–6, 7–17, 18–44 [reference], 45–64, 65+), sex (reference = female), education level (completed high school versus no high school [reference]), and country of birth (born in India, other foreign-born persons, and native-born [reference]).
While the SPARCS dataset includes information on patients' age, sex, race, and ethnicity, it does not contain information on country of origin, income, or education level. To obtain country of origin and income data, we matched each patient's zip code from SPARCS hospitalization records to information obtained from the Census 2000 Long Form [14].
For instance, to calculate age-specific hospitalization rates, all hospitalizations among persons aged 65 and older residing within a particular zip code were divided by the number of persons aged 65 and older living within that same zip code. We then entered the proportion of foreign-born persons and Indian-born persons living within each zip code in New York City, as well as the proportion graduating from high school as independent variables in our regression analysis.
The model took the general form:
Logistic models were constructed for each category of hospitalization and the variables representing the proportion of persons from each region of the world. We tested the models using Hosmer and Lemeshow goodness of fit test, and index plots of the residuals.
Results
In 2000, there were 70,598 persons in New York City who were born in India (see Table 1). Of these, 45% were female, 63% were married, and 14.4% were living below the poverty line. Table 2 presents the relationship between age, sex, and education level on the risk of hospitalization for heart disease alone. As expected, there is an increase in heart disease risk with increasing age, male sex, and lower educational attainment.
Table 1 Socio-demographic characteristics of Indian-born persons in New York City.
Number Percent
Total Population 70,598 -
Age
0–17 years 7,489 10.6%
18–24 years 7,681 10.9%
25–34 years 17,950 25.4%
35–44 years 15,938 22.6%
45–54 years 11,789 16.7%
55–64 years 6,499 9.2%
65–74 years 2,320 3.3%
75 + years 932 1.3%
Sex
Female 31,749 45.0%
Male 38,849 55.0%
Marital Status
Now married 44,665 63.3%
Widowed 2,093 3.0%
Divorced 1,352 1.9%
Separated 561 0.8%
Never married* 21,927 31.1%
Poverty status
Group quarters 583 0.8%
Below poverty line 10,150 14.4%
100–200% poverty line 11,797 16.7%
>200% poverty line 48,068 68.1%
*Includes persons < age 15.
Table 2 Risk ratios of hospitalization for heart disease by age, sex, and education (reference levels and 95% confidence interval are indicated in brackets)
Variable Female Male Total
Age
0–6 0.218 (0.153, 0.310) 0.534 (0.361, 0.788) 0.299 (0.230, 0.388)
7–17 0.192 (0.130, 0.285) 0.336 (0.126, 0.892) 0.219 (0.152, 0.316)
18–44 (ref) 1.00 1.00 1.00
45–64 13.58 (12.99, 14.19) 15.42 (14.42, 16.48) 13.98 (13.47, 14.51)
65+ 44.75 (42.84, 46.75) 77.37 (72.55, 82.50) 55.67 (53.71, 57.70)
Male Sex (Female) N/A 1.62 (1.60, 1.65) N/A
High School Grad. (Non-Graduate)* 0.943 (0.935, 0.951) 0.857 (0.850, 0.865) 0.901 (0.896, 0.906)
*Because some values were small, risk ratios for this category were rounded to 3 decimal places.
Table 3 presents the risk of hospitalization for heart disease, stroke, and diabetes mellitus by country of birth controlling for age, sex, education, and percentage of foreign-born persons in zip code. After controlling for covariates, foreign-born persons were less likely than native-born persons to be hospitalized for heart disease (RR = 0.70, 95% confidence interval 0.67, 0.72). As with native-born persons, foreign-born women (RR = 0.67, 95% confidence interval 0.63, 0.71) were at lower risk of hospitalization for heart disease than foreign-born men (RR = 0.74, 95% confidence interval 0.70, 0.78); however, the difference was much smaller than it is with native-born persons (RR = 1.62, 95% confidence interval 1.60, 1.65, see Table 1). Persons born in India were at similar risk of hospitalization as native-born persons for heart disease (RR = 1.02, 95% confidence interval 1.02, 1.03), but 47% more likely to be hospitalized for heart disease than other foreign-born persons (data not shown). Differences in the risk of hospitalization between India-born men and women were minimal.
Table 3 Risk ratios of hospitalization for heart disease, stroke, and diabetes among Indian persons after controlling for age, sex, education, and percentage of foreign-born persons in zip code (reference levels and 95% confidence interval are indicated in brackets)
Variable Male Female Total
Heart Disease
Foreign-born (Native-born) 0.74 (0.70, 0.78) 0.67 (0.63, 0.71) 0.70 (0.67, 0.72)
Born in India (Native-Born) 1.03 (1.02, 1.04) 1.01 (1.00, 1.02) 1.02 (1.02, 1.03)
Stroke
Foreign-born (Native-born) 0.75 (0.68, 0.83) 0.80 (0.73, 0.87) 0.78 (0.73, 0.83)
Born in India (Native-Born) 1.00 (0.98, 1.02) 0.99 (0.98, 1.01) 1.00 (0.99, 1.01)
Diabetes Mellitus
Foreign-born (Native-born) 0.37 (0.33, 0.40) 0.42 (0.38, 0.47) 0.39 (0.37, 0.42)
Born in India (Native-Born) 0.97 (0.95, 0.99) 0.95 (0.92, 0.97) 0.96 (0.94, 0.97)
The risk of hospitalization for stroke is similar among India-born persons and native-born persons, with a risk ratio of 1 (95% confidence interval 0.99, 1.01). Indian-born persons, however, are more likely than other foreign-born persons to be hospitalized for stroke; the average foreign-born person has a risk ratio of 0.75 (95% confidence interval 0.73, 0.83).
While the risk of hospitalization for diabetes similar among Indian-born persons and native-born persons (RR = 0.96, 95% confidence interval 0.94, 0.97), the likelihood of hospitalization for this condition is considerably higher on average than that of other foreign-born persons regardless of sex. Foreign-born persons overall are at 39% the risk of hospitalization for diabetes mellitus (95% confidence interval 0.37, 0.42).
Discussion
While Indian immigrants to industrialized nations are at similar risk of hospitalization for heart disease, diabetes, and stroke as their native-born counterparts, they are at much greater risk of hospitalization for heart disease, stroke, and diabetes than are immigrants from other countries. Although earlier studies have found differences in the risk of heart disease among Indian-born persons to England and Canada by sex, we found that risks were similar among males and females[10,11].
In an earlier paper, a higher age-adjusted mortality rate due to ischemic heart disease and stroke was noted among foreign-born women relative to native-born women [16]. In that study, it was postulated that the higher observed mortality rates might be due to changing demographic trends among recent immigrants. Specifically, it was hypothesized that mortality differences may be attributable to a higher proportion of female immigrants from higher risk countries. This hypothesis does not seem correct in light of current findings, and may be attributable to random error in the census sample.
Our study was subject to a number of important limitations. While the hospitalization data contained insurance status, the census data did not. We were therefore unable to control for insurance status in our study.
Given that foreign-born persons in the U.S. approach three times the rate of lacking health insurance as native-born persons (33.4% versus 12.2% respectively)[17], it is possible that many persons are at relatively lower risk of hospitalization and relatively higher risk of death. Still, since heart disease is a serious and potentially fatal condition, it seems unlikely that many immigrants would forgo hospital-based care for fear of incurring expenses. Moreover, the abundance of public hospitals in New York City reduces the probability that seriously ill persons would avoid seeking hospital care for fear of incurring medical costs. Nonetheless, some Indian-born persons may avoid seeking care due to a lack of health insurance. To the extent that avoidance of care occurs, the risk of hospitalization among Indian-born persons is underestimated in our analysis.
A second limitation was our use of zip-code-only data for the proportion of foreign-born in a neighbourhood, which may be confounded by ecological factors. This was minimized somewhat by examining individual-level hospitalizations for all variables but education and country of birth. It is notable that our data on the foreign-born overall are constant with a growing body of literature showing lower morbidity and mortality among immigrants to the United States than native-born persons[1].
The risk differences we found between native-born persons and persons born in India were not large – for every 100 hospitalizations for heart disease among native-born persons, we would expect 2–3 additional hospitalizations among persons from India. However, the risk of heart disease, cancer and diabetes is significantly higher than those of other immigrant groups with a similarly low prevalence of smoking[1]. Previous studies of behavioural risk factors suggest that diet is the overwhelming risk factor in the India-born population – a finding consistent with our subjects' higher risk of hospitalization for diabetes mellitus relative to native-born persons and other foreign-born persons [5,10,11]. The presence of a single overriding risk factor simplifies public health efforts; it is easier to target diet and exercise regimens, for instance, than a broader range of health behaviours.
Conclusions
Indian-born New Yorkers are at substantially higher risk of heart disease, stroke, and diabetes than other foreign-born persons. However, these risks are comparable to that of heart disease, stroke, and diabetes among native-born persons. It is also conceivable that access to health services plays a role in the risk of hospitalisation for heart disease or related conditions given that higher hospitalization rates have been observed in countries with universal health coverage. Prospective data are needed to refine our understanding of the risk factors associated with heart disease in persons from India. As with interpreting data with any epidemiological study, results should not be generalized to clinical practice without first considering the individual patient's socio-demographic risk profile. Nevertheless, clinicians working in facilities serving foreign-born populations may wish to maintain a higher degree of vigilance for heart disease and its risk factors among persons from India.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
PM planned the analysis, outlined the study, reviewed the data and regression analyses, and contributed to the development of the manuscript.
HJ compiled the data, designed the small area analyses, and conducted the regression analyses.
KK contributed to the planning of the analysis and the preparation of the manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
==== Refs
Singh GK Miller BA Health, life expectancy, and mortality patterns among immigrant populations in the United States Can J Public Health 2004 95 I14 21 15191127
Malhotra P Kumari S Singh S Varma S Isolated lipid abnormalities in rural and urban normotensive and hypertensive north-west Indians J Assoc Physicians India 2003 51 459 463 12974426
Malhotra P Singh MM Kumar R Kumari S Coronary heart disease and its risk factors in first-generation immigrant Asian Indians to the United States of America Indian Heart J 1996 48 737 738 9062034
Dhawan J Coronary heart disease risks in Asian Indians Curr Opin Lipidol 1996 7 196 198 8883493
Uppaluri CR Heart disease and its related risk factors in Asian Indians Ethn Dis 2002 12 45 53 11913608
Enas EA Coronary artery disease epidemic in Indians: a cause for alarm and call for action J Indian Med Assoc 2000 98 694 5, 697-702 11265799
Enas EA Yusuf S Mehta JL Prevalence of coronary artery disease in Asian Indians Am J Cardiol 1992 70 945 949 1529952 10.1016/0002-9149(92)90744-J
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| 15509299 | PMC529471 | CC BY | 2021-01-04 16:30:03 | no | BMC Cardiovasc Disord. 2004 Oct 27; 4:19 | utf-8 | BMC Cardiovasc Disord | 2,004 | 10.1186/1471-2261-4-19 | oa_comm |
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PLoS MedPLoS MedpmedplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 1556232210.1371/journal.pmed.0010060EssayOtherClinical PharmacologyClinical TrialsDrugs and Adverse Drug ReactionsRegulationA Taxpayer-Funded Clinical Trials Registry and Results Database EssayTurner Erick H Erick H. Turner is a former clinical reviewer of psy-chotropic drugs at the United States Food and Drug Administration. He is currently the medical director of the Mood Disorders Program at the Portland Veteran Affairs Medical Center, assistant professor of psychiatry, and assistant professor of pharmacology and physiology at Oregon Health and Science University, Portland, Oregon, United States of America. E-mail: [email protected]
Competing Interests: The author is on the speaker's bureaus of Eli Lilly, AstraZeneca, and Bristol-Myers Squibb. He has provided outside consulting to Bristol-Myers Squibb, Eli Lilly, GlaxoSmithKline, and Sepracor. He has also received funding for clinical drug trials, which can be spent only for research purposes and which has no effect on his income, from Abbott Laboratories, AstraZeneca, Bristol-Myers Squibb, and DOV Pharmaceuticals.
12 2004 30 11 2004 1 3 e60Copyright: © 2004 Erick H. Turner.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.It already exists within the US Food and Drug Administration, argues a former clinical reviewer of psychotropic drugs at the FDA
It already exists within the US Food and Drug Administration
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Over the past several years, there has been growing concern about selective publication of clinical trial results [1,2]. The debate has intensified since New York State Attorney General Elliot Spitzer filed suit against GlaxoSmithKline on June 2, 2004, alleging that the company was hiding data regarding the efficacy and safety of selective serotonin reuptake inhibitors in pediatric patients with depression [3].
The two most frequently suggested remedies for the selective reporting of clinical trials results have been to register all clinical trials and to make their results publicly available. Registries have been called for at least as far back as 1974; hundreds have in fact already been established [4]. Shortcomings of registries include the fact that they are often not coordinated and that participation is often voluntary and—in cases where they are mandated by legislation—difficult to enforce. For example, ClinicalTrials.gov, a registry authorized by the Food and Drug Modernization Act of 1997, appears not to be comprehensive. One study found that, of 127 cancer protocols sponsored by pharmaceutical companies that met criteria for inclusion, only 48% were in fact submitted to the registry [5]. Thus, one can check a number of registries and still have little assurance that all the relevant trials of interest have been included.
The public would benefi t from more freedom of information at the FDA
(Illustration: Margaret Shear)
Increasing the pressure on pharmaceutical companies to include more trials in registries, the International Committee of Medical Journal Editors has announced that, as a condition of considering a trial for publication, member journals will require its registration in a public trials registry [6]. Further, at the American Medical Association (AMA) Annual Meeting of the House of Delegates in June 2004, the AMA called on the Department of Health and Human Services to establish a comprehensive national registry. In September 2004, an AMA trustee testified in a United States Congressional hearing, outlining elements necessary to make such a registry effective [7]. Momentum for a comprehensive clinical trials registry is also building internationally [8].
In this essay, I argue that a highly valuable but underused registry and results database for US trials already exists within the Department of Health and Human Services, specifically within the Food and Drug Administration (FDA).
New Drug Applications
Before a pharmaceutical company can conduct a US trial that it intends to use in support of a new drug application (NDA), it must first register that trial with the FDA. Because the NDA forms the basis for marketing approval, it seems likely that the percentage of industry-sponsored trials that are registered with the FDA is very high. This registration takes the form of an investigational new drug (IND) application [9]. The IND contains a trial protocol; protocols for additional studies within the same clinical trials program are submitted as amendments to the IND. Later, when the sponsor has completed its clinical trials program and wishes to apply for marketing approval, it submits its NDA.
The FDA then begins the NDA review process, during which a physician, a statistician, and a pharmacologist, among others, generate lengthy review documents [10]. These reviews not only address the sponsor's analyses of the data on pivotal studies, but they often also include reanalyses by the reviewers using raw data obtained from the sponsor. These analyses are conducted in adherence to the statistical methods set forth a priori in the original trial protocols. (By contrast, with most journal publications, it is usually not possible for the reader to verify whether what is presented as the main finding is consistent with the original hypothesis or whether it was a post hoc finding.) After the primary reviewers have written their reviews, shorter reviews are written by their superiors, with the process culminating in a decision about whether to approve the drug for the proposed indication.
Paroxetine for Anxiety Disorders: Checking the Published Literature against the FDA Reviews
A Cochrane systematic review of antidepressants for generalized anxiety disorder [15] lists only one double-blind placebo-controlled study of paroxetine [16], a positive study. A PubMed search reveals no additional double-blind placebo-controlled studies. In accessing the review from Drugs@FDA (approval date April 2001), we learn that there were in fact three pivotal double-blind placebo-controlled studies. One of these studies corresponds to the published positive study noted above. Of the remaining two studies, both apparently unpublished, one was positive while the other was marginally positive.
Turning to the controlled-release formulation of paroxetine (Paxil CR) for panic disorder, a review article states in its abstract that the drug “demonstrated efficacy in three well designed studies in patients with panic disorder with or without agoraphobia” [17]. In reading the corresponding FDA statistical review, we verify that there were indeed three studies. However, the FDA statistical reviewer found that only one of these studies was strongly positive. A second study, judged “supportive” of efficacy, had a marginally significant (p = 0.039) result on a secondary observed-cases analysis, but a nonsignificant (p = 0.38) result on the primary efficacy analysis defined a priori. The third study was clearly negative, with p-values of 0.33 and 0.57 on the primary and secondary analyses, respectively.
A Semi-Public Database
This process occurs entirely outside of the public domain. However, in the interest of making the FDA more “transparent,” and in accordance with the Electronic Freedom of Information Act [11], the FDA has, for the past several years, posted selected NDA reviews for approved drug–indication combinations on the FDA Web site Drugs@FDA (http://www.accessdata.fda.gov/scripts/cder/drugsatfda/index.cfm). These NDA review documents are much more detailed than the resulting package insert and often more detailed than corresponding journal publications.
For example, while the clinical trials section of the package insert is typically a few paragraphs long, the efficacy portion of the clinical review usually runs tens of pages. Because the FDA is made aware of all studies that the sponsor plans to use in support of the NDA before they are conducted, and thus before there can be any selection based on outcome, these reviews cover not only studies that are positive (and more likely to be published in journals), but also studies whose outcome was negative or indeterminate. The sidebar gives an example of how NDA review documents at the FDA give valuable information about paroxetine for anxiety disorders.
FDA Reviews for All Approved Drugs Should Be Made Public
In the examples discussed in the sidebar, our having access to the FDA review documents allows us to become aware of, and see beyond, apparent publication bias. It is in the best interest of the public health for the FDA to make as many reviews available as possible. According to the FDA Web site, “As FDA continues to be one of the world's leading agencies in its emphasis on openness and transparency, it is aware that making even more information available to the public will further the Agency's mission to protect and promote public health and improve its credibility. For example, FDA has aggressively implemented the Electronic Freedom of Information Act…” [11].
Unfortunately, the availability of review documents on Drugs@FDA is sporadic. To take additional examples from psychiatry, NDA reviews have been posted on Drugs@FDA for some approved drug–indication combinations, such as fluoxetine for pediatric depression, and aripiprazole and quetiapine for schizophrenia. However, NDA reviews for many other drug–indication combinations have not been posted: the Prozac Weekly formulation of fluoxetine, clozapine for suicidal behavior in patients with schizophrenia or schizoaffective disorder, and quetiapine for mania, among others. A review on paroxetine for pediatric depression, the subject of Elliot Spitzer's suit against GlaxoSmithKline, is not posted. This is probably because this drug–indication combination was not approved; in fact, it is possible that GlaxoSmithKline did not file an NDA to be reviewed. However, I do not understand why, in cases where NDAs were both submitted and approved, such as the ones listed above, some reviews are posted while others are not.
I therefore suggest that we increase access to the clinical trials registry and results database that already exist within the FDA. The agency could expand its implementation of the Electronic Freedom of Information Act and make all NDA reviews, at least for approved NDAs, available in the public domain. The act is written into the FDA portion of the Code of Federal Regulations as follows: “The Food and Drug Administration will make the fullest possible disclosure of records to the public, consistent with the rights of individuals to privacy, the property rights of persons in trade secrets and confidential commercial or financial information” [12].
Obstacles and Limitations
There would surely be obstacles. The pharmaceutical industry would vigorously invoke Exemption 4 of the Freedom of Information Act, the exemption for trade secrets and confidential business information [13]. However, the FDA Freedom of Information Office already deals with confidential and proprietary information by redacting or editing it out of the review documents before making them available. Within the FDA's Freedom of Information Office, staffing would need to be greatly increased. Some oversight might be necessary to ensure that the taxpaying public has been granted the fullest possible access and that unwarranted redaction does not occur. Unless the Freedom of Information Act is modified, access would still likely be limited to approved NDAs. Data would remain unavailable for trials that did not lead to an approved NDA.
It should be clarified that this resource does not compete with proposals by the AMA and other groups for clinical trial registries—rather, it complements them. The AMA has proposed the creation of a registry that is comprehensive in scope. The FDA's registry and results database are restricted to those trials aimed at supporting US marketing approval or a change in labeling in the US. While data from many studies conducted abroad are submitted to the FDA for this purpose, this is not the case for drugs for which the sponsor has elected not to seek approval for marketing in the US. Nor does the FDA review data from most trials funded by other US government agencies, such as the National Institutes of Health, or by foundations. And drug companies fund investigator-initiated trials that are often not registered with the FDA.
To make the FDA review data more accessible and user-friendly, simple formatting changes would be needed. For those (few) reviews that are currently posted on Drugs@FDA, one can determine the indication being evaluated only after opening the document and paging through it. (Descriptive titles would be helpful, and these could be linked to ClinicalTrials.gov. Further, the trials reviewed could be identified with a unique international identifier, as promoted by the World Health Organization [14].) Despite the fact that the reviews are created in Microsoft Word and converted to PDF, the versions that appear on the Web site are no longer in a searchable text format. While the reviews tend to be well organized, the posted versions are difficult to navigate because there is no hyperlinked table of contents. In addition to having these formatting issues addressed, clinicians and patients might benefit from brief summaries, the writing of which might require the addition of new FDA staff.
Conclusion
Despite the limitations of the FDA's database, making it public is a strategy that could be implemented both rapidly and easily by building upon existing infrastructure. While we await the creation of a clinical trials registry and results database that is truly comprehensive, we already have at our disposal one that could serve as a trove of in-depth and unbiased information on many, if not most, drugs currently marketed in the US.
Citation: Turner EH (2004) A taxpayer-funded clinical trials registry and results database. PLoS Med 1(3):e60.
Abbreviations
AMAAmerican Medical Association
FDAFood and Drug Administration
INDinvestigational new drug
NDAnew drug application
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American Medical Association AMA offers guidelines for clinical trial registry 2004 September 9 Available: http://www.ama-assn.org/ama/pub/article/1615-8833.html . Accessed 26 September 2004
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Center for Drug Evaluation and Research Investigational new drug (IND) application process Food and Drug Administration 2004 Available: http://www.fda.gov/cder/regulatory/applications/ind_page_1.htm . Accessed 26 September 2004
Center for Drug Evaluation and Research NDA review process Food and Drug Administration 2004 Available: http://www.fda.gov/cder/handbook/nda.htm . Accessed 26 September 2004
Food and Drug Administration Executive summary of the Food and Drug Administration's consumer roundtable on consumer protection priorities 2001 Available: http://www.fda.gov/ohrms/dockets/dockets/00n_1665/mm00001.htm . Accessed 22 June 2004
Food and Drug Administration Chapter I—Food and Drug Administration, Department of Health and Human Services. Part 20—Public information Code of federal regulations, 21CFR20.20. Title 21—Food and drugs 1999 Available at: http://www.access.gpo.gov/nara/cfr/waisidx_04/21cfr20_04.html . Accessed 26 September 2004
House of Representatives Committee on Government Reform A citizen's guide on using the Freedom of Information Act and the Privacy Act of 1974 to request government records First report 2003 Washington (D. C.) U. S. Government Printing Office Available: http://www.fas.org/sgp/foia/citizen.html . Accessed 26 September 2004
World Health Organization WHO leads drive for international coordination of clinical research 2004 April 2 Available: http://www.who.int/mediacentre/news/releases/2004/pr23/en/ . Accessed 16 October 2004
Kapczinski F Lima MS Souza JS Schmitt R Antidepressants for generalized anxiety disorder Cochrane Database Syst Rev 2003 2003 CD003592
Pollack MH Zaninelli R Goddard A McCafferty JP Bellew KM Paroxetine in the treatment of generalized anxiety disorder: Results of a placebo-controlled, flexible-dosage trial J Clin Psychiatry 2001 62 350 357 [Erratum: J Clin Psychiatry 62: 658.] 11411817
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| 15562322 | PMC531613 | CC BY | 2021-01-05 10:38:00 | no | PLoS Med. 2004 Dec 30; 1(3):e60 | utf-8 | PLoS Med | 2,004 | 10.1371/journal.pmed.0010060 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1556231510.1371/journal.pbio.0020412Research ArticleBioinformatics/Computational BiologyBiophysicsCell BiologyEubacteriaIn Silico Reconstitution of Listeria Propulsion Exhibits Nano-Saltation In Silico Listeria ReconstitutionAlberts Jonathan B [email protected]
1
Odell Garrett M
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1Center for Cell Dynamics, University of WashingtonFriday Harbor, WashingtonUnited States of America12 2004 30 11 2004 30 11 2004 2 12 e41231 5 2004 28 9 2004 Copyright: © 2004 Alberts and Odell.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Computer Simulation of the Movement of Listeria Bacteria
To understand how the actin-polymerization-mediated movements in cells emerge from myriad individual protein–protein interactions, we developed a computational model of Listeria monocytogenes propulsion that explicitly simulates a large number of monomer-scale biochemical and mechanical interactions. The literature on actin networks and L. monocytogenes motility provides the foundation for a realistic mathematical/computer simulation, because most of the key rate constants governing actin network dynamics have been measured. We use a cluster of 80 Linux processors and our own suite of simulation and analysis software to characterize salient features of bacterial motion. Our “in silico reconstitution” produces qualitatively realistic bacterial motion with regard to speed and persistence of motion and actin tail morphology. The model also produces smaller scale emergent behavior; we demonstrate how the observed nano-saltatory motion of L. monocytogenes, in which runs punctuate pauses, can emerge from a cooperative binding and breaking of attachments between actin filaments and the bacterium. We describe our modeling methodology in detail, as it is likely to be useful for understanding any subcellular system in which the dynamics of many simple interactions lead to complex emergent behavior, e.g., lamellipodia and filopodia extension, cellular organization, and cytokinesis.
A detailed computer simulation explicitly simulates monomer- scale biochemical and mechanical interactions to characterize bacterial motion
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Introduction
Cellular processes generally involve interactions among 101 to 105 gene products. These interactions can be both biochemical, as in the activation of one protein by another, and mechanical, as in the application of force between bodies. Even when each individual interaction is simple and understood in detail, neither intuition nor qualitative description can forecast the emergent behavior of the whole system. We describe a methodology to characterize such emergent behavior using a detailed computer simulation of both biochemical kinetics and mechanical dynamics. In this paper, we apply the technique to the motility of the bacteria Listeria monocytogenes, a well-studied system in which actin network growth produces a force that moves the bacterium inside of cells. We discuss the model design, compare behaviors of the computational and biological systems, use the model to explain observed features of the bacterial motion, and identify observable experimental correlates of our hypotheses through which our interpretations may be confirmed or rejected.
L. monocytogenes is a pathogenic rod-shaped bacterium that invades cells, reproduces, and spreads to neighboring cells, never exposing itself to the extracellular environment, thus avoiding a humoral immune response (Tilney and Portnoy 1989). By expressing the protein ActA (Domann et al. 1992; Kocks et al. 1992, 1995), L. monocytogenes bypasses the host cell's normal controls on actin network growth to produce a dense “comet tail” of actin. This actin tail generates a ram force, by rectifying thermal motion, to both propel the bacterium within a cell and push the bacterium into neighboring cells through distension of the cell plasma membranes.
Among experimental advances thus far made to understand this motile system are identification of the purified proteins required to reconstitute motion in vitro (Loisel et al. 1999), an ability to mimic this motion using polystyrene beads coated with the bacterial ActA protein (Cameron et al. 1999, 2001, 2004), and experiments that have revealed a discrete step-like motion on the nanometer scale (Kuo and McGrath 2000; McGrath et al. 2003). A series of complementary theoretical models have been proposed to account for some observed features of bacterial and bead motion (Peskin et al. 1993; Mogilner and Oster 1996, 2003; Gerbal et al. 2000a, 2000b; van Oudenaarden and Theriot 2000). These studies, taken together, show that L. monocytogenes' actin structures, first described by Tilney and Portnoy (1989), are created from the same protein components and perform a function similar to the actin machinery in the lamellipodia of motile cells. The dendritic nucleation model for actin network growth (Mullins et al. 1998; Pollard et al. 2000, 2001; Pollard 2003) offers a qualitative description of this biochemical network. In the absence of the bacterium, specific signals activate WASP/Scar proteins, and these in turn activate the Arp2/3 protein complex to provide new filamentous actin (F-actin) nucleation sites at or near the barbed (plus) end of existing filaments (Higgs and Pollard 2001). These new filaments form at a characteristic 70° angle to the parent filament, creating dense, highly branched networks (Mullins et al. 1998). Filament barbed ends are rapidly capped with high affinity by capping protein, making the creation/maintenance of free barbed ends critical for continued network growth. Hydrolysis of the ATP that was bound to each actin monomer favors filament disassembly, returning actin monomers to the pool of polymerization-ready G-actin. Cofilin aids in this disassembly by fragmenting F-actin, binding with much higher affinity to ADP actin than to ATP or ADP-Pi actin. The motion of L. monocytogenes exploits all of these actin network features, except that the bacteria's ActA replaces the host cell's WASP/Scar proteins and all the associated upstream signaling mechanisms that normally activate WASP/Scar to control actin polymerization (Welch et al. 1998; Zalevsky et al. 2001).
Our model differs in several ways from previous attempts to generate mathematical or physical models for L. monocytogenes motility (though see Carlsson 2001, 2003). We simulate explicitly a large number of detailed interactions of both a biochemical and mechanical nature, representing all protein–protein binding interactions with on-rate and off-rate kinetic equations. The simulation of actin filament polymerization, for example, depends on the local concentration of actin monomers and the association and disassociation rate constants (which have been experimentally determined), modulated by the steric accessibility of free barbed ends. Together these factors determine the binding/dissociation probabilities for each filament at each simulation time-step. Bulk properities of our actin “gel” arise from the contributions of the many individual parts of the actin network. Our model can thus accomodate arbitrary geometries, explicit stochastic input, and specific small-scale events. Mechanical interactions, which resolve collisions and accommodate the stretching of protein–protein linkages, follow Newton's laws.
We can represent any particular interaction in as coarse or detailed a fashion as desired, subject to the availability of computer resources, and each of these can be based either on experimental information or on simple postulates. We can determine the emergent behavior of the system, which is the dynamical outcome of all the particular interactions, only by running the computer program for many hours or days. In such a model it is neither possible nor desirable to include all details. If our model fails to characterize experimentally observed behavior, then something is missing. If our model does capture an emergent behavior, however, then we can study how quantitative changes in the underlying details (e.g., protein concentrations or specific rate constants) affect this larger scale behavior. The exploration of putative mechanisms is also straightforward, as it is easy to add, remove, or modify each individual interaction.
With our approach, we formalize experimentally based models of specific protein–protein interactions and biochemical kinetics in a direct and flexible way, but there are drawbacks. The theoretical approaches used to analyze the Brownian ratchet model and its refinements (Peskin et al. 1993; Mogilner and Oster 1996, 2003) facilitate the derivation of equations that describe important system characteristics, such as force–velocity curves. No such equations are available in our stochastic, individual molecule-based model; instead, we must distill parametric relationships from ensembles of many repeated simulations. Completing these parametric studies in reasonable human time requires considerable computer resources.
The biochemical and mechanical interactions near the bacterial surface are stochastic processes involving hundreds of filaments. We model dynamic processes on a per filament basis, rather than through bulk network properties and average filament growth. The growth of any particular filament depends upon that filament's precise location, orientation, and biochemical state, all of which change through time. There is no better way to simulate such a system than with a model that tracks each of these variables for each individual filament. In the future, this type of detail will be essential to capture (and thus explain) many observed biological phenomena.
The trajectories generated by this model of L. monocytogenes motility display repeated runs and pauses that closely resemble the actual nanoscale measurements of bacterial motion (Kuo and McGrath 2000; McGrath et al. 2003). Further analysis of the simulation state at the beginning and ends of simulated pauses suggest a new interpretation of the experimental results. We show that there is no characteristic step-size or pause duration in these simulated trajectories and that pauses can be caused by both correlated Brownian motion and by synchronously-strained sets of ActA–actin filament mechanical links.
The Model
We model the molecular mechanics of the growth/disassembly of an actin network as it interacts with a moving rod-shaped bacterium to whose surface many ActA proteins are bound at specific locations. We distinguish molecular mechanics (Howard 2001) from molecular dynamics: we are not concerned with van der Waals forces and hydrogen bonds or with conformational changes during protein–protein interactions. Our model is different from a purely continuum model, in which state variables (those dependent variables which, together, fully describe the state of a system) would characterize only the bulk properties of an actin dense tail, using average compliance and polymerization values. We instead separate the cellular world into two basic classes of entities, those that are relatively large and present in small numbers (e.g., actin filaments, a bacterium) and those that are very numerous and small (e.g., actin monomers and other diffusible proteins). We simulate the former entities, which we call “explicit players,” as individuals; our state variables keep track of the position, orientation, and biochemical state of each individual and its change with time according to appropriate physical laws (e.g., Newtonian force balance laws). Those entities that are more numerous we will call “implicit players”: we represent them with continuum field state variables, i.e., molar concentrations that vary with time and place. We use standard partial differential reaction–diffusion conservation equations to express how these continuum fields change with time as the implicit players interact with each other and with the explicit (individual) players (Figure 1A). Dataset S1 is a simplified psuedo-code of our simulation software.
Figure 1 Model Schematic
(A) shows a simple cartoon of the bacterium and some actin filaments (explicit players) against a backdrop of a moving diffusion–reaction grid attached to the bacterium. We use this spherical coordinate grid, whose origin moves with the bacterium, to keep track of the diffusion and biochemical interactions of the scalar protein concentrations fields that characterize implicit players. Protein size greatly impacts diffusion of proteins in a cellular environment (Luby-Phelps 1994, 2000), so we modify the nominal diffusion coefficients of implicit players accordingly. The dotted line is the path trajectory in 3D space of the bacterium.
(B) illustrates the origin of forces that act on a bacterium in our simulation. Actin filament α is bound to an ActA protein on the bacterial surface, generating a link force that acts to hold the two objects together. Filament β is shown colliding with the bacterium, generating a collision force that acts to push the two objects apart. The barbed end of filament γ is nominally too close to bacterial surface to allow addition of an actin monomer, but Brownian motion might bend the filament into the dotted configuration, thus allowing polymerization and creating a collision. We model our filaments as rigid rods, but simulate this filament flexibility with a polymerization probability function that approaches zero with the filament–bacterium gap; i.e., it is possible for a filament to add a monomer even if the nominal filament–bacterium gap is less than 2.7 nm (the filament length increase per actin monomer). The bacterium, actin filaments, and structures of actin filaments are all subject to Brownian simulation forces and torques, represented by δ.
Various different forces impinge on the simulated bacterium. Forces move two objects apart if they happen to collide at the end of a time-step. Likewise, elastic bonds linking two objects (e.g., an actin filament–ActA bond) exert equal and opposite forces that hold those objects together; these links break under sufficient strain. Forces of random orientation act on every explicit player to simulate Brownian motion (i.e., the sum of all the many collisions with small molecules that, in biological reality, contribute to the Brownian motion is represented in our model by a single vector force and a single vector torque). This system never approaches an equilibrium; Brownian motion and biochemical events ceaselessly create collisions and perturb protein–protein links. Thus, we must compute new forces, exchanged between new neighbors, in each time-step. Figure 1B illustrates the set of mechanisms that combine to generate the net force on the bacterium in our simulation.
At the heart of this simulation is the dendritic nucleation model of actin dynamics (Mullins et al. 1998; Pollard 2003). Asynchronously, each individual actin filament can grow or shrink at either end by actin monomer polymerization/depolymerization; hydrolyze the ATP bound to one or more of its actin monomers to ADP-Pi; dissociate the Pi from one or more such monomers; be severed by ADF/cofilin; bind Arp2/3 to an ATP-actin subunit in the filament; be capped or uncapped at either end; and nucleate new filaments through Arp2/3 initiated side-branches. Repeated nucleation of new branches from existing filaments leads to a dense meshwork of actin in the comet-tail. Besides Arp2/3 mediated branching, all other cross-linking and adhesions involving actin filaments are implicit in the age and length dependent anchoring of f-actin in our simulation space. All actin filaments accumulate adhesions that gradually increase drag coefficients and eventually lock each filament into a fixed position. Figure 2, a video frame from a typical simulation, introduces some of the explicit and implicit players.
Figure 2 A Simulation Video Frame Showing Actin Filament Branching near the Bacterial Surface
Arp2/3 seeds branches off of existing filaments at a characteristic 70° angle. Different ATP/ADP forms of actin have differing affinities for proteins such as ADF/Cofilin. Filament barbed tips can be capped unless they are protected through interaction with an ActA molecule; as indicated, surface-bound ActA molecules elastically link the bacterium to an actin filament near the barbed end.
The simulation time-step has a subtle effect on the simulation of Brownian motion for constrained objects (that is, objects linked to other objects). Applying the same forces and torques that are appropriate for free objects exaggerates the simulated Brownian motion of constrained objects since the motion restriction that results from those constraints can only be resolved over several time-steps, and those time-steps are large relative to the intrinsic time-scale of the constraints. Experimental measurements (Kuo and McGrath 2000) show very little Brownian motion (relative to similarly sized nearby vesicles) of L. monocytogenes associating with their actin tails; to match the biological reality, we need to modify our simulation of Brownian motion, since we cannot yet use much smaller time-steps. We compensate for this technical problem by carrying out simulations both for the two extreme cases (with Brownian motion appropriate for a free bacterium and with no Brownian motion of the bacterium at all) and for an intermediate degree of Brownian motion. Advances in computer processing speeds will, most likely, make such attenuation unnecessary in the near future. We will henceforth use the term “Brownian simulation force” to refer to the forces and torques that we apply to the bacterium to simulate its Brownian motion.
For our model, we use typical physiological concentrations for each of the proteins involved; these are listed in Table 1. Table 2 summarizes the reaction rate constants we used. Some crucial parameters and protein functions are as yet incompletely known. These include the exact pathways and rate constants associated with the stimulation of local actin filament polymerization by the bacterial surface-bound ActA protein. This protein has binding sites for a host of proteins, including G-actin, F-actin, Arp2/3, and Ena/VASP. In addition, while the affinity between free ActA and Arp2/3 has been measured (Zalevsky et al. 2001), that value (KD = 0.6 μM) does not sufficiently characterize that interaction since the interaction rates may be limited by the flux of Arp2/3 or G-actin onto the surface. We have calculated the flux of Arp2/3 and G-actin onto the bacterium's surface to determine the expected equilibrium number of ActA–Arp2/3 and ActA–actin complexes there, as explained in Dataset S2. We tune these approximate rate constants to create actin networks with biologically representative side-branch separation and filament numbers. Because the rate constants that we have obtained in this way will depend on the concentrations of ActA, Arp2/3, and actin monomers in the model, the rates given for ActA–Arp2/3 and ActA–actin interactions in Table 2 apply to the concentrations given in Table 1.
Table 1 Values and References for the Concentrations of Proteins We Used
These values are not for any specific cell type, but are typical biological concentrations and similar to those used for in vitro reconstitutions of bacterial motility
Table 2 The Biochemical Rate Constants Incorporated in Our Model
A hydrolysis rate is given for a vectorial ATP hydrolysis model; experimental evidence currently supports the random hydrolysis model but we have, for simplicity, implemented a vectorial scheme for this analysis. That is, we assume that there is a distinct border within each filament between the ATP actin, ADP-Pi actin, and ADP actin regions; only monomers adjacent to a these borders can transition from ATP actin to ADP-Pi actin or from ADP-Pi actin to ADP actin. We can readily switch to a random hydrolysis model in future studies. The values in angle brackets, for the interactions between ActA, Arp2/3, and actin monomers, are calculated considering the diffusive flux onto the bacterium's surface (see Dataset S2). These values are thus dependent upon ActA density, the concentrations of Arp2/3 and actin, and a heuristic adjustment of these rates to balance new filament nucleation and side-branching in order to achieve realistic tail morphologies. The on-rates in brackets listed here apply to the concentrations in Table 1
The ActA protein is distributed asymmetrically on the bacterial surface, with more ActA near the rearward tail-forming end. The unipolar shape of our distribution is based on measurements of the fluorescence signal from RFP-labeled ActA along the bacterial length for newly divided bacteria (S. Rafelski and J. A. Theriot, unpublished data). New filaments are produced by two pathways. By activating Arp2/3, ActA is thought to catalyze the creation of new actin filaments and side-branches. We simulate the co-binding of ActA, Arp2/3, and an existing actin filament, allowing binding in any order. This complex leads to a new side-branch on the existing filament. Binding of ActA to the actin filament can occur only at specific ATP or ADP-Pi actin sites and is binding site limited, meaning that each bound ActA occludes a linear region of five monomers on the filament from further binding. Creation of a new filament de novo in our model involves the co-binding by ActA of G-actin and Arp2/3, in any order (Boujemaa-Paterski 2001).
In conjunction with other proteins (e.g., Ena/VASP), ActA may also regulate actin dynamics in other important ways (Goldberg 2001). In this version of our model, we do not explicitly simulate Ena/VASP molecules, which can regulate actin networks by binding profilin, competing with capping protein, and regulating Arp2/3 spacing (Krause et al. 2003). Instead we assume that ActA itself can uncap any actin filament barbed end to which it binds closely (within one ActA length) and ignore the other possible Ena/VASP functions. We find that this uncapping function is necessary to obtain persistent motion with a low nucleation rate of new filaments. For the values in Tables 1 and 2, our simulated bacterium do move without this uncapping, but more slowly. Loisel et al. (1999) have reported a similar dependence in their experiments, finding that Ena/VASP is not required for bacterial motion, but that it improves the efficiency of the motion observed.
Results
The gross behavior of our simulated bacterium is life-like; model bacteria move in a qualitatively similar way to wild-type L. monocytogenes. Average speeds of motion varied from ten to hundreds of nanometers per second, as do real observed bacterial speeds in different experiments (40 nm/s in purified proteins in Loisel et al. 1999; 140 nm/s in cytoplasmic extracts in Cameron et al. 2004; 1.4 μm/s in vivo in Dabiri et al. 1990). Videos at any scale may be rendered from our simulations (Videos S1 and S2; other full-length simulations at www.celldynamics.org). Figure 3 merges several frames from one of those movies, showing the large-scale formation, hydrolysis, and depolymerization of the actin comet-tail.
Figure 3 A Simulation Time Series
Several video frames from one simulation show, on a gross scale, the de novo ActA filament nucleation and Arp2/3 branching from existing filaments that form a comet-tail, the hydrolysis of actin filaments in the comet-tail, and the subsequent pointed end depolymerization of ADP-actin (filament severing by ADF/Cofilin is not active in this simulation).
The microrheology experiments of Kuo and McGrath (2000) and McGrath et al. (2003) present an opportunity to illustrate the utility of our stochastic model founded on small-scale details. They reported that L. monocytogenes motility is multiphasic; motion of the bacterium that appears smooth on the micrometer length-scale actually consists of pauses that last many milliseconds, discrete nanometer-scale steps, as well as uninterrupted runs. No current model of this bacterial motility has fully explained these discrete steps, though numerical simulations with the tethered ratchet (Mogilner and Oster 2003) can exhibit saltatory motion. Possible explanations involving strained links between ActA and actin filaments and nucleotide dependent filament templates are discussed in McGrath et al. (2003).
Figure 4 shows the distribution of step sizes and pause durations that our model produces (using the values of Tables 1 and 2 and one set of mechanistic hypotheses) with three different assumptions for the Brownian motion of the bacterium (see under The Model). In all cases, we find that there is no characteristic step-size, but rather a continuum of steps with the smaller steps being more probable than larger ones. The qualitative shapes of these histograms are insensitive to changes in all parameters we might reasonably vary, barring values that disrupt persistent bacterial motion. The parameters we have varied include link characteristics (e.g., link length, link force, and maximum link strain), Arp2/3 branching rate, and temperature. In fact, even doubling the size of each actin monomer (easily done in a simulation) does not change these histograms significantly (data not shown).
Figure 4 Step-Sizes, Pause Durations, and Speeds of Motion with Different Brownian Simulation Force Attenuation
The legend shows the multiplier by which the Brownian simulation force on the bacterium is attenuated, such that a multiplier of 1 corresponds to a Brownian simulation force appropriate to an unconstrained bacterium and a multiplier of 0 signifies no simulated Brownian motion of the bacterium (see discussion of the relationship between applied Brownian simulation force and numerical time-step in the model description). We line-fit and filter trajectories by slope (speed) to identify pauses in the bacterial motion. Any line with slope less than the pause threshold might indicate a pause. The distance between adjacent pause locations is the step-size, assembled into a histogram in (A). No characteristic step-size is evident; smaller steps are simply more probable than larger ones. We exclude steps shorter than 0.2 nm. (B) shows a pause duration histogram; here we exclude pauses <8 ms in duration. (C) shows run speed histograms for runs >50 ms in duration. For a Brownian multiplier of 1, we used 30 simulations with 56,239 pause events for (A) and (B) and 6,098 runs for (C) (1,929 s total simulation time, 644 s total paused time, 116 nm/s average speed, pause threshold 40 nm/s). For a multiplier of 0.5, we used 13 simulations with 49,207 pause events and 2,287 runs (1,534 s total simulation time, 700 s total paused time, 93 nm/s average speed, pause threshold 30 nm/s). For a multiplier of 0, we used 18 simulations with 9,748 pause events and 1,179 runs (940 s total simulation time, 612 s total paused time, 56 nm/s average speed, pause threshold 20 nm/s).
These results suggest that our simulated bacterium does not move with steps of any prefered size, and specifically not with a step-size related to the actin monomer dimensions. The pause in forward progress might equally be considered the defining event in the bacterium's motion; a “step” in this case is just a run made along the path between adjacent pauses. But what physical process stalls the actin polymerization ram to initiate pauses, and what physical process breaks the bacterium out of each paused state into a run? To answer these questions, we need to examine how key descriptive system features vary before, during, and after pauses in our simulations. We do this by looking both at individual pauses and at the average of these system outcomes for many thousands of pauses (see Materials and Methods).
In Figure 5, we follow actin polymerization, link formation and breakage, link number, and path-directed forces for several sequential pause events during a single simulation. Owing to the stochasticity introduced by the Brownian simulation forces in this simulation, it is difficult to find trends in such single simulation profiles. We can learn more by turning off the Brownian simulation forces on the bacterium as has been done in figure 6. Now frequent long pauses are observed that clearly reveal the force relationships during pause initiation and termination. Both filament link force and collision force increase in magnitude synchronously during a pause, indicating resistance to forward progress by a population of ActA–actin filament links. The bacterium moves rapidly forward only when the total filament link force suddenly plummets. This sudden decrease in link force can only be attributed to a cascade of link breakage. This result indicates that the highly strained filament links that had balanced the filament collision force during a pause are rapidly exchanged for unstrained links when a pause ends.
Figure 5 System Outcome Profiles for Several Adjacent Pauses
Pauses are shown by red horizontal lines in a single simulation run in which the Brownian force multiplier was 1 (unconstrained bacterium). Listed for each pause are the pause duration (δt) and distance to the following pause (δs) as reported by our line-fitted analysis. The vertical line segments in the lower half of the figure show discrete events. From the bottom, these are the number of polymerization events on filaments very close to the bacterium, the number of new links formed between the bacterium and filaments, and the number of these bacterium–filament links broken. Above these are plotted the total number of bacterium–filament links. At the top, the net filament link force and the net filament collision force are given in picoNewtons (see Figure 1B), along with the total force. Some general trends aligned with pauses are apparent, such as decreased actin and link dynamics during a pause, but any characteristic biochemical or force response is obscured by the Brownian agitation of the bacterium.
Figure 6 System Outcome Profiles for Several Adjacent Pauses—No Brownian Motion of the Bacterium
Long pauses during this run without simulated Brownian motion of the bacterium (Brownian force multiplier of 0) reveal the force balance/imbalance conditions that cause pause–run behavior. System outcome profiles are shown for several adjacent pauses (red horizontal lines) with their duration (δt) and distance to the following pause (δs) displayed as reported by our line-fitted analysis. For a description of each of the entities plotted here, see the legend to Figure 5. Note that the bacterium pauses in its forward progress when the filament collision and link forces cancel each other; then, the collision forces tend to rise during a pause as filaments in the tail catch-up with the bacterium and generate new filament collisions. Runs are coincident with the breakage of many highly strained filament links which are quickly replaced by unstrained links; note that the filament link number does not change greatly during such an avalanche of link breakages and that it is steadily climbing over this time range in this particular simulation.
The average of these system outcomes further clarifies pause causality and reveals differences between the two cases illustrated in Figures 5 and 6. From Figure 7 we see that pauses can occur with or without Brownian motion of the bacterium. But when we simulate the Brownian motion of the bacterium, we observe that pauses are correlated with an accidental sequence of similarly directed Brownian simulation forces (forward to initiate a pause, backward to sustain a pause, and again forward to break out of a pause). Note that any individual pause event in our average ensemble may experience only a subset of the correlated Brownian simulation force profile presented in Figure 7. On that figure we have partitioned this correlated Brownian simulation force into three temporal regions, labelled A, B, and C. An individual pause event might correlate with the Brownian simulation force sequence of A, or only A and C, or only B and C, or any other combination. Additionally, some pause events might be entirely uncorrelated with any Brownian simulation force trend. In other words, this averaging method reveals system trends that occur frequently, but need not be present in every contributing event.
Figure 7 Anatomy of a Pause
An ensemble of average system outcome analyses, with (solid lines) and without (dotted lines) simulated Brownian motion of the bacterium (see discussion on the relationship between applied Brownian simulation force and numerical time-step in the model description). These averages were compiled from pauses with duration greater than 10 ms, using 10,365 pauses with, and 4,358 pauses without, simulated Brownian motion (there are fewer, longer pauses without Brownian motion of the bacterium). Only sufficiently time-separated pauses contributed to these averages, so that the 10 ms preceding the pause start and the 10 ms following the pause stop are guaranteed not to include the effects of any adjacent pause. The Brownian simulation force trends can be read from the total force curve, which is only slightly offset by the link and collision forces in the case where Brownian movement of the bacterium is simulated (solid lines). Here, initiation of a pause follows a large forward-directed Brownian simulation force on the bacterium (segment A), which increases link turnover and produces a large population of synchronously strained links. Backward path-directed Brownian simulation forces (segment B) maintains the pause until, aided again by forward-directed Brownian simulation forces (segment C), the bacterium transitions back into a run. The Brownian simulation force trends can be read from the total force curve, which is only slightly offset by the link and collision forces. Without simulated Brownian motion of the bacterium (dotted lines), a pause is initiated and maintained when a population of ActA–actin filament links can resist the essentially constant total filament collision force. A pause terminates in this case when these links break en masse. Any individual pause in the averaged set of pauses might not demonstrate all of these response features.
We contrast the Brownian/no Brownian motion cases to better understand pause initiation, maintenance, and termination. Our most realistic simulation will incorporate effects from each extreme case. With simulated Brownian motion of the bacterium, we suggest the following causal temporal sequence for pause initiation, maintenance, and termination (with the caveat that most individual pause events will experience only a subset of this sequence):
A particularly large Brownian simulation force (or accidentally correlated sequence of forces) in the forward path direction causes an unusually rapid but small forward displacement of the bacterium (region A).
The steady-state rate of filament link turnover increases slightly as highly strained links break and are replaced by an ensemble of new links that all form nearly simultaneously in an unstrained state, thus creating a larger than steady-state population of coordinately unstrained links.
A particularly large Brownian simulation force, or correlated sequence of forces, opposite to the path direction forces the bacterium backward against the population of linked barbed end actin filaments; filament collision force increases, filament link force falls, and actin polymerization near the surface decreases. A pause ensues.
During the pause new filaments form and existing but distant barbed ends “catch up” with the bacterium, thus increasing the filament collision force forward which the links restrain. The pause terminates when a particularly large Brownian simulation force (or an accidentally correlated sequence of forces) in the forward path direction is sufficient to break a few of the strain-synchronized filament links. As these links break, the force stretching each remaining link increases, setting in motion an avalanche of cooperative link breakage and initiating a run.
We are justified in interpreting these correlations of Brownian simulation forces as causal because those forces are generated in our simulations so as to be random in direction and magnitude (representing a Gaussian distribution). Nothing in our model can cause such Brownian simulation force “accidents.” Their correlation with pause initiation or termination must therefore be causal.
Absent Brownian simulation forces on the bacterium, the system response throughout the course of a pause is very different. In this case, a pause occurs only when a population of synchronized filament links is able to balance the filament collision forces, which on average increase only slightly during a pause, until a cascade of breaking links allows the bacterium to run again. Judging from the shape of the step-size histograms in Figure 4, the generation of a set of strain-synchronized links that initiate a pause is likely a random event. That figure reveals a Poisson process-like distribution of step-sizes with weak or no Brownian simulation forces; moreover, the step termination (and therefore pause initiation) appears to occur with a constant probability through time. This should be contrasted with the case of simulated Brownian motion appropriate for an unconstrained bacterium, in which step-termination (pause initiation) is correlated with forward path-directed Brownian simulation forces.
The small amplitude of experimentally measured fluctuations of bacteria in vivo (Kuo and McGrath 2000) suggest that the simulations absent Brownian motion of the bacterium come closest to representing the biological reality; the coincidence of similarly directed Brownian movements is probably less important than the balance between filament–bacterium collision and link forces.
Discussion
We have used our model to ask how L. monocytogenes motility is mediated by actin-mediated forces. Building a simulation from basic, well-understood structures and interactions, we have reconstituted bacterial motility in silico. Appropriate speeds and persistence of motion emerge, reproducing experimentally observed values. Additionally, our simulation yields as an emergent behavior the nanometer-scale saltatory motion reported by experimentalists. We can analyze details of the simulated bacterial trajectories to investigate characteristics of this saltation: what is the mechanism behind the stepping, and is there a favored step-size?
Our computational experiments lead us to conclude that the tethered-ratchet model is an inherent “pauser” with several important attributes. First, there is no characteristic step-size or pause length; shorter steps and pauses are more frequent than longer ones. Second, the intensity of Brownian agitation of the bacterium influences average pause duration and frequency, but this agitation is not necessary for persistent saltatory motion. Third, pauses start when a population of filament links happen to form nearly simultaneously with low strain to balance filament collision forces. Pauses end when those links catastrophically break.
To produce nanometer-scale pauses and runs, no special function need be attributed to the bacterial bound ActA protein, beyond an elastic linkage to actin filaments and some mechanism that prevents barbed end filament capping. Specifically, the ActA protein does not need a motor-protein-like stepping ability, nor need it act as a clamped-filament elongation motor (Dickinson and Purich 2002). Given the large number of filaments near the surface of the bacterium and the wide variation in angle of those filaments, it is not clear that we would expect any step-size, even if ActA were motor-like with a discrete working stroke.
The speed of motion during a run is variable, but it is mostly confined to a narrow range of speeds that depends on the parameter set (see Figure 4C). Pauses are a significant feature in our simulated trajectories; bacteria spend large fractions of their time paused (from 33% to 65% in the simulations presented here).
Lastly, we have explored the variation of key biochemical events and mechanical interactions during a typical nanometer-scale saltation, looking at both individual pause events and averages of many such events, in an effort to uncover the causal factors. We conclude that the tethered-Brownian ratchet model is an inherent pauser; forward motion is temporarily halted whenever a population of synchronously strained filament links balances filament collision forces. Different mechanisms cause pause initiation/termination with and without simulated Brownian motion of the bacterium. With simulated Brownian motion of the bacterium, we find that pauses events are largely driven by coordinated Brownian simulation forces: a series of forces in the forward direction helps establish a set of coordinately-strained links, forces in the backward direction can help maintain a pause, and lastly forces in the forward direction help break links to terminate a pause. Without simulated Brownian motion of the bacterium, we find that a coordinately strained set of filament links balances the filament collision forces and that a pause will ensue until those links break en masse. Formation of such a set of filament links is an accidental, but frequent, occurrence, explaining the shape of the step-size and pause duration histograms (see Figure 4).
That we find no characteristic step-size in our simulated nanoscale stepping constrasts with experimental results (Kuo and McGrath 2000; McGrath et al. 2003). Restricted by experimental noise, those researchers cannot see steps smaller than about 2.5 nm, if they indeed exist. Without those steps, about 30% of our simulated steps would be between 4 nm and 6 nm. We are presently sharing trajectories with the Kuo laboratory to directly compare model and experiment.
Mogilner and Oster (2003) explore stepping behavior of their elastic tethered Brownian ratchet model and, for low filament tether numbers and particular capping rates and tether stiffness, observe step sizes similar to those reported by Kuo and McGrath (Kuo and McGrath 2000; McGrath et al. 2003). A step in that model occurs when one filament tether breaks and the remaining tethers all stretch in response to the new force balance. By constrast, steps in our model occur following a catastrophic breakage of many coordinately strained tethered filaments and are not highly dependent upon tethered filament number or capping rate. Because of the method by which we resolve collisions and strained links (see Figure 8), we do not prescribe the elastic properties of the filament–ActA links, and so our stepping is also independent of those values. Dickinson and Purich (2002) proposed a completely different mechanism for nanoscale stepping, involving a putative elongation motor that demonstrates approximately 5.4-nm stepping in simulations. In their model, most filaments are in compression while a few lagging filaments prevent the bacterium from moving forward. It is the “release and relocking” of a single lagging actin filament by the elongation motor that allows a 5.4-nm step. While the mechanisms are severely different, the basic behavior of this elongation motor model is similar to ours. Of the population of filaments interacting with the bacterium in our simulations prior to a step, a subset generates collision forces and a coordinately strained subset attached to ActA proteins balances those collision forces, resisting forward motion. The concurrent breakage of this linked subset allows a step forward, analogous to the “release and relocking” in the elongation motor model, though many more filaments are involved in our “release,” and the step distance before these filaments rebind to ActA, and thus “relock”, is not prescribed.
Figure 8 A Simple Collision Rule
We calculate the magnitude, F, of equal and opposite forces applied to colliding objects such that they no longer collide after a time-step of δt. This force is calculated by considering the maximal distance, δ, of object intersection and the shape-based viscous drag, γ, for each object. In this example, we use Stokes' law for the viscous drag on spheres. We are assuming that the time-scale for a collision to resolve itself is much shorter than the discrete time-step used in the computation. We make similar calculations for more complex shapes and collisions.
The computational analysis of L. monocytogenes motility described here represents a new tool that should be useful for understanding many complex subcellular systems. The construction of this computational model requires experimental measurements of the biological details in L. monocytogenes propulsion and actin dynamics in general. Only in the last several years have crucial biological details come to light, e.g., the role of Arp2/3 in filament branching, or the binding sites and functionality of the ActA protein. Additionally, the implementation of the model in silico requires significant computational power, now affordable in the form of clusters of “off-the-shelf” machines (we estimate use of 30 cpu years on a 2.8 Ghz Pentium 4 in the development and exploration of this model, 3.5 cpu years of which directly contribute to this report). Powerful object-oriented languages are also recently mature (we use Java™), making it possible to write computer code to implement such models. We believe that the confluence of detailed biological information and computational power/software heralds a new approach for understanding subcellular systems in which many thousands of simple biochemical and mechanical interactions lead to complex emergent behavior.
The biological systems in which this approach will be useful are, by definition, rich in detail. This complexity favors collaborations between modelers and the experimentalists who discover and quantify the molecular details without which this study would be specious. Creating a simulation environment that makes intuitive sense to experimentalists, i.e., one in which there is clear correspondence between biological entities and their modeled counterparts, greatly facilitates communication between modelers and biologists, and it ensures appropriate refinement of the model as new biological facts are uncovered.
There are many future refinements and research directions for this model. We can incorporate a more sophisticated representation of the actin hydrolysis cycle (Bindschadler et al. 2004) and include specifics of the interactions between ActA and proteins such as Ena/VASP. Recent work with ActA-coated beads (Cameron et al. 2004) characterizes the relationships between several biophysical parameters and motion initiation, speed, and persistence; these experimental findings can also be explored in silico. Extended with additional cellular components (e.g., a dynamic cortex, microtubules, motor proteins), the model might also be used to explore any of a number of cellular phenomena, including whole cell motility and cytokinesis. Our model, encoded in an object-oriented manner, is structured in a way that is strongly delimited by nature—so while we must still embrace approximation, we can minimize abstraction.
Materials and Methods
A large set of differential equations determine how our state variables change with time. We solve these equations numerically, but not in a standard way because discontinuities in time occur frequently as objects collide suddenly and as objects suddenly spring into existence or disappear (due to new filament nucleation and depolymerization). To solve these thousands of differential equations, we divide time into discrete steps (typically tens of microseconds) balancing the necessities of capturing the system dynamics and accomplishing the simulation in reasonable human time (typically 3–5 d). At the beginning of each time-step, the biochemical events and forces experienced during the last time-step will have changed the state of the system. New collisions and link forces may have arisen, as well as new objects. Existing links may break if they experience excessive strain for several consecutive time-steps. Each explicit player thus experiences a net force vector; in the next step, we move each explicit player in this vector direction so as to reduce or eliminate the strain energy associated with its collisions and links. To accomplish this practically, we calculate the forces required to resolve each individual collision (or strained linkage) in a single time-step. Figure 8 demonstrates this calculation for a collision between two spherical bodies; a similar approach is taken for all pair-wise collisions or links. In brief, we sum all forces, attenuating all their magnitudes by the same factor without changing their directions, so that acting during the time-step they produce just enough displacement to separate objects that collided in the prior time-step. This process avoids prescription of elastic constants and is equivalent to proceeding through a series of quasi-static equilibria, a formally valid approach if the biochemical dynamics are slow relative to the resolution of force imbalance.
Each individual computer run simulates bacterial motion for a period of up to many minutes. We run hundreds of such simulations and then statistically analyze the ensemble of runs. Fitting straight line segments to each trajectory and filtering those segments by slope (speed of motion) reveals that each simulated bacterial trajectory is composed of a sequence of pauses (of varying duration) separated by near-constant speed runs between pause locations. After we identify all the pauses, we measure the distances between adjacent pauses; these are the putative step-sizes. Histograms of pause duration and step-size, distilled from multiple simulations, then allow comparison with experimental observations and reveal whether there exists a preferred step-size or pause duration. Figure 9 shows a segment of trajectory data and the progressive stages of our line-fitting analysis.
Figure 9 Slope-Based Analysis of Bacterial Trajectories
A brief sample of a bacterial trajectory from one simulation run (black), a smoothed approximation to that trajectory (blue), line segments of near zero velocity fit to the smoothed trajectory (green), and the summary of a pause event assembled from those lines (red). Our analysis software seeks nearly horizontal segments of the trajectory (i.e., pauses), of maximal possible duration, in which excursions away from pause location lie with a jitter tolerance that we specify. Trajectories are curves in 3D space with curvature and torsion. To simplify the analysis, we use a path position variable (on the vertical axis), projecting each displacement onto the path tangent vector, instantaneously defined by the bacterial orientation. Then, we identify pauses in the resulting time series to specify displacement along the smoothed trajectory. Labels on each pause report pause durations, δt, and the displacement to the next pause (step-size), δs. Random thermal agitation forces act to buffet the bacterium, and every individual part, in our simulations; this is what makes the trajectories jagged.
We average many thousands of pause events into portraits characterizing system behavior preceding, during, and following the typical pause. To do this, we align pauses, time and space shifting short sections of the path projected trajectories that span a single pause event so as to superimpose their starting or stopping points (Figure 10). These pauses are of different duration, so our average response will be most meaningful near the alignment point. To improve this analysis, we can also select and average only pauses of similar duration or create ensemble portraits from start-aligned and stop-aligned analyses.
Figure 10 Calculating an Average System Response
(A) shows a segment of simulated bacterial trajectory with five identified pauses shown in red; δt is the pause duration, and δs is the distance along the path to the next pause. To obtain the average response of any system outcome, we align the pauses at their stopping (or starting) points as shown in (B).
Any trend remaining after the averaging of many thousands of events will reveal significant system behavior near the alignment point. Any individual event, however, might not exhibit all the trends revealed in such an average, so that the interpretation of these average profiles should be tempered accordingly.
Not all of the capabilities of our model have been enabled in the simulations contributing to this study. Our calculations show that the local depletion of the implicit players, due to their incorporation into a larger assembly, is not significant for the concentrations, rate constants, and geometries of this system (data not shown). Thus, we do not simulate the diffusion of any of the implicit players (proteins), but rather assume that each exists at a constant concentration (see Table 1). With this assumption, we need not accurately represent the depolymerization of the bacterium's comet tail in modeling the movement of the bacterium. (This depolymerization could otherwise have had an important role in regenerating depleted stocks of some implicit players.) We therefore depolymerize F-actin in the most computationally efficient way: we assume an artificially high pointed end depolymerization and ignore cleavage by ADF/Cofilin.
In addition, F-actin interacts with cellular components in vivo that are not explicitly represented in either the dendritic nucleation model or our simulations (e.g., with other cytoskelet al.filaments). Some of these interactions have the effect of locking down actin tail in cellular space. We approximate their effect with a time- and actin length-dependent application of adhesions that eventually fix F-actin and the actin tail in our simulation space.
Supporting Information
Dataset S1 Psuedo Code for an Actin Filament
(29 KB DOC).
Click here for additional data file.
Dataset S2 Steady-State Number of ActA–Arp2/3 Complexes on the Bacterium
(52 KB DOC).
Click here for additional data file.
Video S1 One Actin Filament Interacting with the Bacterium
A close-up look at the interaction of a single polymerizing filament with the bacterium. This filament has an artificially durable link with an ActA protein on the bacterium's surface; these links are typically very transient. The tip-clearance (drawn with a cyan line), the polymerization probability, the capping probability, and the Arp2/3 binding probability (set to zero for this demonstration) are reported at each simulation time-step.
(1 MB MOV).
Click here for additional data file.
Video S2 An Animated Simulation: Motion Initiation and Persistence
An animation rendered from the output of one simulation of L. monocytogenes motility. Microscale hops, as opposed to the nanoscale steps we investigate in this paper, are apparent at this scale view. The bacterium induces an actin tail of variable density and demonstrates persistent motion.
(9.8 MB MOV).
Click here for additional data file.
We thank Julie Theriot, Bruce Alberts, and members of the Center for Cell Dynamics (www.celldynamics.org) for comments on the focus of this model and content of this manuscript, Susanne Rafelski for measurements of ActA distributions, and Paul Mutton for Java EPS figure generation freeware. Initial conception and development of this work was supported by a postdoctoral fellowship in computational biology from the Alfred P. Sloan Foundation (JBA); for that unique opportunity to “cross over” from engineering to biological modeling JBA is especially grateful. This work was further funded by the National Institutes of Health grant NIGMS 5P50 GM 666050-02 (JBA and GMO).
Conflicts of interest. The authors have declared that no conflicts of interest exist.
Author contributions. JBA and GMO conceived and designed the experiments. JBA performed the experiments. JBA and GMO analyzed the data. JBA and GMO wrote the paper.
Academic Editor: Thomas Pollard, Yale University
Citation: Alberts JB, Odell GM (2004) In silico reconstitution of Listeria propulsion exhibits nano-saltation. PLoS Biol 2(12): e412.
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| 15562315 | PMC532387 | CC BY | 2021-01-05 08:28:09 | no | PLoS Biol. 2004 Dec 30; 2(12):e412 | utf-8 | PLoS Biol | 2,004 | 10.1371/journal.pbio.0020412 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1556231610.1371/journal.pbio.0020418Research ArticleCell BiologyImmunologyInfectious DiseasesMicrobiologyEubacteriaDrosophilaSecreted Bacterial Effectors and Host-Produced Eiger/TNF Drive Death in a Salmonella-Infected Fruit Fly Salmonella Infections in DrosophilaBrandt Stephanie M
1
Dionne Marc S
1
Khush Ranjiv S
1
Pham Linh N
1
Vigdal Thomas J
1
Schneider David S [email protected]
1
1Department of Microbiology and Immunology, Stanford UniversityStanford, CaliforniaUnited States of America12 2004 30 11 2004 30 11 2004 2 12 e41822 8 2003 5 10 2004 Copyright: © 2004 Brandt et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
When Immune Defenses Turn Traitor
Death by infection is often as much due to the host's reaction as it is to the direct result of microbial action. Here we identify genes in both the host and microbe that are involved in the pathogenesis of infection and disease in Drosophila melanogaster challenged with Salmonella enterica serovartyphimurium (S. typhimurium). We demonstrate that wild-type S. typhimurium causes a lethal systemic infection when injected into the hemocoel of D. melanogaster. Deletion of the gene encoding the secreted bacterial effector Salmonella leucine-rich (PslrP) changes an acute and lethal infection to one that is persistent and less deadly. We propose a model in which Salmonella secreted effectors stimulate the fly and thus cause an immune response that is damaging both to the bacteria and, subsequently, to the host. In support of this model, we show that mutations in the fly gene eiger, a TNF homolog, delay the lethality of Salmonella infection. These results suggest that S. typhimurium-infected flies die from a condition that resembles TNF-induced metabolic collapse in vertebrates. This idea provides us with a new model to study shock-like biology in a genetically manipulable host. In addition, it allows us to study the difference in pathways followed by a microbe when producing an acute or persistent infection.
A lethal infection of Drosophila is abrogated by removing a secreted Salmonella effector, demonstrating that the fly's immune system, which although initially protective, is subsequently harmful to the host
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Introduction
A hallmark of pathogens is their ability to manipulate their hosts at both a cellular and organismal level. This exploitation of the host can involve the production of specific toxins, secretion of virulence effectors into host cells via type III and type IV secretion apparatuses, or mimicry of host signaling molecules (Young et al. 2002; Gruenheid and Finlay 2003). The host organism can combat the infection with an array of innate and adaptive immune responses. Because there are so many possible combinations of thrusts, feints, and parries, there are many potential outcomes to this battle, ranging from a simple skirmish to defeat for one or the other of the combatants. As a result, microbes can produce acute and virulent infections in diseases such as cholera or plague or can take a path to cause persistent infections as in tuberculosis, Lyme disease, or host-adapted salmonellosis. It is important to understand which bacterial and host factors determine whether a microbe will cause epidemic disease, be a brief nuisance infection, or become a commensal.
Salmonella enterica serovar typhimurium (S. typhimurium) is naturally infectious to mice through an oral route and causes a systemic disease resembling human typhoid fever (Lucas and Lee 2000). S. typhimurium crosses the gut epithelium by entering and then killing M cells of the Peyer's patch. Once across the epithelial barrier, Salmonella infect macrophages and spread to the mesenteric lymph nodes and subsequently to other organs. S. typhimurium has two type III secretory apparatuses (TTSAs) that translocate effector proteins across both bacterial and host membranes into the host cell cytoplasm. One TTSA encoded by Salmonella pathogenicity island 1 (SPI1) plays an important role in cell entry, whereas a second TTSA, SPI2, alters the intracellular environment of the host cell to permit Salmonella growth. Salmonella likely does not find itself in the hemolymph of Drosophila in nature, but by placing it there we can ask some questions that are difficult to approach using other techniques.
The fly is able to fight invading microorganisms via an innate immune response that is composed of at least three arms (Khush and Lemaitre 2000). First, there is an inducible humoral immune response, which involves the secretion of antimicrobial peptides by a liver-like organ called the fat body. Second, there is a melanization response that exposes microbes to reactive oxygen as melanin is deposited on the invader. Third, there is the cellular immune response, which can result in the phagocytosis of relatively small organisms like bacteria or the encapsulation of larger parasites such as nematodes or parasitoid wasp eggs. The humoral immune functions are currently the most thoroughly characterized part of the fly's immune system. This aspect of the immune response is triggered when microbial molecules are recognized by fly pattern recognition receptors and signals are transmitted through the Toll and immune deficiency (IMD) pathways to activate three nuclear factor κB-like transcription factors, dorsal, Dorsal-related immunity factor, and Relish. It was work in this area that led to the discovery of Toll's central role in vertebrate immune recognition (Medzhitov et al. 1997).
Little is known about how Drosophila phagocytes affect the course of infections. It has been demonstrated that these cells act in concert with the humoral immune response to destroy invading bacteria (Braun et al. 1998; Elrod-Erickson et al. 2000), and it is well established that they are involved in the encapsulation of parasites (Carton and Nappi 2001). A potential phagocytic receptor for gram-negative bacteria as well as molecules involved in phagocytosis in cultured Drosophila cells have been identified (Ramet et al. 2002). Still, we have much to learn about how the phagocytes find and phagocytose microbes, kill microbes, or send signals to the body to indicate that an infection is in progress.
To focus attention on the cellular immune response, we have been characterizing the interaction between Drosophila phagocytes and pathogens that are specialized at growing within phagocytes. These bacteria have evolved methods of defeating phagocytes. By using a combination of wild-type and mutant bacteria, we can probe the function of the Drosophila phagocyte. We have shown previously that the two intracellular pathogens Mycobacterium marinum and Listeria monocytogenes can infect Drosophila phagocytes, and that some of the pathogenesis mechanisms developed by these bacteria for use in vertebrate phagocytes also function in the fly (Dionne et al. 2003; Mansfield et al. 2003). In this work we chose to study S. typhimurium because of its well-characterized secreted effector proteins. We found that the mutation of S. typhimurium effectors led to increased fly survival but, paradoxically, also increased bacterial survival. We propose a model that suggests the immune response of the fly can be deleterious to its health. This model predicts that the fly produces damaging immune effectors. We show that the fly homolog of tumor necrosis factor (TNF), encoded by the eiger gene, is involved in this process. Flies homozygous for mutations in eiger outlive wild-type flies infected with Salmonella.
Results/Discussion
We attempted to infect Drosophila with S. typhimurium by feeding bacteria to flies and by injecting bacteria into the hemocoel. Flies were resistant to feeding (unpublished data), but direct injection of approximately 10,000 bacterial cells resulted in an infection that caused death in 7–9 d, while injection of sterile medium led to a mean time of death of approximately 20 d (Figure 1A).
Figure 1 Growth of Salmonella in Drosophila melanogaster
(A) Survival of wild-type flies injected with S. typhimurium. Three sets of 60 flies were injected with approximately 10,000 cfu of SL1344 (Hoiseth and Stocker 1981) from an overnight 37 °C standing culture. Injected flies were incubated at 29 °C. Survival was monitored daily. Circles, S. typhimurium-injected; squares, LB-injected; triangles, uninjected.
(B) Survival of immune pathway mutant flies injected with S. typhimurium. Three sets of 20 flies were injected with approximately 100,000 cfu SL1344 and incubated at 29 °C. Fly survival was monitored at 0, 12, and 24 h postinfection. Flies infected: Black, wild-type (Oregon R); gray, imd1/imd1; white,Dif1/Dif1.
(C) Salmonella growth in infected immunocompromised flies. Flies were injected with approximately 100,000 cfu SL1344 and incubated at 29 °C for the indicated times. Flies were homogenized in LB with 1% Triton X-100 to release bacteria from cells, and the homogenates were plated on LB-streptomycin plates. Only living flies were homogenized. Flies infected: Black, wild-type; gray, imd1/imd1; white, Dif1/Dif1.
(D) Effects of gentamicin on S. typhimurium growth in the fly. Wild-type flies were injected with 10,000 cfu of S. typhimurium and then incubated at 29 °C for 7 d. Flies were then injected with either a solution containing 50 nl of 1 mg/ml gentamicin (black) or water (gray). A second group of previously uninfected flies was preinjected with gentamicin or water 15 min before bacterial challenge to determine the effects of the drug on bacteria before they were phagocytosed. All flies were then incubated after gentamicin or water injection at 29 °C for 4 h. Flies were then homogenized and plated. All error bars show standard deviation.
(E) Effects of S. typhimurium inoculation size on bacterial growth in the fly. Flies were injected with SL1344 over a 1,000-fold dilution range. Flies were incubated at 29 °C. Flies were homogenized immediately after injection or following 7 d of incubation before plating. Injected concentrations: Black, 0.1 optical density at 600 nm (OD600); dark gray, 0.01 OD600; light gray, 0.001 OD600; white, 0.0001 OD600.
The most thoroughly characterized immune response in the fly is the humoral immune response, which involves the secretion of antimicrobial peptides into the hemocoel by an organ called the fat body (Khush and Lemaitre 2000). Two highly conserved signaling systems, the Toll and IMD pathways, have been shown to control the transcription of the antimicrobial peptide genes (Lemaitre et al. 1996, 1997). The IMD pathway is implicated in raising a response to gram-negative bacteria such as Salmonella. Flies homozygous for an imd mutation succumbed to Salmonella infections rapidly, dying within 12 h of infection (Figure 1B). Bacterial numbers exceeded 100,000,000 per fly as the flies died (Figure 1C), and Salmonella in dying flies were found circulating in the hemolymph (unpublished data). Circulating bacteria were not seen in wild-type flies. In contrast to imd homozygotes, flies homozygous for a mutation in Dif, a transcription factor in the Toll pathway, did not die rapidly (Figure 1B and 1C). This experiment shows that mutations in the IMD but not the Toll pathway sensitize the fly to Salmonella. Further, these results suggest that the fly's humoral immune response limits the growth of free Salmonella in the hemocoel.
In mammals and birds, S. typhimurium is largely an intracellular pathogen. To determine whether S. typhimurium was indeed growing in a protected intracellular niche in the fly, we measured the in vivo sensitivity of Salmonella to gentamicin, an antibiotic that does not cross host-cell plasma membranes. To determine whether gentamicin could kill S. typhimurium in flies, 50 nl of 1 mg/ml gentamicin was injected into flies to load them with the drug. S. typhimurium was injected into the flies 15 min later and incubated for 4 h to allow the antibiotic time to act. Flies were homogenized and plated on selective medium to determine Salmonella colony forming units (cfu) (Figure 1D). Under these conditions, bacterial loads were 100-fold lower in antibiotic-injected flies than control flies, showing that the antibiotic could kill Salmonella immediately following injection. To determine whether S. typhimurium in an established infection were protected from gentamicin killing, we injected flies that had been infected with S. typhimurium for 7 d with gentamicin or water, and incubated the flies for 4 h before homogenizing and plating them. The day 7 time point was chosen throughout this paper, as it is the latest time point we can use before most flies begin to die from the infection. This experiment showed that bacteria that had been in the fly for 7 d were protected from the antibiotic and suggests that the bacteria were located in an intracellular location.
In other bacterial infection models in the fly, death of the host occurs only when bacterial numbers increase beyond 106 bacteria per fly. This is the case for infections both of wild-type flies with pathogens such as Pseudomonas aeruginosa and L. monocytogenes (D'Argenio et al. 2001; Lau et al. 2003; Mansfield et al. 2003) and of mutant flies with nonpathogenic bacteria such as Escherichia coli (Lemaitre et al. 1996; Elrod-Erickson et al. 2000). This is not the case for Salmonella in a wild-type fly. To determine the growth characteristics of S. typhimurium in Drosophila, bacteria were injected over a 1,000-fold dilution range; 7 d after infection, flies were homogenized and plated. At this time, bacteria were found to cover only a 5-fold range (Figure 1E). Bacteria injected at low densities grew to levels between 10,000 and 100,000 bacteria per fly, but those injected at higher levels did not pass this threshold. This final number is at least 1,000 fold lower than the number of bacteria found during fatal L. monocytogenes and E. coli infections in the fly. Salmonella thus reaches a population ceiling in flies, and the flies die containing relatively small numbers of bacteria.
To determine the location of bacteria within the fly, we examined flies infected with S. typhimurium expressing green fluorescent protein (GFP). Bacteria carrying the plasmid containing the macrophage-inducible gene (pmig-1) (Valdivia and Falkow 1997), which induces GFP expression upon entry into the phagosome, or the strain smo22 (Vazquez-Torres et al. 1999), which constitutively expresses GFP (unpublished data), were injected into larvae and flies. GFP-expressing S. typhimurium could be seen within hemocytes bled from infected larvae (Figure 2A–2D). Hemocytes in adults are mostly sessile and cannot be easily removed from the fly. However, these cells can be observed through the cuticle, and clusters of these cells are found on the dorsal surface of the abdomen, along the dorsal vessel (Elrod-Erickson et al. 2000; Dionne et al. 2003). Salmonella were found associated with these cells (Figure 2E–2H). The distribution of bacteria seen using the two GFP constructs was similar (unpublished data). This experiment suggests that, similar to the situation in mice, Salmonella are found within phagocytes. Furthermore, the Salmonella in fly hemocytes induced the expression of GFP from a Salmonella promoter normally induced upon phagocytosis by a mouse macrophage. This demonstrates that an S. typhimurium infection-related gene is induced even when the bacteria are infecting an animal at 29 °C instead of 37 °C.
Figure 2 Location of S. typhimurium in the Fly
(A–D) Induction of pmig-1 in Drosophila hemocytes. SL1344 carrying pmig-1 grown standing at 37 °C overnight were dried to a slide, fixed with formaldehyde and photographed using (A) differential intereference contrast (DIC) optics and (B) GFP optics. The bacteria are not highly fluorescent under these conditions. SL1344 carrying pmig-1 and grown as described above were injected into D. melanogaster larvae and the hemocytes were isolated and fixed after 24 h incubation at 29 °C. A hemocyte is shown in (C) DIC and (D) GFP optics using the same exposures as in A and B. Intensely fluorescent bacteria in the hemocytes are visible. Bar equals 5 um.
(E–H) S. typhimurium growth in living flies. SL1344 carrying pmig-1 were injected into wild-type flies and incubated for 2 d at 29 °C. (E and F) Uninfected flies are compared to (G and H) infected flies with (E and G) DIC and (F and H) GFP optics. The arrowhead highlights GFP-expressing Salmonella associated with hemocytes on the dorsal side of the fly.
S. typhimurium carrying mutations in a gene encoding a regulator of virulence, phosphatase P
(phoP) (Rathman et al. 1996), or blocking the function of either SPI1 (orgA::Tn10) (Jones and Falkow 1994) or SPI2 (ssrA::miniTn5) (Shea et al. 1996), were injected into flies. All of these mutants killed flies more slowly than wild-type bacteria (Figure 3A, graph area “i”). However, analysis of bacterial growth within infected flies revealed a more complicated story. PhoP mutants produced infections that were less lethal than wild-type Salmonella, but the infecting bacteria grew to the same levels seen in wild-type infections (Figure 3B). In contrast, a SPI1 or SPI2 mutant caused little death, and the bacteria grew to an average of 149,000 cfu/fly (SPI2 mutants) instead of the average of 40,000 cfu per fly found in wild-type infections (Figures 1D and 3B). Thus, the level of bacterial growth of this mutant is almost 4-fold higher than for wild-type Salmonella. This suggests that if S. typhimurium cannot manipulate Drosophila cells by secreting effectors, the survival of both pathogen and host is dramatically improved.
Figure 3 Effects of Salmonella Virulence Mutations on Disease in the Fly
(A) Mean time to death for infected flies. Identical quantities of S. typhimurium strains were injected into Oregon R flies and survival was monitored daily (i). Infections with slrP and rescuing construct were performed separately and are thus reported separately (ii).
(B) Growth of mutant Salmonella in the fly. Approximately 10,000 cfu of each bacterial strain were injected into flies. Flies were then homogenized and plated at the time of injection (black bars) or following a 7-d incubation (white bars). All error bars show standard deviation.
(C–H) Phagocytosis assays in living Drosophila. To assay the effects of Salmonella infections on phagocyte function, flies were injected with approximately 10,000 cfu of each strain of S. typhimurium. Following a 7-d incubation, the flies were first injected with FITC-labeled dead E. coli and incubated for 60 min to permit them to be phagocytosed. Trypan blue was then injected to quench the fluorescence of extracellular bacteria. The area boxed in (C) was photographed using FITC optics (D–H). The flies were injected with the following bacterial strains: (D) SL1344; (E) LB (control); (F) BJ66 (SPI1); (G) P3F4 (SPI2); (H) slrP (Table 1).
Table 1 Primers for Mutagenesis and Testing of S. typhimurium
PSLT, plasmid Salmonella typhimurium; Sit, Salmonella iron transport; Sfb, Salmonella ferric binding; Sod, superoxide dismutase; Ssp, Salmonella secreted protein
Table 1 Continued
To determine which of the known TTSA effector proteins contributed to this phenotype, flies were injected with Salmonella carrying in-frame deletions of these genes as well as several others we suspected might be involved in virulence in the fly (Figure 3A) (Datsenko and Wanner 2000). Deletions of spiC, Salmonella secreted effector A (sseA), sseB, sseC, and sseD all resembled a SPI2 knockout as expected because the proteins encoded by these genes are implicated in building the SPI2 translocation machinery (Nikolaus et al. 2001; Freeman et al. 2002; Ruiz-Albert et al. 2003; Zurawski and Stein 2003). Deletion of the gene encoding the Salmonella leucine-rich repeat-containing protein (PSlrP) produced a phenotype similar to that of the knockout of the entire SPI2 TTSA in terms of fly death (Figure 3A, graph area “ii”). Wild-type virulence could be restored to the slrP mutant by expressing slrP, controlled by its native promoter, in trans on a single-copy plasmid, p.slrP (Figure 3A, graph area “ii”). Because SPI1 and SPI2 bacteria accumulate to higher levels than wild-type strains, in the fruit fly, we measured growth of slrP-knockout bacteria. Mutation of slrP did not significantly alter the growth of Salmonella (Figure 3B). These experiments suggest that slrP is a determinant of Salmonella virulence in the fruit fly, but that mutation of slrP alone is not sufficient to alter Salmonella growth. The protein encoded by slrP is suspected to be a specific effector translocated directly into the host cytoplasm. Its function there remains unknown (Miao et al. 1999; Miao and Miller 2000; Waterman and Holden 2003).
The results we obtain during fly infection differ from what is found in mammalian and avian hosts. SPI1 is required by Salmonella for breaching the gut barrier but is dispensable if the bacteria are introduced systemically by intraperitoneal or intravenous injection. Both phoP- and SPI2-mutant Salmonella are highly attenuated even when injected into mammalian hosts; host survival increases, and the mutant bacteria do not readily replicate and are cleared during infections. In the fly, not only does host survival increase during infection by SPI1 or SPI2 mutants but, paradoxically, the bacteria are not cleared and replicate better than their wild-type parents. This is an interesting difference, because it separates mere bacterial presence from pathogenesis and disease. It remains to be determined why the fly does not clear the mutant bacteria in the manner that is seen in the mammalian host.
The analysis of adult hemocytes in the fly is difficult, because we lack good molecular markers for them, and the cells are sessile and rare enough to make them difficult to find regularly in tissue sections. We therefore turned to a functional assay to monitor the behavior of these infected cells by determining their ability to carry out one of their definitive behaviors, phagocytosis. Flies were infected with wild-type and mutant Salmonella and then assayed for the phagocytic capacity of the hemocytes on their dorsal surface. Fluorescein isothiocyanate (FITC)-labeled dead E. coli were injected into flies and incubated for 60 min to allow time for phagocytosis to occur. Trypan blue was then injected into the flies to quench the fluorescence of extracellular bacteria. Uptake of the E. coli was then monitored by observing the flies under FITC illumination with a dissecting microscope. At day 1 post-Salmonella infection, hemocytes infected with wild-type, SPI1-mutant, SPI2-mutant, and SlrP bacteria showed similar phagocytic activities (unpublished data). Following 7 d of infection, flies infected with wild-type or slrP-mutant bacteria had greatly reduced numbers of phagocytic cells (Figure 3C–3H). In contrast, phagocytes remained active during the course of infection by SPI1 or SPI2 mutants. Our interpretation of this experiment is that the wild-type Salmonella infection results in death of phagocytes or a reduced capacity for phagocytosis. This reduction is not dependent on SlrP function.
In mammals, cytokines such as TNF and interferon relay information about infection through the body. Cytokine expression, in particular TNF expression, is responsible directly and indirectly for a significant degree of the pathology observed during microbial infection (Beutler and Rietschel 2003). Flies have one known TNF-like protein, encoded by the gene eiger (Igaki et al. 2002; Moreno et al. 2002). Overexpression of eiger can induce cell death, but no phenotype had been identified for loss-of-function mutants thus far. We infected wild-type and eiger– flies with Salmonella to determine whether this fly TNF homolog played a role in the pathogenesis of Salmonella infections in Drosophila. The mean time to death was lengthened by 3 d in the two eiger mutants tested (Figure 4A). This experiment suggests that eiger/TNF signaling during a Salmonella infection contributes to the rate of death of the fly. Accumulation of Salmonella did not differ between eiger mutants and the background fly strain (Figure 4B). This suggests that eiger mutants do not directly affect Salmonella growth, but do markedly affect host survival. RNA transcripts of eiger were not significantly altered during Salmonella infection in comparison to Luria broth (LB)-injected controls (Figure 4C). This suggests that if eiger is regulated during Salmonella infection, the transcriptional changes are too small to be seen relative to expression in whole flies or the changes are posttranscriptional.
Figure 4 Effects of Eiger Mutations on S. typhimurium Infections in the Fly
(A) Survival of eiger-mutant flies infected with S. typhimurium. Three sets of 20 flies were infected with 10,000 cfu of SL1344 and incubated at 29 °C. Two eiger mutants (eiger1 and eiger3) and the background strain (w118) were assayed. Survival was monitored daily. Circle, eiger1/eiger1; square, eiger3/eiger3; triangle, w118. Solid shapes indicate LB-injected flies; open shapes indicate SL1344-injected flies. Mantel-Cox analysis demonstrated p < 0.001 when comparing infected wild-type to eiger-mutant flies.
(B) Growth of S. typhimurium in eiger-mutant flies. Flies were infected with 10,000 cfu of SL1344 and plated at the time of injection or following a 7-d incubation. Black, time = 0; white, time = 7 d. All error bars show standard deviation.
(C) Effects of Salmonella infection on eiger RNA transcript levels. Total RNA was extracted from five flies per sample on days 0, 1, 3, 5, and 7 postinjection with (open circles) SL1344 or (closed squares) LB. Quantitative real-time RT-PCR was performed. Relative eiger transcript quantity is expressed as the fold-difference in comparison to the day 0 value. All error bars show the standard deviation of three RNA preparations.
In human disease, morbidity and mortality are often the result of immune responses to invading pathogens rather than to direct action of the pathogens themselves (Beutler and Rietschel 2003). Fever, inflammation, and shock are examples of such processes. In plants, a model is emerging in which host cells monitor important cell functions and respond to their perturbations by pathogens (Staskawicz et al. 2001; Schneider 2002). We suggest the infection caused by Salmonella in the fly has attributes of both models. Salmonella growth appears to be restricted to hemocytes by action of the humoral immune response. Perhaps the manipulation of these hemocytes by secreted effectors induces additional immune responses that limit bacterial growth. This resembles what is seen in a resistant plant infected by a bacterium expressing the appropriate avirulence protein. Such proteins are secreted into the plant cell's cytoplasm via a TTSA, and they alter the host cell's physiology, presumably to make growth conditions for the bacteria more favorable. Resistant plants can sense this physiological change and raise a type of immune reaction called a hypersensitive response. In the case of Drosophila, we suggest that the fly's immune response not only limits the growth of the pathogen but also damages the fly and ultimately leads to its death. When S. typhimurium is prevented from using its secreted effectors, the bacteria survive in the phagocyte and grow to higher numbers, possibly because the bacteria are not being attacked as intensely by the host. The result is that there are more bacteria in the fly and host death is greatly delayed. These two properties appear to be separable. The mutations in eiger suggest that there are signaling events that increase the death rate in the fly but do not alter the numbers of Salmonella. These experiments provide a new genetic model to explore microbial choice of pathogenic lifestyles in addition to a potential new genetic model for the study of TNF-induced metabolic collapse.
Materials and Methods
Injection assays
Bacteria and medium were injected in a volume of 50 nl through a pulled glass needle. The injection volume was regulated using a Picospritzer III injector (Parker Hannifin, Rohnert Park, California, United States). The needle was placed in the anterior abdomen on the ventrolateral surface. One-week-old male flies were used for all experiments. All experiments were performed in triplicate with at least 20 flies in each replicate. Oregon R flies were used as our wild-type strain.
Determination of CFUs in flies
Infected flies were homogenized in LB containing 1% Triton X-100. Three infected flies were homogenized together either with a small pestle or by shaking with 0.4 mm glass beads. Diluted homogenates were plated on LB-agar with 50 μg/ml streptomycin.
In vivo phagocytosis assay
This assay was performed essentially as described previously (Elrod-Erickson et al. 2000). Infected flies were injected with 50 nl of 1 mg/ml FITC-labeled dead E. coli (Molecular Probes, Eugene, Oregon, United States) and incubated for 1 h at 25 °C to permit phagocytosis of the bacteria. Trypan blue (4%) was then injected into the flies. Enough dye was injected to turn the entire fly blue. Flies were observed under a Leica MZ3 fluorescent dissecting microscope (Leica, Wetzlar, Germany) using GFP epifluorescence optics, and photographed with an ORCA camera (Hamamatsu, Osaka, Japan) using Openlab software (Improvision, Coventry, UK).
Generation of Salmonella knockouts and slrP complementation plasmid
Isogenic gene knockouts were made in Salmonella strain 14028s/pKD46 (Datsenko and Wanner 2000). Transformants were verified by PCR using primers with homology to flanking regions of the target gene. The deleted gene region was transferred to our test strain, SL1344, using standard P22 lambda phage transduction. Transductants were selected on LB agar containing kanamycin and 10 mM EGTA overnight at 37 °C, and mutations were verified by PCR. Primer information is provided in Table 1. Primers designated “Salmonella typhimurium (STM)# 5′ + P1” and “STM# 3′ + P4” were used to generate the kanamycin insert specific to the target gene, using pKD13 as the template. Primers designated “STM# 5′ PCR” and “STM# 3′ PCR” were used to verify recombinants. The bacterial strains and plasmids used for cloning are listed in Table 2. The slrP complementation plasmid, p.slrP was constructed using the single copy plasmid pDM.2002 (Detweiler et al. 2003). SlrP along with its native promoter was amplified from SL1344 genomic DNA using the following primers: 5′-
CGCGGATCCAGCGTTGCAGCAGAAAAT-3′ (slrP 5′ BamHI) and 5′-
CGCGGATCCTGGGTTAAGCCCGTTTAC-3′ (slrP 3′ BamHI) (Miao and Miller 2000).
Table 2
S. typhimurium Strains and Plasmids
Mig, macrophage-inducible gene; org, oxygen-related protein; rps, ribosomal protein subunit; Tn, transposon
Eiger RNA quantitation
Total RNA was extracted from five flies per sample using a Qiagen RNeasy Kit (Qiagen, Valencia, California, United States). Quantitative real-time RT-PCR was performed with rTth polymerase (Applied Biosystems, Foster City, California, United States) and the following eiger primers: 5′-
GATGGTCTGGATTCCATTGC-3′ (5′ oligo), 5′-
TAGTCTGCGCCAACATCATC-3′ (3′ oligo) and 5′-6FAM-
GACGACGAGGACGACGACGTTAGCT-TAMRA-3′ (hybridization oligo). Concentrations of eiger transcripts were normalized to the expression of the D. melanogaster ribosomal protein 15a transcript in each sample (Schneider and Shahabuddin 2000). All experiments were performed in triplicate.
We thank the Falkow lab and CS Detweiler for Salmonella strains and M. Miura for eiger mutants. We thank Stanley Falkow, Hirotaka Kanuka, Mary Beth Mudgett, and Louisa Wu for comments on the manuscript, and the members of the Falkow and Tan labs for many useful discussions. This work was supported by NIH RO1 AI053080-01, a Pilot Project Grant from the Stanford Digestive Disease Center (NIH DK56339), NSF Graduate Research Fellowships (SMB and LNP), NIH 5 T32 A107328 (MSD), Stanford Medical School Deans Fellowship (MSD), and NIH 5 T32 GM07276 (TJV).
Conflicts of interest. The authors have declared that no conflicts of interest exist.
Author contributions. SMB, MSD, RSK, LNP, TJV, and DSS conceived and designed the experiments. SMB, MSD, RSK, LNP, TJV, and DSS performed the experiments. SMB, MSD, RSK, LNP, TJV, and DSS analyzed the data. SMB, MSD, RSK, LNP, and DSS contributed reagents/materials/analysis tools. SMB and DSS wrote the paper.
Academic Editor: Shizuo Akira, Osaka University
Citation: Brandt SM, Dionne MS, Khush RS, Pham LN, Vigdal TJ, et al. (2004) Secreted bacterial effectors and host-produced eiger/TNF drive death in a Salmonella-infected fruit fly. PLoS Biol 2(12): e418.
Abbreviations
cfucolony-forming unit
DICdifferential intereference contrast
DIFDorsal-related immunity factor
FITCfluorescein isothiocyanate
GFPgreen fluorescent protein
IMDimmune deficiency
LBLuria broth;mig
OD600optical density at 600 nm
pho
phosphatase
pmigplasmid containing the macrophage-inducible gene
slr
Salmonella leucine-rich
SPI
Salmonella pathogenicity island
sse
Salmonella secreted effector
STM
Salmonella typhimurium
TNFtumor necrosis factor
TTSAtype III secretory apparatus
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| 15562316 | PMC532388 | CC BY | 2021-01-05 08:21:17 | no | PLoS Biol. 2004 Dec 30; 2(12):e418 | utf-8 | PLoS Biol | 2,004 | 10.1371/journal.pbio.0020418 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1556231710.1371/journal.pbio.0020421Research ArticleEcologyEvolutionPaleontologyZoologyPrimatesHomo (Human)Modern Humans Did Not Admix with Neanderthals during Their Range Expansion into Europe No Admixture between Humans and NeanderthalsCurrat Mathias
1
2
Excoffier Laurent [email protected]
1
1Computational and Molecular Population Genetics Lab, Zoological Institute, University of BernBernSwitzerland2Genetics and Biometry Laboratory, Department of Anthropology and Ecology, University of GenevaGenevaSwitzerland12 2004 30 11 2004 30 11 2004 2 12 e42112 5 2004 6 10 2004 Copyright: © 2004 Currat and Excoffier.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Cro-Magnons Conquered Europe, but Left Neanderthals Alone
The process by which the Neanderthals were replaced by modern humans between 42,000 and 30,000 before present is still intriguing. Although no Neanderthal mitochondrial DNA (mtDNA) lineage is found to date among several thousands of Europeans and in seven early modern Europeans, interbreeding rates as high as 25% could not be excluded between the two subspecies. In this study, we introduce a realistic model of the range expansion of early modern humans into Europe, and of their competition and potential admixture with local Neanderthals. Under this scenario, which explicitly models the dynamics of Neanderthals' replacement, we estimate that maximum interbreeding rates between the two populations should have been smaller than 0.1%. We indeed show that the absence of Neanderthal mtDNA sequences in Europe is compatible with at most 120 admixture events between the two populations despite a likely cohabitation time of more than 12,000 y. This extremely low number strongly suggests an almost complete sterility between Neanderthal females and modern human males, implying that the two populations were probably distinct biological species.
A model of human expansion into Europe reveals almost complete sterility between Neanderthal females and modern human males, implying that the two populations were probably distinct biological species
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Introduction
The “Neanderthals” or Homo sapiens neanderthalensis (HN) constitute a group of hominids, whose particular morphology developed in Europe during the last 350,000 y under the effect of selection and genetic drift, reaching its final form approximately 130,000 y ago (Klein 2003). This subgroup of hominids populated Europe and western Asia until the arrival of the first modern humans, Homo sapiens sapiens (HS), approximately 45,000 y ago (Mellars 1992). This arrival coincided with the beginning of Neanderthal decline, a process that occurred in less than 15,000 y and that is still not fully understood (Stringer and Davies 2001). An important question which remains to be assessed is whether Neanderthals could hybridize with modern humans and if they left some traces in the current modern human gene pool. While this hypothesis is excluded under the Recent African Origin Model (RAO), which postulates a complete replacement of former members of the genus by H. sapiens, it is central to the tenets of the multiregional hypothesis (Eckhardt et al. 1993; Wolpoff et al. 2000), which assumes a gradual transition from H. erectus to modern humans on different continents. From a paleontological and archaeological point of view the debate is still open, even if the supporters of the RAO (Stringer and Davies 2001; Rak et al. 2002; Schmitz et al. 2002) are gaining momentum over those supporting European regional continuity (Duarte et al. 1999; but see also Tattersall and Schwartz 1999). Recent morphological studies support a clear distinction between Neanderthals and modern humans (Harvati 2003; Ramirez Rozzi and Bermudez De Castro 2004), and genetic evidence, such as the clear divergence and monophyly of the HN mitochondrial DNA (mtDNA) control region (Krings et al. 1997, 1999; Ovchinnikov et al. 2000), suggested a long separation of the HN and HS female lineages (Krings et al. 2000; Scholz et al. 2000; Schmitz et al. 2002; Caramelli et al. 2003), with a divergence time estimated to lie between 300,000 and 750,000 y ago (Krings et al. 1997, 1999). The complete absence of Neanderthal mtDNA sequences in the current European gene pool, attested from the study of more than 4,000 recorded sequences (Richards et al. 1996; Handt et al. 1998) supported the absence of Neanderthal mtDNA leakage in the modern gene pool, but it was argued that even if some HN genes could have passed in the ancient Cro-Magnon gene pool, they could have been lost through genetic drift (Relethford 2001; Hagelberg 2003). Recently, several attempts were made at circumventing the drift problem by the direct sequencing of modern human fossils contemporary with the last Neanderthals. Cro-Magnon sequences were found very similar to those of current Europeans (Caramelli et al. 2003), even though contamination from modern DNA could not be completely excluded (Serre et al. 2004). All studies nevertheless agreed in showing the absence of Neanderthal sequence motifs among early modern human fossil DNA (Caramelli et al. 2003; Serre et al. 2004), but only Neanderthal contributions larger than 25% to the modern gene pool could be statistically excluded under a simple model (Figure 1A and 1B) of instantaneous mixing of Neanderthals and modern humans (Nordborg 1998; Serre et al. 2004). Thus, the problem of the genetic relationships between Neanderthals and modern humans remains fully open.
Figure 1 Different Models of the Interactions between Neanderthals and Modern Humans
(A) Model of instantaneous mixing of unsubdivided Neanderthal and modern human populations.
(B) Same as (A), but with an exponential growth of the modern human population having started before the admixture with Neanderthals.
(C) Model of a progressive range expansion of modern humans into Europe. This model is spatially explicit, and the modern human population occupies a different range than the Neanderthal population before the admixture. Under this model, admixture is progressive and occurs because modern humans move into the territory of Neanderthals, a territory that shrinks with the advance of modern humans.
In order to further investigate this issue, we have developed a more realistic modeling of the admixture process between Neanderthals and early modern humans. In brief, the differences with previous approaches are the following (see Figure 1 and the Materials and Methods section for further details): (1) Europe is assumed to be subdivided into small territories potentially harboring two subpopulations (demes): an HN and an HS deme; (2) Europe is settled progressively by modern humans, resulting in a range expansion from the Near East. This range expansion implies also a demographic expansion of early modern Europeans, which stops when Europe is fully settled; (3) local population size is logistically regulated for both Neanderthals and modern humans; (4) we assume there is competition between modern humans and Neanderthals, resulting in the progressive replacement of Neanderthals by modern humans due to their higher carrying capacity caused by a better exploitation of local resources (Klein 2003); (5) Consequently, admixture between the two populations is also progressive and occurs in subdivisions occupied by both populations, in a narrow strip at the front of the spatially expanding modern human population (Figure 2); (6) coalescent simulations are used to estimate the likelihood of different rates of local admixture between modern humans and Neanderthals, given that Neanderthal mtDNA sequences are not observed in current Europeans.
Figure 2 Range Expansion of Modern Humans into Europe from the Near East
Simulations begin 1,600 generations ago, with the area of Europe already colonized by Neanderthals shown in light gray, and an origin of modern human expansion indicated by a black arrow (lane A). Lanes (B–F) show the progression of the wave of advance of modern humans (dark gray) into Europe at different times before present. The black band at the front of the expansion wave represents the restricted zone of cohabitation between modern humans and Neanderthals.
The additional realism of this model makes it also more complex, and the range expansion and admixture processes will depend on several parameters, like the carrying capacities of the local populations, their intrinsic growth rate, the amount of gene flow between adjacent demes, the local rate of admixture between populations, or the geographical origin of the range expansion. Since it is difficult to explore this complex parameter space, we used archeological and paleodemographic information to calibrate the values of these parameters. For instance, the estimated duration of the replacement process (about 12,500 y, Bocquet-Appel and Demars 2000a) was used to adjust the speed of the expansion of modern humans and, thus, provided strong constraints on local growth and emigration rates. Based on available information, we thus defined a set of plausible parameter values considered as a basic scenario (scenario A). Local admixture rate, which is the parameter of interest here, was then varied, and its effect on the estimated contribution of Neanderthals to the current modern human gene pool was recorded. The sensitivity of admixture estimates to alternative parameterization of our model was studied in eight alternative scenarios (scenarios B to I), by varying each time the values of a few parameters.
Results
Expected Neanderthal Contribution to the Current European Gene Pool as a Function of Admixture Rates
The description of the nine envisioned scenarios for the colonization of Europe by modern humans is reported in Table 1. For each of these scenarios, the admixture rate, which is the parameter of interest in this study, was allowed to vary and only marginally influenced the cohabitation period and the replacement time of HN by HS (Table 1). Note that the cohabitation period at any given place (shown as a narrow black band on Figure 2) is limited to 6–37 generations, depending on the scenario.
Table 1 Expected Proportion of Neanderthal Lineages in the Present Modern Human Gene Pool under Different Demographic Scenarios
The expected contribution of Neanderthal lineages in the current gene pool of modern humans (over all the simulated demes) was obtained from 10,000 simulations. Standard deviations are shown in italic. Demographic scenarios: (A) The basic scenario with realistic parameters; (B) identical to (A), with an origin in Iran at the extreme east of the simulated area; (C) identical to (A), but with a diffused source area consisting of 25 demes at KHS = 40, instead of only one deme; (D) identical to (A), but HS occupied all the south of the Neanderthal range (North Africa and North of the Arabian Peninsula) before the onset of the expansion, which corresponds to an HS initial population size of 14,000 breeding females; (E) identical to (A), with rHS = 0.8, and KHN = 25; (F) identical to (A), with mHS = 0.5 and KHN = 25 as in (D); (G) identical to (A) with a faster colonization time, due to larger growth and migration rates (mHS = 0.35 and rHS = 0.6); (H) identical to (A), with interbreeding resulting in symmetrical transfer of genes between modern humans and Neanderthals; (I) identical to (A), but with carrying capacity KHS being reached instantaneously and a local recruitment of γKHS Neanderthal lineages. In this latter scenario, there is thus a single event of admixture at demographic equilibrium and no logistic growth
a
KHN: carrying capacity of Neanderthal demes; KHS: carrying capacity of modern human demes; rHS: intrinsic rate of growth of modern humans per generation; mHS: migration rate between adjacent modern human demes
bIn generation
cThe different rates of admixture are given in number of admixture events per deme. For instance, a value of 1/10 implies an average of one admixture event for ten demes for the whole period of cohabitation between Neanderthals and modern humans
The expected proportion of Neanderthal genes in the gene pool of modern humans was estimated by coalescent simulations and is reported in Table 1 for different rates of admixture between Neanderthals and modern humans. At odds with previous estimates (Nordborg 1998 ; Gutierrez et al. 2002; Serre et al. 2004), our simulations show that even for very few admixture events, the contribution of the Neanderthal lineages in the current gene pool should be very large (see also Figure S1). For instance, in scenario A, with a 4-fold advantage in exploitation of local resources by modern humans, a single fertile admixture event in one deme out of ten over the whole period of coexistence between HN and HS should lead to the observation of 38% of HN genes in the present mtDNA HS gene pool (scenario A in Table 1). This proportion would be lower but still amount to 15% if the advantage of modern humans was reduced to 1.6 times over Neanderthals with the same admixture rate (scenario F in Table 1). With higher but still relatively low levels of admixture, a majority of Neanderthal genes should be expected in the current European gene pool (Table 1). For instance, with as much as two admixture events per cell over the total coexistence period of Neanderthals and modern humans, more than 95% of the current HS gene pool should be tracing back to Neanderthals, for all scenarios with logistic demographic regulation described in Table 1 (scenarios A to H). As shown on Figure 3, the proportion of current lineages that can be traced to Neanderthals is, however, not uniformly distributed over Europe in scenario of moderate or low interbreeding. A gradient should be visible from the source of the range expansion (which shows the largest proportion of modern human genes) toward the margins of the expansion (the British Isles and the Iberian Peninsula), which should then be expected to harbor a larger proportion of Neanderthal genes than the rest of Europe (Figure 3). However, this gradient would be relatively weak, and the expected proportion of HN lineages at any position is primarily affected by the degree of admixture between the two populations.
Figure 3 Expected Proportion of Neanderthal Lineages (in Black) among European Samples under Demographic Scenario A (Table 1) at Different Geographic Locations, for Different Interbreeding Rates
(A) One admixture event on average per 50 demes over the whole period of cohabitation between Neanderthals and modern humans; (B) one admixture event per five demes; (C) one admixture event per two demes; (D) one admixture event per deme.
The finding that even minute amounts of interbreeding between Neanderthals and modern humans should lead to a massive introgression of Neanderthals' mtDNAs into the Cro-Magnon gene pool is somehow counterintuitive and deserves further explanations. The successful introgression of Neanderthal mtDNAs is due to a massive dilution of the modern human mtDNA gene pool into that of the pre-existing population (Chikhi et al. 2002) and to a low probability of being lost by drift at the time of introgression (see below). The dilution process can be seen as follows: An HN gene entering the HS gene pool at an early stage of the colonization process will lower the frequency of HS genes in the HS deme; the migrants sent from this deme to colonize an adjacent new territory can themselves harbor HN genes, so that a further HS deme can be founded by a mixture of HS and HN genes; additional admixture events will further lower the proportion of HS genes in HS demes. The repetition of these admixture and migration steps will thus rapidly dilute HS genes. Under this process, the European HS population can be fully introgressed by HN genes under scenarios A to H, if two or more admixture events occurred in each deme (see Table 1, last two columns). For such large rates of admixture, the fraction of HS genes in demes adjacent to the source of HS expansion is already diluted by more than 28% with HN genes (results not shown). Therefore, in the absence of counteracting selective forces, the dilution process repeated over several demes would hinder the spread of HS genes away from the source of the colonization. The range expansion would thus be mainly carried out by individuals having HN genes, explaining why the HS European population would appear fully introgressed by HN genes. The success of introgressing HN genes is also due to their integration into the HS deme while it is in a period of demographic (logistic) growth (see Figure S2), so that these introgressing genes are unlikely to be lost by genetic drift, and will, rather, be amplified by the logistic growth process occurring in the HS deme. In order to assess the importance of the period of logistic growth relative to the dilution process, we have modeled a range expansion process where a newly founded deme reaches instantaneously its carrying capacity, and where a given proportion of genes is recruited from the local Neanderthal gene pool. The results of those simulations (reported in Table 1 as scenario I) show that without logistic growth much larger interbreeding rates would be necessary to have the same impact on current human diversity. Indeed, the occurrence of two admixture events per deme over the whole cohabitation period would only lead to 5% of the current gene pool being of Neanderthal ancestry, instead of 100% when logistic growth is implemented.
Estimation of Admixture Rates between Neanderthals and Modern Humans
The present results show that if Neanderthals could freely breed with modern humans, having progressively invaded their territory, their contribution to our gene pool would be immense. Since no Neanderthal mtDNA sequence has been observed so far among present Europeans, it is of interest to estimate the maximum admixture rate between Neanderthals and modern humans that would be compatible with an absence of Neanderthal genes, accounting for the current sampling effort and genetic drift over the last 30,000 y. This assessment was done by coalescent simulations. The likelihoods of different admixture rates are reported in Figure 4 for each scenario. Maximum-likelihood estimates are obviously obtained for a total absence of interbreeding between HS and HN, but here the interest lies in the upper limit of a 95% confidence interval. We see that the scenarios A to H can be divided into three groups. Scenarios A, C, G, and H lead to very similar upper bounds for the estimation of the maximum admixture rate (approximately 0.015 admixture events per deme; see Table 2). Similarity of results obtained for scenarios A and C show that the fact that the origin of the spread of modern humans was diffused over a large area or concentrated at a single point does not substantially influence our results. A shorter duration of the colonization of Europe by HS (approximately 8,000 y; scenario G) leads to an estimation very similar to that obtained under scenario A. Also the implementation of fully symmetric interbreeding between HN and HS (scenario H) leads to results almost identical to those obtained when we only allow breeding between HN females and HS males (scenario A). The place of origin for modern humans seems more important, as a putative origin in Iran (scenario B) or in North Africa (scenario D) leads to even lower maximum interbreeding rates (approximately 0.01 admixture events per deme) than if the source is located closer to Europe as in scenario A. Moreover, scenario D also shows that a much larger initial size of the HS population (14,000 breeding females instead of 40 in scenario A) does not reduce the final Neanderthal contribution to the HS gene pool. This is because we model local (at the deme level) and not global contacts between the two populations. Finally, scenarios E and F, corresponding to larger carrying capacities of Neanderthals, would be compatible with a larger amount of admixture between the two species (approximately 0.03 admixture events per deme), which is understandable given the longer cohabitation times under these scenarios (21–37 generations) than under scenarios A–D and G–H (6–12 generations). The estimates of the average number of admixture events per deme can be translated into a maximum number of interbreeding events having occurred over all Europe during the whole replacement process of Neanderthals by modern humans, as reported in Table 2. We find that, depending on the scenario, these maximum estimates range between 34 (scenario B) and 120 (scenario E) admixture events over the whole of Europe , which are extremely low values given the fact that the two populations have certainly coexisted for more than 12,000 y in that region.
Figure 4 Likelihood of Different Rates of Interbreeding under the Nine Scenarios Described in Table 1
The horizontal bold dashed line corresponds to 14.7% of the maximum likelihood, defining the upper limit of a 95% confidence interval for the interbreeding rates (see, e.g., Kalbfleisch 1985).
Table 2 Measure of Genetic Interaction between Neanderthals and Modern Humans
aUpper limit of a 95% confidence interval
bThis figure is computed from the previous column by assuming that there were a total of 140,000 reproducing females in the total modern human population in Europe (see Materials and Methods)
Discussion
Our simulations show that the mitochondrial evidence in favor of no, or very little, interbreeding between Neanderthals and modern humans is much stronger than previously realized (Wall 2000; Nordborg 2001). We indeed find that the current absence of Neanderthal mtDNA genes is compatible with a maximum admixture rate about 400 times smaller than that previously estimated (Nordborg 1998; Serre et al. 2004). This initial estimate (25%) was, however, based on a simple but unrealistic model of evolution, assuming no population subdivision, constant population size, and a single and instantaneous admixture event between Neanderthals and modern humans. Taking into account the progressive nature of the range expansion of modern humans into Europe, the maximum initial input of Neanderthal genes into the Paleolithic European population can thus be estimated to lie between only 0.02% (scenario B) and 0.09% (scenario E) (Table 2). Our simulations of alternative scenarios of HS range expansion into Europe suggest that our results are not very sensitive to local HS growth rates, level of gene flow between neighboring HS demes, or the geographical origin of HS range expansion. It is also worth emphasizing that the final HN contribution to the European gene pool does not really depend on the size and spread of the population at the source of the range expansion (compare scenario A to C and D in Table 1). This is logical since the colonization process starts from a restricted number of demes at the edge of the pre-existing range in our model of subdivided population (see Figure 1C). If this model is correct, it implies that the current European genes should have coalesced in a small number of individuals present in the demes at the source of the colonization of Europe, or, in other words, that there was a bottleneck having preceded the range expansion into Europe. Available data on European mtDNA diversity indeed support this view, since most European populations do present a signal of Paleolithic demographic expansion from a small population, which could be dated to about 40,000 y ago (Excoffier and Schneider 1999).
Additional complexities of the simulation model could have been envisioned, like the possibility for long-range dispersal, some heterogeneity of the environment leading to different carrying capacities and preferential colonization routes, or uneven migration rates. However, these extra parameters would have been very difficult to calibrate due to the scarcity of paleodemographic data. Moreover, it is likely that they would not have lead to qualitatively different results. For instance, since long-range dispersal speeds up the colonization process (Nichols and Hewitt 1994), short range migration rates would need to be reduced, in order to preserve a realistic colonization time. But this reduction would have no effect on local cohabitation time, which is the important factor affecting admixture rates (Table 2). Another source of realism could be the implementation of a recent Neolithic expansion wave on top of a Paleolithic substrate. This additional expansion wave has not been implemented here, as it is clearly beyond the scope of the present study. However, our present results suggest that small amounts of admixture between the Paleolithic and the Neolithic populations would lead to a massive contribution of Paleolithic lineages among the current Europeans. This point is important as it implies that if Neanderthal lineages had been present among the Paleolithic populations, they would not have been erased by the spread of the Neolithic in Europe. If we were using previous estimations of the Neolithic contribution to the current European genetic pool of about 50% (Barbujani and Dupanloup 2002; Chikhi 2002), the effect of a Neolithic expansion would require our estimates of the initial input of HN into the modern pool to be roughly multiplied by two, but still be very small (0.07% for scenario A). Note also that the simulation of a pure acculturation process, which amounts to increasing the carrying capacity of populations after the Neolithic by a factor 250 has virtually no effect on the expected proportion of Neanderthal genes in current Europeans (see Figure S1). Another argument against a major influence of the Neolithic expansion stems from mtDNA studies, since the demographic expansion inferred from mtDNA diversity and dated to about 40,000 y ago (Excoffier and Schneider 1999) implies that most of the mtDNA lineages of current Europeans result from a Paleolithic range expansion (Ray et al. 2003). If the expansion of Neolithic settlers had fully erased Paleolithic mtDNA diversity, one would indeed not expect to see this Paleolithic expansion signal. It thus argues in favor of a minor contribution of Neolithic genes to the current European gene pool, as expected under our model of progressive range expansion with continuous mixing.
Compared to previous models assuming an instantaneous mixing of HN and HS populations (Nordborg 1998; Serre et al. 2004) (see Figure 1A), we find that extremely small Neanderthal contributions should still be visible in the European gene pool. It implies that HN genes have a much larger probability of persisting when entering a progressively invading HS population than when entering a stationary population. This is because HN genes enter the HS population in demes that are still growing in size (see Figure S2), which prevents them from being lost by genetic drift and which amplifies their absolute number in the deme, making it likely they will persist and reach observable frequencies in the global population. This process is actually similar to that occurring in an unsubdivided growing population (e.g., Otto and Whitlock 1997). Actually, if HN genes were to directly enter an unsubdivided HS population that grew exponentially until today (see Figure 1B), the current absence of HN genes would also imply a very small amount of Neanderthal introgression into our gene pool (Nordborg 1998; Serre et al. 2004). However, this continuous and global exponential growth process appears difficult to justify (Serre et al. 2004) and does not really apply to the late Pleistocene human population (Weiss 1984; Biraben 2003).
Under our model, the progressive range expansion (Figure 1C) and the local logistic growth contribute to reduce the probability of losing introgressed HN genes. Without logistic growth, much larger interbreeding rates would be necessary to have the same impact on current human diversity (see scenario I in Table 1 and in Figure 4). Under this scenario, the absence of Neanderthal mtDNA sequences in present Europeans is still compatible with a maximum of about 1,850 fertile breedings between Neanderthal females and Cro-Magnon males, corresponding to a maximum initial input of 1.2% Neanderthal genes into the European Cro-Magnon population (Table 2). This figure being 20 times larger than when assuming an initial logistic growth of newly founded populations, it shows that the local logistic growth and the progressive range expansion contribute equally to reducing the inferred admixture rate compared to the simple model assuming a single admixture event and an instantaneous settlement of Europe by modern humans (see Figure 1A) (Serre et al. 2004). However, because new territories are often colonized by a few migrants and not by whole populations, local logistic growth has been incorporated into most models of range expansion (e.g., Fisher 1937; Skellam 1951; Shigesada and Kawasaki 1997). It should thus be considered as a normal feature of range expansions.
Another important result of this study is to show that an expanding population or species is likely to have its own genome invaded by that of the invaded population if interbreeding is possible and gradual, which could explain some documented cases of mtDNA introgression (e.g., Bernatchez et al. 1995; Shaw 2002). Our results indeed suggest that introgression should occur preferentially in species having gone through a range expansion, and that the introgressing genome would be that of the invaded population and not that of the invasive species. Of course this result should only apply to the part of the genome that is not under selection or that is not linked to the selective advantage of the invaders. If the mitochondrial genome of modern humans was involved in their higher fitness, the absence of observed mtDNA introgression would not necessarily be due to an absence of interbreeding, but would rather result from an active selection process against crosses between Neanderthal females and modern human males, and one would therefore expect to see potential leakage of Neanderthal genes in our nuclear genome. While some evidence for the differential fitness of some mtDNA human genomes in distinct climates has been recently found (Mishmar et al. 2003; Ruiz-Pesini et al. 2004), it is unlikely that such differences were involved in the selective advantage of modern humans over Neanderthals. It is indeed doubtful that modern humans coming from the Middle East would have had mitochondria better adapted to the colder environment of Europe than Neanderthals, who had spent tens of thousands of years in such a climate (Tattersall and Schwartz 1999; Klein 2003). It is therefore more likely that modern humans' higher technology and higher cognitive abilities (Klein 2003), resulting in better resource processing and environmental exploitation, have allowed them to out-compete Neanderthals, and that mtDNA was selectively neutral in that respect. It should however be kept in mind that our conclusions assume no sex bias in interbreeding rates. Studies of fossil Y chromosome or nuclear DNA would be needed to examine the basis of this assumption, but it seems difficult to imagine why interbreeding between Neanderthal men and modern human females resulting in the incorporation of Neanderthal genes would have been more frequent than the reverse situation.
Even though our model of interaction and competition between Neanderthals and modern humans may not entirely correspond to the reality, it captures two important historical aspects that were neglected in previous studies. The first one is the documented progressive spread of modern humans in Europe (see Figures 1 and 2), and the second is the local and progressive demographic growth of Paleolithic populations, with density-dependent interactions with Neanderthals. The incorporation of these additional sources of realism cannot be handled by current analytical models, but it can be readily integrated into a coalescent simulation framework, showing that it will be possible in the future to predict patterns of molecular diversity among populations or species belonging to a particular ecological network. Given the long period of cohabitation of the two populations in Europe and ample opportunities to interbreed, the absence or extremely low number of admixture events between Neanderthals and modern humans is best explained by intersterility or reduced fitness of hybrid individuals, promoting these populations to the status of different biological species. No interbreeding between the two populations also strongly argues in favor of a complete replacement of previous members of the genus Homo by modern humans and against a multiregional evolution of H. sapiens (Eckhardt et al. 1993; Wolpoff et al. 2000). It thus gives more credit to the RAO hypothesis (Excoffier 2002; Stringer 2002), since some very divergent H. erectus mitochondrial sequences should have also been observed if interbreeding had occurred during the colonization of Eurasia by modern humans from Africa.
Our conclusions about the genetic incompatibility between modern humans and Neanderthals would however be wrong if the absence of Neanderthal mtDNA genes in the current gene pool of modern Europeans was due to some processes that were not incorporated into our model. For instance, a range expansion of Neolithic populations without genetic contacts with Paleolithic could have erased both Paleolithic and remaining Neanderthal genes, but as discussed above, there are evidences for a substantial contribution of Paleolithic populations to the current gene pool (Barbujani and Dupanloup 2002; Chikhi et al. 2002; Dupanloup et al. 2004), invalidating this theory. Also an extremely rapid range expansion of a very large and unsubdivided modern population would also be compatible with an absence of Neanderthal genes despite considerable admixture, like in the scenario shown in Figure 1A (Nordborg 1998; Serre et al. 2004), but the long duration of the replacement process would be difficult to justify in that case. Finally, the occurrence of a cultural or ecological barrier, and not necessarily of a genetic barrier, could have prevented the realization of biologically possible hybridizations. Under this scenario, Neanderthals and early modern humans would have just avoided each other, which is contradicted by the observation of technological exchanges between Neanderthals and Cro-Magnons (e.g., Hublin et al. 1996). Moreover, the fact that the two populations had a very similar economy (Klein 1999, p. 530), indicates they had occupied an overlapping ecological niche and had thus ample opportunities to meet. It therefore seems that our model of subdivided population and progressive range expansion, implying local contacts, competition, and potential hybridization is quite plausible. One of its merits is also to explain both the replacement of Neanderthals by modern humans through a better exploitation of local resources, but also the late colonization of Europe by modern humans, which would have been possible only after the emergence of refined Upper Paleolithic technologies giving a competitive edge over Neanderthal industries (Klein 1999, pp. 511–524).
Materials and Methods
Digital map of Europe
The simulated region corresponds to the geographical region encompassing Europe, the Near East and North Africa. It has been modeled as a collection of 7,500 square cells of 2,500 km2 each, arranged on a two-dimensional grid, with contours delimited by seas and oceans. Each cell harbors two demes, one potentially occupied by modern humans (HS) and one potentially occupied by Neanderthals (HN). Given the estimated range distribution of Neanderthals (Klein 2003), HN demes were allowed in only 3,500 cells, mainly located in the lower part of Europe and in the Near East (see Figure 2A). Three land bridges have been artificially added to allow the settlement of Great Britain and Sicily.
Simulation of the colonization of Europe by modern humans
The simulation of the colonization process in Europe is an extension of that described in absence of competition in a homogeneous square world (Ray et al. 2003). At the beginning of the simulation, 1,600 generations ago (corresponding to 40,000 y ago when assuming a generation time of 25 y), the HN demes are all filled at their carrying capacity, KHN, and, in the basic scenario, the population HS is assumed to be restricted to a single deme in the Near East at a position corresponding approximately to the present border between Saudi Arabia and Jordan. Note that alternative locations and a more widespread distribution are also envisioned in other scenarios (see Table 1).This source for the spatial and demographic expansion of modern humans into Europe has been chosen arbitrarily, as its exact origin is still debated (Bocquet-Appel and Demars 2000a; Kozlowski and Otte 2000). Since we model the evolution of mtDNA, we only simulate the spread of females, but we implicitly assume that there are the same number of males and females in each deme. The source deme for HS is assumed to be at its carrying capacity KHS of 40 females, corresponding to a density of about 0.06–0.1 individuals per km2 (including males and juveniles), in agreement with density estimates for Pleistocene hunter-gatherers (Steele et al. 1998; Bocquet-Appel and Demars 2000b). HS individuals can then migrate freely to each of the four neighboring HS demes at rate m/4. When one or more HS individuals enter an empty deme, it results in a colonization event, which initiates a local logistic growth process, with intrinsic rate of growth rHS per generation, and with limiting carrying capacity KHS. Interactions between the HS and the HN demes of the same cell are described below in more detail, and its combination with migrations between HS demes results in a wave of advance progressing from the Near East toward Europe and North Africa.
Demographic model incorporating competition and admixture
We describe here a demographic model of interaction between populations, incorporating competition and interbreeding between individuals of the HN and HS populations, as well as migration between neighboring demes from the same subdivided population. We distinguish here migrations events between HN and HS populations from migrations between neighboring HN or HS populations. We model the former ones as admixture events, whereas the latter ones correspond to true dispersal events. The life cycle of a population at a given generation is as follows: admixture, logistic regulation incorporating competition, followed by migration. This life cycle thus assumes that migration is at the adult stage. In line with previous work (Barbujani et al. 1995), the frequency of admixture events is assumed to be density-dependent. Within a given deme, each of the Ni individuals from the i-th population has a probability
to reproduce successfully with one of the Nj members of the j-th population, and γ
ij represents the probability that such a mating results in a fertile offspring. Alternatively, γ
ij could represent the relative fitness of hybrid individuals or an index of disassortative mating. Following admixture, population densities are then first updated as
Our model of density regulation incorporating competition is based on the Lotka–Volterra interspecific competition model, which is an extension of the logistic growth model (Volterra 1926; Lotka 1932). For each population, a new density N
″
i is calculated from the former density as
where ri is the intrinsic growth rate of the i-th population, Ki is its carrying capacity, and αij is an asymmetric competition coefficient (Begon et al. 1996, pp. 274–278). An αij value of 1 implies that individuals of the j-th population have as much influence on those of population i as on their own conspecific, or that competition between populations is as strong as competition within a population. Lower values of αij indicate lower levels of competition between populations than within populations; a value of zero implies no competition between individuals from different populations. We have decided here not to fix αij values, but to make them density-dependent as
reflecting the fact that the influence of the members of a population on the other grows with its density. An example of the demographic transition between HN and HS is shown in Figure S2, together with the amount of admixture between the two populations. In the migration phase, each population of each deme can send emigrants to the same population in neighboring demes at rate m.
N
″
i
m emigrants are thus sent outward each generation, and distributed equally among the four neighboring demes, as described previously (Ray et al. 2003). If a gene is sent to an occupied deme, the migration event results in gene flow; otherwise, it results in the colonization of a new deme. This latter possibility only exists for the population of modern humans, since we assume that Europe was already fully colonized by Neanderthals. Finally, the densities of the two populations are updated as a balance between logistic growth, migration, and admixture as
where Ii is the number of immigrants received from neighboring demes.
Parameter calibration
We have calibrated the parameters of our simulation model from available paleodemographic information and from the estimated colonization time of Europe by modern humans. Estimates of the total number of hunter-gatherers living before Neolithic times range between 5 and 10 million (Coale 1974; Hassan 1981; Weiss 1984; Landers 1992; Chikhi et al. 2002), of whom about 1 million individuals were living in Europe. Taking a carrying capacity KHS of 40 females would imply the presence of 220,000 effective mtDNA genes in the 5,500 demes occupied by modern humans in Europe and the Middle East. Since this number represents only females, the total number of individuals living over Europe was multiplied by four to include men and juveniles, leading to a total density of about 880,000 HS individuals. This value of KHS corresponds to a density of 0.064 individuals per square kilometer, which is close to the value (0.04) used by some previous simulation of modern humans (Rendine et al. 1986; Barbujani et al. 1995) and well within the range obtained from actual hunter-gatherer groups (0.01–0.35; Binford 2001) or that estimated for ancient hunter-gatherers (0.015–0.2; Steele et al. 1998; Bocquet-Appel and Demars 2000b). The time required for the colonization of Europe by modern humans is the other information that was used to calibrate the growth rates, rHS, the rate of migration, mHS, and the Neanderthal carrying capacity (KHN), as these three parameters have an influence on the speed of the migration wave (Fisher 1937; Skellam 1951). Since modern humans arrived in Europe approximately 40,000 y ago and occupied the whole continent by 27,500 before present (BP) (Bocquet-Appel and Demars 2000b), the colonization process lasted approximately 500 generations, assuming an average generation time of 25 to 30 y (Tremblay and Vezina 2000; Helgason et al. 2003 ).
Scenarios of modern human range expansion in Europe
Among the many sets of parameter values leading to the appropriate colonization time and the complete disappearance of Neanderthals, we have retained the following scenarios. Scenario A: Origin of HS in a single deme of the Near East at the border between Saudi Arabia and Jordan, mHS = mHN = 0.25, rHN = 0.4, and KHN = 10, rHS = 0.4, KHS = 40. Note that a value of KHN of ten corresponds to a total density of about 140,000 Neanderthals over Europe (0.016 individuals per km2), which is of the same order of magnitude as the rare available estimates (250,000 Neanderthals, Biraben 2003). Under this scenario, we have only considered admixture events between HN females and HS males, such that γHS,HN = 0 . Eight alternative scenarios have been considered by using extreme values of the parameters of the model (m, r, K, European colonization time, place, and size of initial HS population). Scenario B is identical to scenario A, except that the HS origin is located in Iran. Scenario C uses the same parameters as scenario A, but the HS source is more diffuse and corresponds to a subdivided population of 25 demes (1,000 breeding females) surrounding the source deme defined in scenario A. Scenario D is identical to A, except that the initial HS population is even much more numerous (14,000 breeding females located in 1,400 demes) and occupies all the south area of the HN occupation zone. Scenario E is identical to A, but rHS is here equal to 0.8, which is the maximum growth rate estimated for the Paleolithic human population (Ammerman and Cavalli-Sforza 1984; Young and Bettinger 1995). Scenario F is identical to A, except that mHS is here much higher and equal to 0.5, implying that 50% of the women are recruited in adjacent demes. The carrying capacity of Neanderthals KHN had to be readjusted for scenarios E and F, which may appear as extreme, in order to maintain a colonization time of about 500 generations. It was indeed set to 25, giving a total density of HN of 350,000 individuals over Europe. Scenario G is identical to A, except that rHS is here equal to 0.6 and mHS is equal to 0.35, leading to a shorter colonization time of the European continent by HS. Under scenario G, the colonization time of Europe is approximately 8,000 y, which would correspond to the minimum colonization time estimated from direct fossil evidence, since the first European HS fossil is dated to about 36,000 y BP (Trinkaus et al. 2003), and the latest HN is dated around 28,000 y BP (Smith et al. 1999). Scenario H is identical to A, but admixture can occur between HN males and HS females as well, such that γHS,HN = γHN,HS
. Finally, scenario I uses the same parameters as A, but a different demographic model. When a cell is colonized by HS, it is directly filled at KHS with an initial proportion γ of Neanderthals. Admixture thus occurs when demographic equilibrium is already reached, and not during the demographic growth as in the other models.
While the γ values are the true parameters of our model, they may not be very telling per se, and we have therefore chosen to quantify levels of interbreeding between populations using another parameterization, which is the average number of admixture events per deme between modern humans and Neanderthals. By performing a large series of simulations, we could find the values of γ leading to a given average number of admixture events per deme (e.g., 1/500, 1/100, 1/10, 1, 2, etc.). For instance, a value of 1/10 means that one admixture event occurred on average in one deme out of ten during the whole cohabitation period between HN and HS.
Coalescent simulations
For each scenario and for different interbreeding values, γij, the demography of the more than 14,000 demes is thus simulated for 1,600 generations. The density of all demes, the number of migrants exchanged between demes from the same population, and the number of admixture events resulting in gene movements between Neanderthals and modern humans are recorded in a database. This demographic database is then used to simulate the genealogy of samples of 40 genes drawn from 100 demes, representing a total of 4,000 modern human genes distributed over all Europe and corresponding approximately to the current sampling effort of European mtDNA sequence (Richards et al. 1996; Handt et al. 1998). The coalescent simulations proceed as described previously (Ray et al. 2003; Currat et al. 2004). The average proportion of sampled genes whose ancestors can be traced to some Neanderthal lineages was then computed over 10,000 simulations. The likelihood of each interbreeding coefficient, γij, is estimated for the different scenarios by the proportion of 10,000 simulations that lead to a Most Recent Common Ancestor of all 4,000 sampled mtDNA sequences being of modern human origin.
Supporting Information
Figure S1 Proportion of Neanderthal Lineages in the European Population as a Function of the Average Number of Admixture Events per Deme between HN and HS
These values are given for the nine scenarios (A–I) listed in Table 1, and for a new scenario A+Neol. This latter scenario is similar to A, except that the carrying capacity of the modern humans is increased by a factor 250 at the time of the Neolithic transition (320 generations BP). The influence of this demographic increase on the simulated HN proportion is very weak, as shown on this figure.
(357 KB TIF).
Click here for additional data file.
Figure S2 Evolution of the densities of demes HN (in black) and HS (in gray) within a cell simulated under demographic scenario A for γij = 0.4. The cell is colonized by HS at time −1520 ( 0 = present). The thin black line with white circles represents the distribution of admixture events, whose numbers are reported on the right axis
(322 KB TIF).
Click here for additional data file.
Thanks to Nicolas Ray and Pierre Berthier for computing assistance. We are grateful to Monty Slatkin, Arnaud Estoup, and Grant Hamilton for their critical reading of the manuscript, and to four anonymous reviewers for their helpful comments. This work was supported by a Swiss NSF grant No 3100A0–100800 to LE.
Conflicts of interest. The authors have declared that no conflicts of interest exist.
Author contributions. MC and LE conceived and designed the experiments. MC performed the experiments. MC and LE analyzed the data. MC and LE wrote the paper.
Academic Editor: David Penny, Massey University
Citation: Currat M, Excoffier L (2004) Modern humans did not admix with Neanderthals during their range expansion into Europe. PLoS Biol 2(12): e421.
Abbreviations
BPbefore present
HN
Homo sapiens neanderthalensis
HS
Homo sapiens sapiens
mtDNAmitochondrial DNA
RAORecent African Origin Model
==== Refs
References
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| 15562317 | PMC532389 | CC BY | 2021-01-05 08:21:17 | no | PLoS Biol. 2004 Dec 30; 2(12):e421 | utf-8 | PLoS Biol | 2,004 | 10.1371/journal.pbio.0020421 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1556231810.1371/journal.pbio.0020422Research ArticleBioinformatics/Computational BiologyEvolutionGenetics/Genomics/Gene TherapyYeast and FungiPatterns of Intron Gain and Loss in Fungi Patterns of Intron EvolutionNielsen Cydney B
1
2
Friedman Brad
1
3
Birren Bruce
2
Burge Christopher B [email protected]
1
Galagan James E [email protected]
2
1Department of Biology, Massachusetts Institute of TechnologyCambridge, MassachusettsUnited States of America2The Broad Institute of Massachusetts Institute of Technology and Harvard UniversityCambridge, MassachusettsUnited States of America3Department of Mathematics, Massachusetts Institute of TechnologyCambridge, MassachusettsUnited States of America12 2004 30 11 2004 30 11 2004 2 12 e42222 6 2004 5 10 2004 Copyright: © 2004 Nielsen et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
The History of the Intron - Balancing Gains and Losses
Little is known about the patterns of intron gain and loss or the relative contributions of these two processes to gene evolution. To investigate the dynamics of intron evolution, we analyzed orthologous genes from four filamentous fungal genomes and determined the pattern of intron conservation. We developed a probabilistic model to estimate the most likely rates of intron gain and loss giving rise to these observed conservation patterns. Our data reveal the surprising importance of intron gain. Between about 150 and 250 gains and between 150 and 350 losses were inferred in each lineage. We discuss one gene in particular (encoding 1-phosphoribosyl-5-pyrophosphate synthetase) that displays an unusually high rate of intron gain in multiple lineages. It has been recognized that introns are biased towards the 5′ ends of genes in intron-poor genomes but are evenly distributed in intron-rich genomes. Current models attribute this bias to 3′ intron loss through a poly-adenosine-primed reverse transcription mechanism. Contrary to standard models, we find no increased frequency of intron loss toward the 3′ ends of genes. Thus, recent intron dynamics do not support a model whereby 5′ intron positional bias is generated solely by 3′-biased intron loss.
A comparative study of four fungal genomes reveals the patterns of intron gain and loss over several hundred million years of evolution
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Introduction
Over a quarter of a century after the discovery of introns, fundamental questions about their function and evolutionary origins remain unanswered. Although intron density differs radically between organisms, the mechanisms by which introns are inserted and deleted from gene loci are not well understood. A correlation has been observed between intron density and positional bias (Mourier and Jeffares 2003). Introns are evenly distributed within the coding sequence of genes in intron-rich organisms, but are biased toward the 5′ ends of genes in intron-poor organisms. This bias is particularly pronounced in the yeast Saccharomyces cerevisiae. It has been suggested that both the paucity and positional bias of introns in yeast may be due to intron loss through a mechanism of homologous recombination of spliced messages reverse-transcribed from the 3′ poly-adenylated tail (Fink 1987). This reverse transcription mechanism was first demonstrated in experiments with intron-containing Ty elements in yeast (Boeke et al. 1985). More recently, Mourier and Jeffares (2003) concluded that homologous recombination of cDNAs is the simplest explanation for the positional bias observed in all intron-poor eukaryotes. However, few data exist concerning the actual mechanisms and dynamics of intron evolution.
Fungal genomes are in many ways ideal for exploring questions of intron evolution. The fundamental aspects of intron biology are shared between fungi and other eukaryotes, making fungi appropriate model organisms for intron study. They are gene dense with relatively simple gene structures compared with plants and animals, making gene prediction more accurate. Fungi also display a wide diversity of gene structures, ranging from far less than one intron per gene for S. cerevisiae, to approximately 1–2 introns per gene on average for many recently sequenced ascomycetes (including the organisms in this study), to roughly seven introns per gene on average for some basidiomycetes (e.g., Cryptococcus). Finally, fungi display a strong 5′ bias in intron positions, enabling us to investigate the processes underlying this phenomenon.
In principle, a 5′ intron bias could arise through various combinations of intron gain and loss, and a complete understanding of intron positional bias requires an assessment of the contributions of both of these processes. A number of studies demonstrate the occurrence of intron gain and loss in individual genes or gene families. Logsdon et al. (1995) offered early examples of well-supported intron gain by comparing triose-phosphate isomerase genes from diverse eukaryotes and demonstrated that numerous introns could be most parsimoniously explained by a single gain with no subsequent losses. O'Neill et al. (1998) later provided evidence for de novo intron insertion into the otherwise intron-less mammalian sex-determining gene SRY. Evidence for the occurrence of multiple independent intron losses has also been reported in studies such as those by Robertson (2000), who inferred gain and loss events in a family of chemoreceptors in Caenorhabditis elegans.
More recently, a number of genome-wide studies of intron dynamics have been conducted. Roy et al. (2003) described genome-wide comparisons between human and mouse (with Fugu as an outgroup) and between mouse and rat (with human as an outgroup), and observed a sparseness of intron loss and complete absence of intron gain in these closely related organisms. On the other hand, Rogozin et al. (2003) observed an abundance of lineage-specific intron loss and gain when analyzing clusters of orthologous genes in deeply branching eukaryotes. Similarly, Qiu et al. (2004) analyzed ten protein families in distantly related eukaryotes, with a single prokaryotic outgroup, and obtained evidence that extant introns are predominantly the result of intron gains. In search of clues to understand the mechanism of intron gain, Fedorov et al. (2003) aligned introns from various eukaryotes, and Coghlan and Wolfe (2004) applied a similar approach in a comparative study of nematodes. None of these studies addressed the positional bias of intron gain and loss events. Here we report the results of a genome-wide comparative analysis of intron evolution in organisms that have a strong 5′ bias in intron location and are at an appropriate evolutionary distance to reveal positional trends in intron gain and loss.
Results
To investigate the roles of both gain and loss in intron evolution, we compared the genomes of four recently sequenced fungi spanning at least 330 million years of evolution (Taylor et al. 1999; Berbee and Taylor 2000; Heckman et al. 2001) (Figure 1): Aspergillus nidulans, Fusarium graminearum, Magnaporthe grisea, and Neurospora crassa. Ortholog sets composed of one gene from each of the four genomes were identified as pairwise best bidirectional BLAST hits satisfying stringent overlap criteria. Orthologs in each set were subsequently aligned, and the locations of introns were marked. These intron positions (regions of the multiple sequence alignment containing an intron in at least one of the four sequences) were subjected to rigorous alignment quality filtering to eliminate alignment and annotation errors (Figure 2A). To set the filtering thresholds, we manually classified ten residue alignment windows on either side of 181 randomly selected intron positions as “clearly homologous,” “possibly homologous,” or “non-homologous.” Requiring 30% identity and 50% similarity in these windows captured 92% of the clearly homologous positions, 29% of the possibly homologous positions, and only 2% of the non-homologous positions (Figure 2B). Passing intron positions were split into five quintiles according to their relative position within the annotated coding sequence.
Figure 1 Phylogenetic Tree and Intron Conservation Patterns
(A) Phylogenetic tree of the four fungal organisms studied (M. grisea, N. crassa, F. graminearum, and A. nidulans) with estimated time scale in millions of years ago. The rooted organismal tree was constructed using an unweighted pair group method using arithmetic averages based on a concatenated alignment of 2,073 orthologous gene sets. Estimated dates of divergence from Taylor et al. (1999), Berbee and Taylor (2000), and Heckman et al. (2001).(B) Classification of intron presence (+) and absence (−) patterns across the four fungal species. A blue “+” indicates a raw intron gain in the corresponding organism, a red “−” indicates a raw intron loss in the corresponding organism, a green “+” indicates a conserved intron, and all other introns are indicated in black.
Figure 2 Alignment Filtering Protocol
(A) Schematic of filtering protocol applied to a ten-residue window on each side of every intron position. If either side failed the filter, the position was discarded.
(B) Distributions of minimum percent identity and similarity in ten-residue windows around 181 randomly selected intron positions, for three manual classifications. The minima were taken between the left and right windows. The yellow lines indicate the chosen thresholds of at least 50% similarity and 30% identity, and bars are colored yellow if they fall above the thresholds (pass) or orange if they fall below the thresholds (fail). Parentheses indicate the number of introns in each class that pass the cutoff and the total number of introns in that class. The five lowest-percent identity and similarity bars, containing 77 positions, in the “non-homologous” plot are omitted so as to not obscure the rest of the histogram.
Genome-Wide Characterization of Intron Conservation
We applied our analysis protocol to 2,073 putative ortholog sets that included 9,352 intron positions. Of these initial intron positions, 5,811 were removed because of low conservation surrounding the intron, or because of an adjacent gap, or both. It is possible that some of the positions neighboring gaps may in fact reflect intron gain or loss events that occurred simultaneously with coding sequence insertion or deletion (Llopart et al. 2002). However, removing these positions did not significantly impact our results, as the number of positions adjacent to gaps was only about one-tenth of the number of positions that passed the quality filter, and the removal of these introns did not alter the apparent positional bias of the overall distribution (Figure 3). An additional 92 introns had nearby introns with insufficient conservation between the two introns and were thus also rejected.
Figure 3 Positional Biases in Intron Gain and Loss
Relative intron positions were defined as the number of bases in the coding sequence upstream of the intron divided by the total length of the coding sequence. These relative positions were binned into five categories (quintiles), each representing one-fifth of the coding sequence length (quintiles numbered 1–5 on the x-axis).
(A) Introns passing quality filter (light blue, back) and introns adjacent to gaps in the protein alignment that were removed by our quality filter (orange, front).
(B) Raw and inferred gains. Raw gains (green, back) are those introns present in exactly one organism (excluding the outgroup, A. nidulans). Inferred gains (blue, front) are corrected for the estimated number of cases that arose by other combinations of gain and loss events. Inferred gains are thus slightly lower than raw gains.
(C) Raw and inferred losses. Raw losses (green, front) are those introns absent in the organism in question but present in at least one of its siblings (descendants of its parent in the phylogenetic tree) and one of its cousins (non-descendants of its parent). Inferred losses (red, back) are corrected for the estimated number of introns lost along multiple lineages, or gained and then lost. Inferred losses are thus slightly higher than raw losses.
(D) Number of introns gained (blue) and lost (red) since last common ancestor (losses shown as negative numbers).
(E) Intron loss rate at each position since last common ancestor (introns lost per ancestral intron). Error bars represent binomial standard deviation.
In the end, a total of 3,450 intron positions (roughly 37% of intron positions considered) passed the quality filter. The complete set of aligned orthologs with passing and failing intron positions is provided in Table S1. These data constitute a genome-wide survey of high-confidence aligned intron positions and their patterns of conservation over at least 330 million years of evolution.
An example of an alignment of putative orthologs with three passing intron positions is shown in Figure 4A. In each passing intron position (black-edged rectangles), individual introns are labeled according to the classes previously outlined in Figure 1B. One intron position is conserved across all four species (green rectangle), one is a raw gain in N. crassa (blue box), and the third is present only in A. nidulans, and, because of the ambiguity in inferring gain or loss in this case, is classified as “Other” (black-edged gray rectangle). Examining the region around the one raw gained intron in N. crassa at the nucleotide level (Figure 4B) reveals a clean insertion of the intron sequence within a highly conserved region. The gained intron has consensus terminal dinucleotides (GT…AG) and a putative branch point sequence that matches the yeast consensus (
TACTAAC) at six of seven positions. In addition, this set of orthologs contained one poorly aligned intron position (Figure 4A, unedged gray rectangle) that was excluded by our filters. All three passing positions (black-edged rectangles) display high amino acid sequence conservation on both sides flanking the intron, supporting the correctness of the alignment. In contrast, the failing intron position (unedged gray rectangle) is adjacent to a region of the alignment that lacks significant conservation. The 3′ flank of this intron position displays considerable variation, especially with respect to the M. grisea gene, which was predicted to have a much longer 3′ coding region. In such an alignment region, it is difficult to distinguish true differences in intron conservation from potential annotation or alignment errors. Our filtering process thus eliminated this position from further analysis.
Figure 4 Example Ortholog Alignment
(A) Alignment of protein sequences for orthologs MG04228, NCU05623, FG06415, and AN1892 with intron characters inserted. “0,” “1,” and “2” indicate the phase of an intron. A black-edged rectangle indicates an intron position passing our quality filters; an unedged gray rectangle indicates an intron position that was removed by our filter. The green rectangle indicates conserved introns, the blue box marks a raw intron gain, and the gray boxes within black-edged rectangles highlight all other introns. The consensus (bottom) line characters are as follows: asterisk, identical residue in all four sequences; colon, similar residue; and period, neutral residue.
(B) Nucleotide alignment of the region flanking the gained intron in (A). Putative 5′ and 3′ splice sites and a branch point sequence are highlighted in blue.
Calculation of Raw Gains and Losses
We calculated “raw gains” and “raw losses” by positional quintile for each organism other than the outgroup, A. nidulans. We defined raw gains as those introns present in only a single organism (see Figure 1B). We defined raw losses as those introns that are absent in the organism in question, present in some other descendant of the organism's parent (a “sibling”), and present in some non-descendant of the parent (a “cousin”) (Figure 1B). Intron positions are considered conserved if present across all four organisms. Patterns of intron presence and absence that are not captured by the above definitions were excluded from the raw counts because of the ambiguity in inferring intron gain or loss events in such cases (marked as “Other” in Figure 1B).
Probabilistic Model of Intron Gain and Loss
Raw gain and loss counts are based on parsimony and may differ somewhat from the true number of gain and loss events. The set of raw gains may include introns that were lost in multiple lineages, thus overcounting the true number of gains in a given lineage. Similarly, the set of raw losses excludes introns lost in the given organism and also lost in all cousins or siblings (marked as “Other” in Figure 1B).
We used a probabilistic model to correct for these inaccuracies. Our model assumes that all loss and gain events occur independently and uniformly within each quintile. In particular, we assume Dollo's postulate (Dollo 1893): any introns that align to the same position must have a common ancestor (no “double gains”), as in Nei and Kumar (2000) and Rogozin et al. (2003). Our method differs from the Dollo parsimony method described in Farris (1977) and applied in Rogozin et al. (2003) in that we do not artificially minimize loss events by assuming that gains occurred at the latest possible point in evolution. It also differs in that we allow different branches of the phylogenetic tree to have different rates of loss and gain. We applied our method separately to each of the five positional quintiles for each organism other than the outgroup, A. nidulans.
First we estimate two types of intron loss rates. The organismal loss rate, q, is calculated by dividing the number of raw losses in an organism by the total number of introns present in at least one sibling and at least one cousin. This represents the fraction of introns in the parent that did not survive to the present day in that organism. For instance, the organismal loss rate in F. graminearum is given by
\eqalignnoq = (AM + AN + AMN) / ((AM + AN + AMN) + \cr(AFM + AFN + AFMN)) (1)
where AM, for example, represents the number of intron positions with an intron present in A. nidulans (A) and M. grisea (M) but absent from F. graminearum (F) and N. crassa (N).
The sibling loss rate, r is defined for a given organism as the fraction of introns in the parent that did not survive in any sibling. We define “sibling raw losses” for an organism as the number of introns that are present in the organism and at least one cousin but in no sibling. This quantity is then divided by the number of introns present in that organism and at least one cousin to give the sibling loss rate. For example, the sibling loss rate for F. graminearum is given by
r = (AF) / (AF + AFM + AFN + AFMN).
We next correct the raw gains for each organism. Raw gains include some introns that were in fact lost in all but one lineage. We use the loss rates to calculate the expected number of these multiple losses, m, and subtract this quantity from the raw gains to obtain “inferred gains.” To calculate m we first count B
0, the number of introns conserved in the organism and at least one sibling, but in no cousin. The quantities m and B
0 are related through the variablen
0, the number of introns present in an organism's parent but not in any cousin, by the equations
m = n0r(1 − q)
and
B0 = n0(1 − r)(1 − q).
This follows from our assumption of independent gains and losses. Thus, we can calculate the expected number of multiple losses as
m = B0r / (1 − r).
We use the loss rates to estimate the number of introns in each organism's parent. To do so, we estimate separately the number of parental introns present in at least one cousin n
1, and the number not present in any cousin n
0 (introduced above). To estimate the size of the set of parental introns present in at least one cousin, we first count the subset of these introns that are presently observable. An intron is in this set if it is present in at least one cousin and at least one sibling, or is present in at least one cousin and in the organism in question. We call this number of introns B
1. By the assumption that gains and losses are independent, we have
B1 = n1(1 − qr).
Using this relation and the one in equation 4 above, we calculate the number of introns in the phylogenetic parent as
Finally, we correct raw losses. Our definition of raw losses undercounts the true number by omitting those introns not conserved in at least one cousin and at least one sibling. Taking F. graminearum as an example, the true number of losses would also include some introns conserved in the patterns A, M, N, and MN. We calculate the number of inferred losses as n
total
q.
This method can be extended to any phylogenetic tree and to any organism with at least one cousin.
Abundance of Intron Gains
One immediate conclusion stemming from our analysis is the importance of intron gain. A summary of all raw and inferred gains and losses is shown in Figure 3. Substantial numbers of gained introns were observed in all three organisms—more than 100 independent inferred gains in each lineage, with over 200 in M. grisea (Figure 3B). The total numbers of gains that have occurred in each genome are likely to be substantially higher, since only predicted orthologs in all four species were considered, and roughly a third of the introns in these genes passed our quality filters. Differences in intron dynamics between lineages are also apparent, with the numbers of gained and lost introns approximately balanced in M. grisea and F. graminearum, but with roughly twice as many losses as gains in N. crassa (Figure 3D). It is thus apparent from these data that the process of intron gain plays a significant role in intron evolution.
1-Phosphoribosyl-5-Pyrophosphate Synthetase Genes Display Lineage-Specific Increases in Intron Gain Rate
A striking example of intron gain occurs in a set of putative orthologous 1-phosphoribosyl-5-pyrophosphate (PRPP) synthetase genes. These genes encode a widely conserved protein that catalyzes the production of PRPP, a precursor in the nucleotide biosynthesis pathway. In contrast to the majority of orthologs that displayed fewer than two gained introns, the set of PRPP synthetase genes displayed a total of 22 raw gains (Figure 5A, blue boxes) that passed our alignment quality filters: six in N. crassa, 14 in M. grisea, and two in F. graminearum. The number of raw gains in the PRPP synthetase genes in M. grisea and N. crassa was significantly higher (p < 3 × 10−22 and p < 4 × 10−9, respectively) than the average for other genes analyzed, resulting in unusually large numbers of introns in these genes (Figure 5B). In comparison, the numbers of introns in PRPP synthetase genes in available animal genomes were within the typical range for the respective organisms, e.g., five in C. elegans, and six in fruitfly, human, mouse, rat, and Fugu. Thus the rate of intron gain for the PRPP synthetase gene in some fungi is unusually high. This gene represents an extreme example of the impact of intron gain and illustrates the variability of gain rates in different lineages.
Figure 5 Intron Conservation in the PRPP Synthetase Gene
(A) Alignment of PRPP synthetase putative orthologs MG07148, NCU06970, FG09299, and AN1965. A black-edged rectangle indicates an intron position passing our quality filters, whereas an unedged gray rectangle indicates an intron position that was removed by our filter. Blue boxes mark raw intron gains, red boxes indicate raw intron losses, and gray boxes within black-edged rectangles highlight all other introns. We manually corrected an annotation error in the first intron of the last row of the alignment.
(B) Phylogenetic conservation pattern of introns in the PRPP sythetase gene. Each passing intron position was categorized as being present in A. nidulans (A), F. graminearum (F), M. grisea (M), N. crassa (N), A. nidulans and N. crassa (AN), F. graminearum and M. grisea (FM), or all four organisms (AFMN). There are no passing cases of conservation in three or four species. The number of introns in each category is shown with a purple line. The black error bar plot shows the mean and standard deviation for each category for all 2,008 ortholog sets after fitting to a Poisson distribution (see Materials and Methods). The number of introns in M. grisea and N. crassa is significantly higher, at the p < 1 × 10−9 level.
Fungal Introns Display Phase Bias, but Lack Observable Sequence Preference
For each in-group lineage (M. grisea,
N. crassa, F. graminearum), we determined the frequency of phase 0, 1, and 2 introns in the set of all intron positions (Table 1). In contrast to recent reports based on a much smaller sample size indicating that phase frequencies for extant fungal introns do not differ significantly from a uniform distribution (Qiu et al. 2004), our genome-wide dataset demonstrates a clear bias for phase 0 introns in each of the three fungal in-group lineages examined (p < 4 × 10−9 for N. crassa and p < 1 × 10−12 for M. grisea and F. graminearum; in Table 1, “all passing,” and similar biases were seen in the unfiltered set). The phase distributions of raw gains and raw losses for each of the three organisms are not significantly different from a uniform distribution at p < 0.01; however, the datasets for these subclasses were much smaller (Table 1). Finally, we examined the exon sequences flanking gained introns, and observed no clear sequence bias (Table 2).
Table 1 Intron Phase Distribution for Filtering and Conservation Classes
aSignificantly different from uniform distribution, at p < 0.01
bOne intron removed because of phase discrepancy across N. crassa, M. grisea, and F. graminearum.
Fg, F. graminearum; Nc, N. crassa; Mg, M. grisea.
Table 2 Exonic Nucleotide Composition near Introns
aFour nucleotides were extracted upstream and downstream of each intron in the specified organism
bFour nucleotides were extracted upstream and downstream of the orthologous site in the other two organisms, consistent with method of Qiu et al. 2004
Fg, F. graminearum; Nc, N. crassa; Mg, M. grisea.
Absence of 3′ Bias in Intron Losses
To determine whether the pattern of intron loss in these fungi might account for the observed bias in intron position, we examined the pattern of loss as a function of position within the gene (see Figure 3E). Contrary to what would be expected if intron loss primarily involved homologous recombination of poly-adenosine-primed reverse transcripts, the rate of intron loss tends to be lower, rather than higher, at the 3′ ends of genes. Moreover, the highest rates of intron loss occur in the middles of genes in all three organisms. We found no evidence that this pattern was affected by our filtering methods. These findings suggest either other mutational mechanisms (e.g., reverse transcription primed internally) or the presence of selective pressure to preferentially conserve introns near the 5′ and 3′ ends of genes.
Discussion
We developed a system that automatically identifies evolutionary and positional patterns of intron conservation on a genome-wide scale. The core of the system is a process for stringently filtering alignments of orthologous genes to exclude potential annotation or alignment errors. The result of the filtering process is a high-confidence set of aligned intron positions. Differences in intron conservation at each individual position can be characterized as gains or losses (or ambiguous) based on parsimony. However, this does not accurately account for the possibility of multiple gain or loss events. We have developed a probabilistic model that allows for multiple events, providing a corrected estimate of the total number of gains and losses within the dataset. Our probabilistic method allows for a more accurate assessment of rates of gain and loss. In our dataset, allowing for multiple events results in only modest corrections to the rates estimated using parsimony.
Our analysis demonstrates a significant role for intron gain over the past few hundred million years in the fungi analyzed. Previous analyses of specific gene families have provided evidence of specific instances of gained introns (Logsdon et al. 1998; Robertson 2000; Hartung et al. 2002; Qiu et al. 2004). However, the relative importance of intron gain versus loss is not well understood. Recent large-scale analyses have suggested that intron gain may play a predominant role in shaping gene structures (Qiu et al. 2004), although lineage-specific differences are apparent (Rogozin et al. 2003). In particular, intron gain appears to occur rarely if at all in mammalian genes (Roy et al. 2003). Our data suggest that intron gain is a significant driving force in the evolution of genes in fungi. In F. graminearum and M. grisea the number of introns gained was on par with the number lost and similar in magnitude to the number of introns gained in N. crassa.
The mechanisms underlying intron gain are not known. We analyzed the set of predicted intron gains for possible signatures that might shed light on this process. No statistically significant bias was detected in the positions of gained introns along the coding sequence (see Figure 3 data not shown). Similarly, no preferred insertion site sequence was detectable (Table 2), and no significant phase bias for gained introns was observed (see Table 1). The lack of an insertion site preference and absence of significant phase bias for gained introns in fungi is consistent with previous investigations and may set fungi apart from other organisms (Qiu et al. 2004).
Our data further indicate that intron gain can vary substantially between different gene families in a lineage-specific fashion. The PRPP synthetase gene is a particularly striking example, exhibiting significant increases in gained introns in two of the four lineages investigated. Moreover, the paucity of intron positions shared between N. crassa and M. grisea suggests the possibility of independent increases in gain rate in the two species. Alternatively, the apparent high intron gain rate exhibited by this gene may have arisen just prior to the last common ancestor of N. crassa and M. grisea. Although it is premature to speculate about possible mechanisms, one possibility is that a factor or factors responsible for intron insertion evolved to associate with the PRPP synthetase gene locus, transcript, or message at this point, leading to a higher rate of intron insertion in this gene.
Finally, our results do not support the mechanism commonly proposed to account for the 5′ positional bias of introns in intron-poor organisms (Mourier and Jeffares 2003). Contrary to what would be expected if intron loss primarily involved recombination of poly-adenosine-primed reverse transcripts, the rate of intron loss tends to be lower at the 3′ ends of genes. Instead, the highest rates of intron loss occur in the middles of genes in all three organisms. (This result is consistent with the results of Roy et al. (2003) in their analysis of intron evolution in mammals. Although their report describes only six instances of loss, in each case it was an internal intron.) The preference for internal introns may reflect a process of reverse transcription primed internally. Alternatively, there may be pressure to preferentially conserve introns near the 5′ and 3′ ends of genes. In particular, there is strong evidence for a functional role for the 5′-most intron in many genes. What remains clear is that the pattern of loss in these fungi over the last 330 million years cannot be explained solely by a mechanism involving 3′-end-primed reverse transcription of spliced messages. Instead, fungal intron dynamics appear to reflect a more complex interplay between intron gain and loss, an interplay that is likely to shape intron evolution in other eukaryotes.
Materials and Methods
Sequences and annotations
All sequences and annotations were taken from the Broad Institute Fungal Genome Initiative website (http://www.broad.mit.edu/annotation/fungi/fgi). The following datasets were used: A. nidulans (Assembly 1, 18 February 2003), N. crassa (Assembly 3, 1 February 2001), F. graminearum (Assembly 1, 11 March 2003), and M. grisea (Assembly 2, 18 July 2002).
Ortholog identification
A group of four proteins, one from each organism, was considered an ortholog set if each pair was a pairwise best bidirectional BLAST hit in the respective genomes, and all the BLAST hits overlapped by at least 60% of the length of the longest protein. This yielded 2,073 sets of orthologs (out of an average of 10,500 genes in the four organisms). We repeated our analysis, requiring that each best bidirectional hit also be the only BLAST hit in each genome (spanning 60% the length of the longest protein). This protocol yielded only 1,178 ortholog sets, but gave qualitatively similar results for intron gains and losses (Figure S1).
Ortholog alignment
The proteins in each ortholog set were aligned using ClustalW 1.82 (Chenna et al. 2003), and intron position characters were inserted into the alignments, using “0,” “1,” or “2” to indicate the intron phase. Phase 0 intron characters were inserted between the amino acids coded for by the codons adjacent to that intron, and phase 1 and 2 intron characters were inserted immediately following the amino acid coded for by the codon interrupted by the intron. If an intron was not present in all the sequences at a given position, special intron gap characters, were inserted in the other sequences in order to maintain the downstream amino acid alignment. A total of 9,352 intron positions were aligned. At only 28 (0.3%) of these positions were introns of different phases aligned, making it reasonable to ignore “phase shifting” in our analysis.
Alignment filtering
Regions of low alignment quality were eliminated with a filter that required at least 30% identity and 50% similarity in a window of ten residues on each side of the intron position. These parameters were determined following manual classification of a set of 181 randomly selected intron positions as “clearly homologous,” “ambiguous/possibly homologous,” or “non-homologous” (see Figure 2B). Using the parameters above, 92% of the homologous positions, 29% of the ambiguous positions and only 2% of the non-homologous positions passed the filter.
To further exclude likely annotation and alignment errors, intron positions were also filtered by eliminating positions adjacent to gaps in the amino acid alignment and by eliminating positions with nearby introns but low evidence of homology in the intervening sequence. It is possible that some of these positions may in fact reflect intron gain or loss events that occurred simultaneously with coding sequence insertion or deletion. However, removing these positions did not significantly impact our results, as the number of positions adjacent to gaps was only about one-tenth of the number of positions that passed the quality filter, and the introns removed did not have an apparent positional bias (see Figure 3A)
Statistical significance of high gain rate in PRPP synthetase
We modeled the number of gains in a particular organism as a Poisson distribution under two different null hypotheses. One null hypothesis was that the gains were spread uniformly across all genes. The other was that the number of gains in each gene was proportional to the length of the gene. In the first case the Poisson parameter λ is given by the total number of raw gains observed in that organism divided by the total number of ortholog sets ( p < 3 × 10−22 for M. grisea,
p < 4 × 10−9 for N. crassa, and p < 0.007 for F. graminearum). In the second case λ is given by the total number of raw gains observed in that organism multiplied by the length of that gene in amino acids and divided by the total number of amino acids in all genes in that organism ( p < 7 × 10−25 for M. grisea,
p < 3 × 10−10 for N. crassa, and p < 0.003 for F. graminearum). We reported the less significant of the two p-values in the results.
Analysis of intron gain phase and sequence preference
For each of the three in-group lineages, the frequency of phase 0, 1, and 2 introns was determined for five different datasets: for each class of conservation (conserved, raw gains, and raw losses), for all introns passing our filter, and for all introns in the ortholog set. The p-value for the significance of phase 0 bias was determined by the χ2 test with two degrees of freedom using equal expected phase frequencies. To detect sequence bias at intron insertion sites, we examined gained introns separately in F. graminearum,
M. grisea, and N. crassa. For each gained intron, we extracted four bases upstream and downstream of orthologous sites in the other two sequences, consistent with Qiu et al. (2004). The results are shown in Table 2.
Supporting Information
Figure S1 Intron Gains and Losses Inferred from Best-Only BLAST Hit Orthologs
Positional biases in intron gain, loss, and current distribution in three fungal genomes determined using orthologs predicted by a “bidirectional only hit” method. (A), (B), and (C) are roughly analogous to (D), (E), and (A), respectively, in Figure 3.
(78 KB DOC).
Click here for additional data file.
Table S1 Database of Alignments of All 1,447 Ortholog Sets with at Least One Passing Intron Position
Also available at http://genes.mit.edu/NielsenEtAl/.
(4.3 MB ZIP).
Click here for additional data file.
We thank S. Calvo, L.-J. Ma, and E. S. Lander for comments. This work was supported by grants from the National Institutes of Health, National Science Foundation, and United States Department of Agriculture (CBN, BB, and JEG) and the Burroughs Wellcome Fund (CBB).
Conflicts of interest. The authors have declared that no conflicts of interest exist.
Author contributions. CBN, BB, CBB, and JEG conceived and designed the experiments. CBN, BF, and JEG analyzed the data. CBN, BF, and JEG contributed materials/analysis tools. CBN, BF, CBB, and JEG wrote the paper.
Academic Editor: Ken H. Wolfe, University of Dublin
Citation: Nielsen CB, Friedman B, Birren B, Burge CB, Galagan JE (2004) Patterns of intron gain and loss in fungi. PLoS Biol 2(12): e422.
Abbreviations
PRPP1-phosphoribosyl-5-pyrophosphate
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Logsdon JM Tyshenko MG Dixon C D-Jafari J Walker VK Seven newly discovered intron positions in the triose-phosphate isomerase gene: Evidence for the introns-late theory Proc Natl Acad Sci U S A 1995 92 8507 8511 7667320
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| 15562318 | PMC532390 | CC BY | 2021-01-05 08:21:18 | no | PLoS Biol. 2004 Dec 30; 2(12):e422 | utf-8 | PLoS Biol | 2,004 | 10.1371/journal.pbio.0020422 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1556231910.1371/journal.pbio.0020427Research ArticleBioinformatics/Computational BiologyGenetics/Genomics/Gene TherapyPhysiologyNephrologyHomo (Human)A Transcriptional Profile of Aging in the Human Kidney Kidney AgingRodwell Graham E. J
1
Sonu Rebecca
2
Zahn Jacob M
2
Lund James
2
Wilhelmy Julie
3
Wang Lingli
4
Xiao Wenzhong
3
Mindrinos Michael
3
Crane Emily
2
Segal Eran
5
Myers Bryan D
1
Brooks James D
6
Davis Ronald W
3
7
Higgins John
4
Owen Art B
8
Kim Stuart K [email protected]
2
7
1Division of Nephrology, Stanford University Medical CenterStanford, CaliforniaUnited States of America2Department of Developmental Biology, Stanford University Medical CenterStanford, CaliforniaUnited States of America3Department of Biochemistry, Stanford University Medical CenterStanford, CaliforniaUnited States of America4Department of Pathology, Stanford University Medical CenterStanford, CaliforniaUnited States of America5Department of Computer Science, Stanford University Medical CenterStanford, CaliforniaUnited States of America6Department of Urology, Stanford University Medical CenterStanford, CaliforniaUnited States of America7Department of Genetics, Stanford University Medical CenterStanford, CaliforniaUnited States of America8Department of Statistics, Stanford University Medical CenterStanford, CaliforniaUnited States of America12 2004 30 11 2004 30 11 2004 2 12 e4279 5 2004 7 10 2004 Copyright: © 2004 Rodwell et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
A Global View of Gene Expression in the Aging Kidney
In this study, we found 985 genes that change expression in the cortex and the medulla of the kidney with age. Some of the genes whose transcripts increase in abundance with age are known to be specifically expressed in immune cells, suggesting that immune surveillance or inflammation increases with age. The age-regulated genes show a similar aging profile in the cortex and the medulla, suggesting a common underlying mechanism for aging. Expression profiles of these age-regulated genes mark not only age, but also the relative health and physiology of the kidney in older individuals. Finally, the set of aging-regulated kidney genes suggests specific mechanisms and pathways that may play a role in kidney degeneration with age.
A study of human aging in the kidney reveals similar changes in the transcriptional profile in cortex and medulla, suggesting that a common underlying aging process is taking place
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Introduction
Aging affects nearly all organisms and is a major risk factor in most human diseases. Recent work has begun to uncover molecular mechanisms that specify lifespan and to identify alterations in cellular physiology that occur at the end of life (Tissenbaum and Guarente 2002). For example, oxidative damage caused by the generation of free radicals in the mitochondria has been found to hasten aging by causing an accumulation of damaged cellular components (Droge 2003). Telomere shortening may also play a role in aging by preventing DNA replication and cell division in later years (Hasty et al. 2003). Genetic studies have identified many genes that play a role in specifying lifespan. For example, mutations in yeast sir2 (chromatin regulator), worm daf-2 (insulin-like growth factor receptor), fly methuselah (tyrosine kinase receptor), mouse p53, and the human Werner's syndrome gene (DNA helicase) cause dramatic changes in lifespan (Guarente and Kenyon 2000). Several aging mechanisms alter longevity in multiple organisms. For example, mutations in the gene encoding insulin-like growth factor receptor alter lifespan in worms, flies, and mice, indicating that an endocrine signaling pathway has a conserved role in aging (Hekimi and Guarente 2003).
Genetic studies have shown that aging can be slowed in mutants that are defective in a wide range of cellular processes (such as mitochondrial function, chromatin regulation, insulin signaling, transcriptional regulation, and genome stability). This indicates that aging is a complex process driven by diverse molecular pathways and biochemical events. As such, a powerful approach to study aging is to use systems biology, which allows a multitude of factors affecting aging to be analyzed in parallel. For example, DNA microarrays and gene expression chips have been used to perform a genome-wide analysis of changes in gene expression in old age. Extensive studies in Caenorhabditis elegans and Drosophila melanogaster have identified hundreds of age-regulated genes (Hill et al. 2000; Zou et al. 2000; Lund et al. 2002; Pletcher et al. 2002; Murphy et al. 2003). Several studies have described age-regulated genes in the muscle and brain of mice (Lee et al. 1999, 2000) and the retina and muscle of humans (Yoshida et al. 2002; Welle et al. 2003, 2004). These age-regulated genes may serve as markers of aging, enabling one to assess physiological age independently of chronological age. Analysis of the functions of these age-regulated genes has identified specific biochemical mechanisms that change toward the end of life.
A key question still unresolved is to what extent the mechanisms of aging are conserved between species with vastly different lifespans. Some studies suggest that similar mechanisms are involved in aging in many species. For example, caloric restriction extends lifespan in yeast, worms, flies, mice, and primates (Weindruch 2003). Additionally, signaling through the insulin-like growth factor pathway, chromatin regulation by sir2, and oxidative damage have each been shown to affect lifespan in diverse model organisms (Tissenbaum and Guarente 2002). Other studies emphasize that changes occurring at the end of life are unlikely to be evolutionarily conserved (Kirkwood and Austad 2000). In the wild, very few animals (including humans) survive to their maximal biological lifespan. Thus, the changes in physiology that occur in very old animals have minimal effects on the fitness of individuals, and are unlikely to be evolutionarily conserved. Therefore, aging is likely to be species-specific, and studies of old age in model organisms are unlikely to be relevant to humans.
We have begun our studies of human aging by focusing on the kidney, an organ that shows a quantifiable decline in function with age. One of the primary functions of the kidney is to remove toxins from the blood, which involves filtering plasma through specialized capillary beds (glomeruli) in the renal cortex. The primary function of the tubules within the medulla is to concentrate or dilute the urine so as to maintain fluid balance. The major age-related change in kidney function is a 25% decline in the glomerular filtration rate starting at age 40 (Hoang et al. 2003). The ability of the medulla to concentrate urine declines progressively with age.
In this study, we present a molecular portrait of the aging process in the human kidney by analyzing gene expression as a function of age on a genome-wide scale. We show that age regulation is similar in the cortex and the medulla, and that age-regulated genes in the kidney are broadly expressed. We show that the expression profiles of age-regulated genes correlate well with the morphological and physiological state of the kidney in old age. Finally, we analyze the set of age-regulated genes to identify specific metabolic processes and cellular functions that change as a function of age, and discuss their possible roles in specifying the functional lifespan of the kidney.
Results
To procure material for analyzing changes in gene expression with age in the human kidney, we obtained kidney samples from normal tissue removed at nephrectomy for either removal of a tumor or for transplantation from 74 patients ranging in age from 27 to 92 y (Tables S1 and S2). We dissected each of the 74 kidney samples into cortex (72 samples) and medulla (62 samples) sections, isolated total RNA from each section, synthesized biotinylated complementary RNA (cRNA), and hybridized the labeled cRNA to Affymetrix high-density oligonucleotide arrays (HG-U133A and HG-U133B, containing a total of 44,928 probe sets corresponding to approximately 33,000 well-substantiated human genes). The level of expression for each gene was determined using DChip (Zhong et al. 2003), and the gene chip data were entered into the Stanford Microarray Database ( http://genome-www5.stanford.edu/) . Using our dataset, the expression level for every gene as a function of age could be plotted. For example, the expression of CDO1 (which encodes a cysteine dioxygenase type 1 protein) tended to increase with age. There was also variation between subjects and between the cortex and the medulla (Figure 1A). Nearly all of the variation represents true differences between samples, as very little variation was observed when we performed repeat hybridizations using the same tissue sample (data not shown).
Figure 1 Age-Regulated Genes
(A) Shown are expression levels for gene CDO1. White and black circles represent expression from cortex and medulla, respectively. The y-axis indicates log2 (expression level), and the x-axis indicates age of patient (years). Dotted and solid lines indicate best fit slopes for the cortex and medulla values, respectively.
(B) For every gene, we calculated a one-sided p˜
-value that its expression changes with age. Shown is a histogram representing all of the genes represented by the Affymetrix DNA chip. Genes that decrease with age have p˜
-values near zero, and genes that increase with age have p˜
-values near one. If there were no age-regulated genes (i.e., the true βkj = 0 for every gene j), then the histogram of p˜
-values would be flat (i.e., have a uniform distribution on the interval from zero to one). The x-axis shows the p˜
-value, and the y-axis shows the number of genes with that p˜
-value. There are 985 genes with a p-value less than 0.001.
We used a linear regression model to identify genes that showed a statistically significant change in expression with age (i.e., were age-regulated). We saw large differences in expression between tissue types and between the sexes. These differences were of similar magnitude for both young and old subjects, so that aging in one tissue (or sex) typically ran parallel to aging in the other (as seen in Figure 1A). Our linear regression model allowed for these parallel trends; reasons for arriving at such a model are given below. Mathematically, our model takes the form
In equation 1, Yij is the base 2 logarithm of the expression level for gene j in sample i, Agei is the age in years of the subject contributing sample i, Sexi is one if sample i came from a male subject (and zero for female), Tissuei is one if sample i was a medulla sample (and zero for cortex), and ɛij is a random error term. The coefficients βkj for k = 0, 1, 2, and 3 are values to be estimated from data. Our primary interest is in β
1j
, which describes how quickly the expression of gene j changes with age, withβ
1j = 0 for genes with no linear age relationship.
In model 1 and others that we considered, the coefficients were estimated by least squares. The estimated values β^kj
can differ from zero, even when the true coefficient is zero. We judged statistical significance through p-values, where a value of p
ij near zero corresponds to a large absolute value |β^kj|
unlikely to have arisen by chance. Such p-values do not distinguish genes that increase with age from those that decrease with age. We also use one-tailed p-values, written p˜kj
, taking values near zero to be significantly decreasing trends and those near one to be significantly increasing trends (see Materials and Methods).
To make p-values comparable over genes, it is essential to use the same model for all genes. Before settling on the common model 1, we considered an alternative that allowed a quadratic trend in age. The p˜
-values for the quadratic coefficient (not shown) gave no reason to suspect that a curved relationship was needed. Similarly, a piecewise linear age relationship (with bends at ages 50 and 70) was not significantly better than a linear one. Large and statistically significant differences in expression were found for the two tissue types, and so the tissue type was included in equation 1. Incorporating tissue type into the model reduces the estimate of the noise variance, leading to greater power for detecting an age relationship. Similarly, a small number of genes were found to have significantly different expression between sexes. Seven genes were found to have a difference at p < 0.001 for both sex and age.
We performed a genome-wide scan for genes that changed expression with respect to age. Age-regulated genes can be identified by plotting p˜
-values for age based on model 1 (Figure 1B). Genes that significantly decrease in expression with age appear in a peak on the left, while those whose expression increases with age are in a peak at the right. Using model 1, we found 985 genes that change with respect to age (p < 0.001), which is considerably greater than would be expected by chance (approximately 45 from a total of 44,928 genes). Of these, 742 genes increase expression with age and 243 decrease expression with age (Table S3).
Most of our samples were taken from patients that underwent nephrectomy for various medical reasons (see Table S1). We evaluated whether pathology, medical history, or medication might be factors that confounded our aging analysis. For example, if old people tend to be hypertensive more often than young, then genes that respond to hypertension may appear to be age-related.
We identified 20 different medical and other factors that might potentially confound our study, including race, blood pressure, diabetes, and type and size of tumor present in the kidney (see Table S1). Fourteen factors (such as diabetes or proteinuria) affected less than ten patients, making it unlikely that they could account for age-related change in gene expression in the 74 patients analyzed. Six factors occurred in ten or more patients (non-white race, two types of tumors, size of tumor, and hypertension), but it is unlikely that these affected our aging study for the following reasons.
First, with the exception of transitional cell carcinoma, none of the other factors were skewed with respect to age, and would not be expected to bias gene expression in an age-related fashion (Figure S1).
Second, the two types of tumors (renal cell carcinoma and transitional cell carcinoma) were localized to an isolated region of the kidney. Our normal samples were obtained from the region of the kidney furthest from the carcinoma, were not directly contaminated with cancer cells, and appeared normal histologically (see Materials and Methods). This procedure for obtaining kidney samples has been used previously to profile gene expression in normal kidney (Higgins et al. 2004) and as a normal control in a kidney cancer study (Higgins et al. 2003).
Third, we used regression models to directly test whether our aging studies were affected by seven medical factors: renal cell carcinoma, transitional cell carcinoma, size of tumor, hypertension, systolic blood pressure, diastolic blood pressure, and diabetes mellitus. For renal cell carcinoma, we used a regression model predicting expression from age, sex, tissue type, and a zero/one variable indicating whether the sample came from a patient with renal cell carcinoma or not. The result gave a p-value for whether renal cell carcinoma affected each of the 44,928 genes present on the Affymetrix DNA chip. The smallest p-value was 0.00013. We would expect to see almost six such p-values by chance alone. This result indicates that the presence of renal cell carcinoma does not significantly affect the expression of any gene in the normal tissue from the same kidney, compared to normal tissues taken from kidneys without renal cell carcinoma.
Next, we plotted the results using only the age-regulated genes, to investigate whether adjustments for renal cell carcinoma could affect their change in expression with respect to age. We used one regression model that included a renal cell carcinoma term and another model that did not have the term. We then selected genes that showed statistically significant (p < 0.001) age regulation using either of these models. Renal cell carcinoma does not significantly affect the age slopes for these genes (Figure S2A), indicating that this medical factor has little effect on age-related gene expression.
We repeated the regression analysis for six other factors that might confound our results (transitional cell carcinoma, size of tumor, hypertension, systolic blood pressure, diastolic blood pressure, and diabetes mellitus). The regression slopes changed very little with and without these factors, indicating that these factors do not strongly affect our analysis of age regulation (Figure S2B–S2G).
Fourth, five of the samples were from kidneys that did not have tumors, and two of these were from donor kidneys used for transplantation that had no associated pathology at all. The expression profile from these five patients was similar to the profile from other samples used in our study. In summary, it is unlikely that these disease and medical factors have confounded our analysis of age-regulated changes in gene expression.
Changes in the expression for some of the 985 age-regulated genes may directly reflect the aging process in the kidney; these genes would serve both as aging markers and provide clues about molecular mechanisms for aging in the kidney. Other changes may result from an age-related change in the relative proportion of cell types within the kidney, such as would result from increased infiltration of immune cells with age. Finally, the expression changes may reflect the downstream response of the kidney to an age-related process elsewhere, such as would result from age-related changes in blood pressure or vascular supply.
Common Mechanisms of Aging in the Cortex and Medulla of the Kidney
Since the cortex and medulla contain different cell types and have distinct functions, it was of interest to test whether they age similarly. It is possible that the pattern of degeneration in a particular cell type reflects those metabolic pathways that are used most heavily by that cell. For example, there could be deterioration in cell adhesion in glomerular epithelial cells that form part of the filtration barrier in the cortex, while there could be an age-related decline in ion traffic or water flow across the apical or basolateral membranes of tubular epithelial cells in the medulla. Alternatively, distinct cell types could show a common pattern of age-related decline involving pathways common to all cells, such as protein synthesis and mitochondrial function. This degeneration of core cellular processes would affect every cell function, including filtration by glomerular epithelial cells and water and solute reabsorption by tubular epithelial cells.
To test whether age-related gene expression changes are different in cortex and medulla, we considered a model in which a term of the form β
4j × Tissue × Age was added to the model in equation 1. In such a model, the change in expression with age is linear within each tissue type, but the slope in the medulla is larger than that in the cortex byβ
4j. Figure 2A shows the histogram of the p˜4j
-values. Genes showing tissue-specific slopes would appear in peaks on the left and right. The figure shows neither of these peaks, indicating there is no statistically significant difference in aging between the two tissue types.
Figure 2 Similar Age-Regulation in Cortex and Medulla
(A) For every gene, we calculated a p˜
-value that there is a Tissuei ×Agei effect, and plotted the results in a histogram. Genes that show different age regulation in the cortex or the medulla would be contained in peaks on the left and right parts of the histogram. The figure shows that the number of genes that have different expression levels in the cortex and medulla is about the same as or less than would be expected by chance. The x-axis shows one-sided p˜
-values for Tissuei ×Agei, and the y-axis shows the number of genes with that p˜
-value. There is a systematic under-representation of the edge regions compared to a random sample of uniform random variables because of correlations among the 44,928 p˜
-values computed from 133 samples.
(B) To show whether aging in the cortex and the medulla is similar, we selected age-regulated genes in the cortex and calculated the one-tailed p˜
-value for age effects in the medulla. The histogram shows these selected p˜
-values. The spike at the right shows genes that increase with age in the medulla. Those genes also increased with age in the cortex.
(C) Shown is a scatterplot of all 684 genes that are age-regulated in either the medulla or the cortex (p < 0.001). The y-axis is the slope for the medulla of the expression change with respect to age, and the x-axis is the slope for the cortex. The solid line is the least squares line, with a slope of 0.58. The dotted line has a slope of one and passes through the origin.
(D) Same as (C) but for 22 genes that are age-regulated in both the cortex and the medulla (p < 0.001).
To further investigate coordinate aging in the cortex and medulla, we searched for age-regulated genes in each of these tissues independently, and then tested whether age-regulated genes in one were also age-regulated in the other. Specifically, to find age-regulated genes in the cortex, we fit the model
using the cortex samples only. To find age-regulated genes in the medulla, we fit the model
using only the medulla samples. We found 634 genes in the cortex samples and 72 genes in the medulla samples that showed significant changes in expression with age (p < 0.001).
Having identified age-regulated genes in the cortex, we next examined whether they were also age-regulated in the medulla. Figure 2B shows the p˜
-values for change with age in the medulla samples, for those genes that are age-regulated (p < 0.001) in the cortex samples. If aging in the medulla were unrelated to aging in the cortex, we would expect to see a flat histogram. The actual histogram has a strong peak of genes on the right, indicating that significantly age-regulated genes in the cortex tend to also be significantly age-regulated in the medulla. Of the 634 genes that increased expression with age in the cortex, 22 also increased expression with age in the medulla, compared with the 0.6 genes expected at p = 0.001. We obtained similar results when we took the converse approach, first selecting the 72 age-regulated genes in the medulla, and then testing whether they were also age-regulated in the cortex (data not shown).
Next, we compared the slope of expression with respect to age in the cortex to that in the medulla (Figure 2C). The results show a strong correlation between age coefficients in cortex and medulla. For the 684 genes age-regulated in at least one of the tissue types, the age coefficients had a correlation of r = 0.487. Models 2 and 3 allow us to investigate whether the cortex and medulla age at the same rate as specified in model 1. For the 22 genes that are significantly age-related in both tissues, the age coefficients have a high correlation (r = 0.96), and the slopes themselves are numerically close (Figure 2D). We found a small mean absolute difference in slopes of 0.00185 (log2 expression per year), corresponding to only a 6% divergence in expression over 50 y. Given the strong similarities in the aging profiles of these two tissue types, we are able to increase the statistical power of our analysis by pooling the cortex and medulla datasets (resulting in model 1).
Increased Expression of Immune Genes in the Kidney in Old Age
We examined the list of 985 age-regulated genes, and immediately found evidence for increased expression of genes from immunocytes. Many of the 985 age-regulated genes are expressed specifically in B cells (e.g., immunoglobulin mu, kappa, and lambda), T cells (e.g., T cell receptor beta), or neutrophils (e.g., neutrophil cytosolic factors 1 and 4) (see Table S3). Nearly all of these immune genes increase expression with age. These results suggest that there are increased numbers of immune cells in the kidney in old age, resulting in an age-related increase in abundance in all genes that are expressed specifically in these cells. Immune function is known to decline with age, and the increased numbers of immunocytes in the kidney might compensate for decreased function in individual immune cells, either for immune surveillance or for responding to low levels of inflammation occurring normally. In addition to increased cell numbers, the apparent increase in expression of the immune genes could also be due to increased expression within the immune cells themselves.
Immunohistochemical experiments using antibodies directed against markers specific for B cells, T cells, or neutrophils showed that the kidney samples contained a small proportion of immune cells (less than 1%) in sporadic clusters scattered throughout each section (data not shown). The number of immune cells varied greatly from section to section, and thus it was not possible to use immunohistochemistry to confirm a quantitative increase in the numbers of immune cells in the kidney with age.
If the number of immune cells increases with age in our kidney samples, then any gene showing an age-related increase in expression might do so because it is expressed in immune cells and not because it is age-regulated in the kidney. As immune cells comprise only a small fraction of the kidney sample, age-regulated genes that are expressed at higher levels in the kidney than the blood are likely to be expressed in kidney cells themselves. To compare gene expression levels between the blood and the kidney, we purified RNA from whole blood from five new individuals, prepared labeled cRNA, and then hybridized it to Affymetrix gene chips in the same manner as before. We computed the log2 of the expression level for each gene, and then calculated an average expression level for the blood (five samples) and the kidney (134 samples). Of the 985 genes that change expression with age, 538 are expressed at higher levels in blood cells than in the kidney samples. Age-related changes in the RNA abundance of these genes may reflect either changes in the fraction of immune cells in the kidney or age-related changes in expression in kidney cells. The remaining 447 are expressed at higher levels in the kidney than in whole blood, and age regulation of these genes is likely to reflect expression changes in kidney cells themselves (Table S4). Of these 447 genes, 257 have increased expression levels in old age (age-induced) and 190 have decreased expression levels (age-repressed) (Figure 3).
Figure 3 Expression of the 447 Genes as a Function of Age
Rows correspond to age-regulated genes, ordered from most highly induced to most highly repressed. Columns correspond to individual patients, ordered from youngest to oldest. The age of certain patients is shown for reference. Left panel refers to data from cortex samples, and right panel depicts data from medulla samples. The first row shows the chronicity index (ChI; morphological appearance and physiological state of the kidney),from blue (healthiest) to yellow (least healthy) as indicated in the scale bar. Key genes discussed in the text are marked. Scale shows log2 of the expression level (Exp). A navigable version of this figure can be found at http://cmgm.stanford.edu/~kimlab/aging_kidney/explorer.html.
Age Regulation Compared to Developmental Regulation
Aging is thought to be caused by slow degeneration of the transcriptome (the entire set of genes expressed in a tissue), rather than a qualitative change in expression, as occurs during tissue specification. As such, changes in gene expression associated with aging should be less than expression differences between different types of tissues. To confirm this idea, we compared the magnitude of gene expression differences due to differentiation (cortex versus medulla) to those due to aging. We used the same approach as before to evaluate differences in expression in cortex versus medulla on a genome-wide scale. For every gene, we calculated the p-value for differential expression in the cortex and the medulla, and plotted the results in a histogram (Figure 4). Genes contained in the peak on the right are more abundant in the medulla whereas genes in the peak on the left are more abundant in the cortex. There were 23,322 genes that were differentially expressed between the cortex and medulla (p < 0.001), indicating that regulation of expression due to differentiation (between the cortex and medulla) is much greater than that related to aging. This result is consistent with the idea that aging results from a slow degeneration of a core transcriptome in the cortex and the medulla of the kidney.
Figure 4 Differential Expression in the Cortex and the Medulla
For each gene, we calculated a p˜
-value for expression differences in the cortex versus the medulla. Shown is a histogram of these p˜
-values. Genes enriched in the cortex are in a peak on the left, and genes enriched in the medulla are in a peak on the right. The x-axis indicates p˜
-value, and the y-axis indicates number of genes.
Majority of Age-Regulated Genes in the Kidney Are Expressed Broadly
To address whether different organs have distinct or common aging profiles, we analyzed whether the 447 age-regulated genes in the kidney were expressed specifically in the kidney or broadly in many tissues. If the kidney has its own specific pattern of aging, one might expect that the set of 447 aging-regulated genes would be enriched for those expressed specifically in the kidney, such as genes that have direct roles in forming the filtration barrier or in regulating ion or water reabsorption. If there is a common profile for aging shared among tissues, one might expect that most of the list of 447 aging-regulated genes would be expressed in many tissues.
We determined the level of expression of the age-regulated genes in different tissues using data from a previous study reporting a genome-wide profile of gene expression in 26 different human tissues with Affymetrix gene arrays (Su et al. 2002). Of the 447 aging-regulated kidney genes, 227 are represented in the previous work. Nearly all of these have general, rather than kidney-specific, expression patterns; specifically, we calculated the median expression level from all tissues and compared this to the average expression level from the kidney samples. We found that only seven of the 227 aging-regulated genes were enriched in the kidney more than 2-fold compared to the median level from all tissues (Figure 5). The observation that nearly all of these 227 age-regulated genes are expressed in many tissues suggests that they act in common cellular pathways. Altered expression of these genes in old age may weaken these common functions, subsequently leading to physiological decline of kidney-specific functions.
Figure 5 Developmental Profile of the Age-Regulated Genes
Shown are the log2 of the expression levels for 227 age-regulated genes in 26 human tissues, using data from Su et al. (2002). Rows correspond to genes, columns correspond to human tissues. a, kidney; b, cerebellum; c, whole brain; d, cerebral cortex; e, caudate nucleus; f, amygdala; g, thalamus; h, corpus callosum; i, spinal cord; j, whole blood; k, testis; l, pancreas; m, placenta; n, pituitary gland; o, thyroid gland; p, prostate; q, ovary; r, uterus; s, salivary gland; t, trachea; u, lung; v, thymus; w, spleen; x, adrenal gland; y, liver; z, heart. Scale shows log2 of the expression level. A navigable version of this figure can be found at http://cmgm.stanford.edu/~kimlab/aging_kidney/explorer.html.
Molecular Markers for Physiological Aging
The expression levels of these 447 age-regulated genes constitute a molecular profile of aging, and we can examine the expression profile of individual patients to observe how they compare to the average for their age group. Older individuals tended to express age-induced genes at higher levels and age-repressed genes at lower levels than younger individuals. However, certain individuals had unusual expression profiles, in which genes were expressed at levels more typical of a different age group. For example, patient 81 was 78 y old but had an expression profile as though she were older (see Figure 3). Her kidney showed very high levels of age-induced genes and very low levels of age-repressed genes. Patient 95 was 81 y old, with an expression profile similar to patients 30 or 40 y younger.
Do the molecular gene expression profiles correlate with the physiological ages of the kidney samples? That is, does patient 81 have a kidney showing excessive age-related damage and does patient 95 have a kidney with unusually good health? To answer these questions, we determined the morphological and physiological states of the kidneys from each of the patients by examining histological stains. As people grow older, there is a general decline in the morphological appearance of the kidney: (1) the glomeruli lose their structure and their capillaries are replaced with fibrous tissue (glomerular sclerosis), (2) the tubules collapse and atrophy, and the interstitial space between them widens and scars (tubular atrophy/interstitial fibrosis), and (3) there is a thickening of the innermost layer of the arteriole wall due to the accumulation of hyaline material (arterial intimal hyalinosis). We gave three scores to each kidney section corresponding to the appearance of the glomeruli, the tubules, and the arterioles. Scores ranged from zero for normal appearance for youthful patients to four for an advanced state of glomerular sclerosis, tubular atrophy/interstitial fibrosis, or arterial intimal hyalinosis (see Table S1). We then added the glomerular, tubular, and arteriolar scores together to form a combined score ranging from zero (best) to 12 (worst), termed the chronicity index. The chronicity index is a quantitative estimate of the morphological appearance and physiological state of the kidney for each of the patients (see Table S1). Figure 6 shows an example of a kidney in good condition from patient 40 (29 y old with a chronicity score of zero) and a kidney showing age-related morphological decline from patient 62 (84 y old with a chronicity score of ten). As expected, the chronicity index shows a strong positive correlation with age showing that morphology and function tend to be worse for older subjects (Figure 7).
Figure 6 Chronicity Index of Kidney Samples
Histology from patient 40 is shown on the left, demonstrating a normal glomerulus (G), tubules and interstitial space (T), and arteriole (A), respectively (chronicity score of zero). Histology from patient 62 is shown on the right, demonstrating glomerulosclerosis (g), tubular atrophy and interstitial fibrosis (t), and arterial intimal hyalinosis (a), respectively (chronicity score of ten). Hematoxylin and eosin staining of paraffin-embedded sections.
Figure 7 Chronicity Index Increases with Age
Shown is the chronicity index versus age for most of the kidney samples used in this study. The line shows the least squared fit through the data points.
We then compared the chronicity index to the gene expression profiles of the 447 age-regulated genes as a function of age (see Figure 3). In general, we found that the gene expression profiles correlated well with the chronicity index. Patients with expression profiles normally associated with people much older also had a high chronicity index; for example, the expression profile of patient 81 was similar to that of patients who were much older, and the chronicity index was also unusually high for the patient's age. Conversely, patients with expression profiles normally associated with younger people tended to have a low chronicity index for their age, such as patient 95. Although the 447 age-regulated genes were selected solely on the basis of their change with chronological age, these results indicate that their expression profiles are able to predict patients that have kidneys exhibiting unusual health or abnormal degeneration for their age. Thus, the 447 age-regulated genes can be used as molecular markers for physiological decline in the kidney during aging.
Age-Regulated Genes in the Kidney
Some of the 447 age-regulated genes may be involved in either causing or preventing aging in the kidney, whereas expression changes for others may be a consequence of age-related cellular changes. A candidate from our list that might promote age-related decline is mortalin-2 (which encodes Heat Shock Protein 70), which decreases expression in the kidney in old age. Heat shock proteins act as protein chaperones, and likely function to counteract cell senescence by alleviating the accumulation of damaged proteins in old cells. In human fibroblasts, overexpression of mortalin-2 extends lifespan in vitro (Kaul et al. 2003). In the nematode C. elegans, overexpression of mortalin or HSP-16 (a related heat shock protein) extends longevity, and several genes encoding heat shock proteins decrease expression in old age (Lund et al. 2002). Reduced expression of mortalin-2 in old human kidneys could increase the accumulation of denatured proteins and thereby reduce general cellular function.
A gene from our list that might function to prevent aging is the gene encoding insulin-like growth factor receptor, which decreases expression in old age. Loss-of-function mutations in this gene result in extended longevity in worms, flies, and mice (Tissenbaum and Guarente 2002). This observation suggests that decreased expression of this gene during normal aging might help prolong the functional lifespan of human kidneys.
We examined the list of 447 age-regulated genes for functional groups showing a consistent change with age. One group includes genes involved in the formation of the extracellular matrix, which show a consistent increase in expression in old age. Seven age-regulated genes encode proteins known to play key roles in maintaining epithelial polarity (three types of claudins, two cadherins, occludin, and a cell adhesion molecule), all but one of which increase expression in old age (see Table S4). Forty-nine age-regulated genes encode protein components of the extracellular matrix, all but four of which increase expression in old age. In the kidney, the extracellular matrix could play a key role in governing the filtration of blood via the basement membrane, a capacity that declines with age. The observation that genes involved in forming the extracellular matrix increase expression in the kidney with age may be directly relevant to the age-related decline in glomerular filtration rate.
Another functional group is a set of 11 genes encoding ribosomal proteins, all of which increase expression with age. Protein synthesis rates are known to decline as animals grow older, and increased expression of these ribosomal protein genes may serve to offset this.
Changes in the expression of regulatory genes with age may have particularly strong effects on kidney metabolism and function, since these changes are likely to initiate cascades of changes in downstream genes. We examined our list of 447 age-regulated genes for those that are likely to function as regulatory genes. Of the 447 age-regulated genes, 15 encode transcription factors and 51 encode proteins that are part of signaling pathways.
Age-Regulated Genes Enriched in the Glomeruli
As filtration of the blood takes place in the glomerulus, age-regulated genes that are enriched in the glomerulus may be especially important for understanding how kidney function declines with age. We identified genes enriched in the glomerulus using data from a previous study, in which cDNA microarrays were used to compare expression levels in the glomeruli relative to the rest of the kidney (Higgins et al. 2004). Of the 447 genes identified in our study, 213 were represented on the cDNA microarrays in the previous experiment, and 19 were enriched greater than 2-fold in the glomeruli relative to total kidney (Table S5). These included four genes that encode proteins involved in the formation of the extracellular membrane (a type 5 collagen, alpha-2 macroglobulin, and two tissue inhibitors of met-alloproteinase), all of which increase expression with age.
Discussion
Old age is associated with a functional decline in a myriad of molecular and cellular processes. To gain a global perspective of the diverse pathways that change with age, we performed a whole-genome analysis of gene expression as a function of age for kidney samples from 74 patients ranging in age from 27 to 92 y. Many factors affect gene expression in addition to age, including variability between individuals, between different tissues within the kidney, and between sexes. The large number of samples in our dataset provided good power for identifying age-regulated genes in noisy data despite small changes in expression, and allowed us to use a statistical linear regression model to identify 985 genes that change expression with age.
The results from this work show that transcriptional differences between young and old individuals involve an accumulation of small changes in expression from many genes, rather than resulting from large expression changes in a small number of genes. This observation suggests that functional decline in old age is not the result of the complete failure of a small number of cellular processes. Rather, it is the slight weakening of many pathways that cumulatively causes a significant decrease in cell function. Studying aging by analyzing one pathway at a time is difficult, because any single pathway might show only a small change with respect to age and might contribute only a small amount to the overall functional decline in old age. By contrast, functional genomics is a powerful approach to study aging, because many genes can be simultaneously scanned in parallel for small changes in expression.
Although the cortex and medulla are comprised of different types of cells and perform different physiological functions, our results suggest that they share a common mechanism for aging. Previous experiments have characterized changes in expression for human fibroblasts, muscle, and the retina with age (Ly et al. 2000; Yoshida et al. 2002; Welle et al. 2003, 2004). We plotted the expression levels of the 985 aging-regulated genes found in this work against the dataset of aging in muscle (Welle et al. 2003), and found that these genes did not show much age regulation in muscle. Specifically, the Pearson correlation (r) of the regression slopes for these 985 genes was only 0.085 between the kidney and muscle aging experiments and hence accounts for only 0.0072 of the variance between these two tissues (Figure S3). It is unclear whether this amount of correlation is biologically relevant. The small sample size used in the study of aging in human muscles might have limited our ability to detect similarities in aging in the two organs. It will be important to use a larger sample size of muscle tissues in future experiments to discern common patterns of age regulation in the kidney and the muscle with higher resolution.
Aging has been best studied in model organisms, and it is thus of great interest to discern whether aging in these species is similar to the aging process in humans. Previous studies have reported gene expression changes associated with old age for worms, flies, and several tissues from mice (Lee et al. 1999, 2000; Hill et al. 2000; Zou et al. 2000; Lund et al. 2002; Pletcher et al. 2002; Murphy et al. 2003). We found no correlation between age regulation in human kidney and age regulation in either worms or flies (Figure S4).
Although our analysis did not show evidence for evolutionary conservation of age regulation, a previous study suggested that there is a small overlap in age-regulated gene expression between flies and worms (McCarroll et al. 2004). However, most of the similarities occured in young or middle-aged animals, rather than old animals. There is thus little evidence for evolutionary conservation of changes in gene expression in old age, emphasizing the need to elucidate mechanisms of aging using human subjects themselves and not model organisms.
Many of the age-regulated genes in the kidney may change in response to declining kidney function. Functional decline of the kidney with age varies between individuals, and these genes could be used as diagnostic markers to evaluate levels of kidney function in older patients. This could provide invaluable information in understanding the clinical course of kidney aging and the suitability of using older kidneys in organ transplants. Other genes may be directly regulated by aging per se, and these genes could pinpoint mechanisms that play key roles in the aging process itself.
Materials and Methods
Samples
Normal kidney samples were obtained either from biopsies of donor kidneys for transplantation or from nephrectromy patients (with informed consent) in which the pathology was localized and did not involve the part of the kidney sampled. Key factors from the medical record for each patient used in this study are listed in Table S1, and include sex, race, age, blood pressure, pathology, medications, serum creatinine, and urinary protein concentrations. Kidney tissue was harvested meticulously with the intention of gathering normal tissue uninvolved in the tumor. Tissue was taken from a point as far away from the tumor as possible. Any samples that showed evidence of pathological involvement or in which there was only tissue in close proximity to the tumor were discarded. Kidney sections were immediately frozen on dry ice and stored at −80 °C until use. The same harvesting sources and techniques have been used previously to profile expression in normal kidney (Higgins et al. 2004) and to provide normal controls in a study on kidney cancer (Higgins et al. 2003).
Histology
Frozen tissues were placed in cryomolds, embedded in Cryo Tissue Tek O.C.T. Compound (Sakura Finetek, Torrance, California, United States) and cut into 4-μm sections (Leica Microsystems, Wetzlar, Germany). Sections were stained with hematoxylin and eosin, and then histologically evaluated to exclude samples showing abnormal histology. Histology slides were also marked into two main functional sections, the cortex and medulla, to help aid in accurate dissection of these two areas. We reviewed radiological findings for all tumors and histology for all slides. We excluded any cases in which radiological imaging, gross examination at the time of resection, or histological review of the removed tissue indicated that there might be tumor involvement of the normal areas. Cases with incomplete or unclear medical records were excluded from this study.
RNA isolation
Frozen kidney tissue samples were dissected into cortex and medulla sections. Portions were weighed (0.05–0.75 g), cut into small pieces on dry ice, and then placed in 1 ml of TRIzol Reagent (Invitrogen, Carlsbad, California, United States) per 50–100 mg of tissue. The tissue was homogenized using a PowerGen700 homogenizer (Fisher Scientific, Pittsburgh, Pennsylvania, United States), and the total RNA was isolated according to the TRIzol Reagent protocol.
High-density oligonucleotide arrays
A standard protocol designed by Affymetrix (Santa Clara, California, United States) for their HG-U133A and HG-U133B high-density oligonucleotide arrays was slightly modified by the Stanford Genome Technology Center (Stanford, California, United States), and all samples were processed in their facility (see Protocol S1). Eight micrograms of total RNA was used to synthesize cRNA for each sample, and 15 μg of cRNA was hybridized to each DNA chip. The samples were done in random order with respect to tissue type and age.
Microarray data normalization and analysis
Using the dChip program (Zhong et al. 2003), microarray data (.cel files) were normalized according to the stable invariant set, and gene expression values were calculated using a perfect match model. All arrays passed the quality controls set by dChip. All of the Affymetrix data are available at the Stanford Microarray Database ( http://genome-www5.stanford.edu/) and at the Web site http://cmgm.stanford.edu/approximatelykimlab/aging_kidney/. The Affymetrix probe IDs and the locus link IDs for the genes discussed in the paper are in Tables S3–S5. The accession numbers for all genes on the Affymetrix arrays can be obtained from the Stanford Microarray Database.
Regression models and p-values
The p-values we use are based on t-tests from standard linear regression theory. Under the hypothesis H0 that βkj = 0, the estimated coefficient β^kj
is a random variable. The least squares value is a particular number, β^LS
kj
. The p-value measures the extent to which the least squares value is surprisingly large assuming H0 holds. Specifically, the two-tailed p-value is
and the one-tailed p-value we use is
Sometimes p˜kj
is employed to test H0 against an alternative hypothesis of βkj < 0. We use it because it distinguishes between significant increasing and significant decreasing coefficients. Under H0, the distribution of p is U(0,1), and so is that of p˜
. Numerically, the equation
holds.
The t-test is derived under an assumption of normally distributed errors. The data showed estimated errors with heavier than normal tails. The t-test is known to be robust against heavy-tailed errors.
A linear regression is more appropriate for these data than is an analysis of variance (ANOVA) on age groups, because the latter is aimed at piecewise constant expression patterns, and it is not plausible that expression should change sharply at a given age. A genome-wide ANOVA (data not shown) did, however, find a similar group of age-related genes. Unlike ANOVA, regression summarizes the age effect in one coefficient. This is advantageous for interpretation and for statistical power when there is little nonlinearity.
The decision of whether to include a variable in model 1 was based on the collection of p-values for all the genes. If the histogram of p˜
values differed sharply from uniform, and if the smallest p-values were small compared to 1/44,928, then the coefficient was included.
Gene lists were made using a threshold p-value of 0.001. Such a gene list can be expected to have about 44 genes in it by chance, even if all of the coefficients are really zero. Thus, of the 985 age-related genes, it is plausible that about 44 of them are false positives. We have chosen to work with a fixed significance level, instead of attempting to fix the false discovery rate, because our test statistics are strongly correlated.
We were concerned that intra-subject correlations might have affected our results. For each of 59 subjects with both cortex and medulla samples, we subtracted log2 expression in the cortex from that in the medulla, and fit a regression of the difference versus age and sex. Such an analysis removes intra-subject correlations. There was again no evidence of genes aging differently in the two tissue types (data not shown).
Supporting Information
Figure S1 Age Distribution of Medical and Related Factors
Each row shows the presence of a medical or related factor. Age of patients is shown on the y-axis. Only transitional cell carcinoma showed a strong age bias. We have identified over 20 different factors that might potentially confound our study on aging, such as race, blood pressure, diabetes, and type and size of tumor adjacent to the normal section (see Table S1).
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Figure S2 Medical Factors Do Not Affect Age Regulation
We used regression models to directly test whether our aging studies were affected by seven medical factors: renal cell carcinoma, transitional cell carcinoma, size of tumor, hypertension, systolic blood pressure, diastolic blood pressure, or diabetes mellitus. Scatterplots show age-related slopes using a regression model that includes a term for the medical factor compared to slopes from a regression model that does not include that medical factor.
(A) Effect of renal cell carcinoma (RCC) on age-related expression. We selected genes that showed statistically significant (p < 0.001) age regulation using either a model with a renal cell carcinoma term or without a renal cell carcinoma term. The vertical and horizontal axes show the slope from a model with and without the renal cell carcinoma term, respectively. The slopes change very little with and without the renal cell carcinoma term. As one might expect, many of the genes that are significant at the 0.001 level are just barely so. There were 866 genes significant in both models, 119 significant only when renal cell carcinoma was not in the model, and 86 significant only when renal cell carcinoma was in the model. The overall picture of age relationship changes very little whether a term for renal cell carcinoma is included in the model or not.
We also used a regression model predicting expression from age, sex, tissue type, and a zero/one variable indicating whether the sample came from a patient with renal cell carcinoma or not. The result gave a p-value for whether renal cell carcinoma affected each of the 44,928 genes present on the Affymetrix DNA chip. The smallest p-value we saw was 0.00013. We would expect to see almost six such p-values by chance alone. This result indicates that the presence of renal cell carcinoma does not significantly affect the expression of any gene in the normal tissue from the same kidney, compared to normal tissues taken from kidneys without renal cell carcinoma.
(B) Effect of transitional cell carcinoma (TCC) on age-related expression. Scatterplot showing age-related slopes with and without a term for transitional cell carcinoma. Transitional cell carcinoma was present in 13 patients, all of whom were old. Thus if transitional cell carcinoma affected gene expression in adjacent normal tissue, then it might bias our results on aging. (B) shows data for presence or absence of transitional cell carcinoma in the model. The gene with the smallest p-value for transitional cell carcinoma had a p-value of 8.8 × 10−6. The expected number of p-values this small in 44,928 trials is 0.4, so the presence of this gene is not particularly compelling evidence that transitional cell carcinoma biased our results. The histogram of p-values looks uniform, as we would expect if transitional cell carcinoma were very weakly related, or not related, to expression changes with age (data not shown). We have not used false discovery rate techniques for this problem, because the age coefficients for different genes are far from independent. The scatterplot shows that transitional cell carcinoma does not affect age-related slopes very much.
(C) Tumor size does not affect age regulation.
(D) Hypertension (HTN) does not affect age regulation.
(E) Systolic blood pressure (SBP) does not affect age regulation.
(F) Diastolic blood pressure (DPB) does not affect age regulation.
(G) Diabetes mellitus (DM) does not affect age regulation.
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Figure S3 Comparison of Age Regulation of Gene Expression between Kidney and Muscle Tissue in Humans
We obtained the muscle dataset from the GEO database (Welle et al. 2003). To compare age regulation in the kidney and muscle, we queried whether the 447 genes identified as age-regulated in the kidney were similarly age-regulated in the muscle. We determined regression coefficients for the 447 genes in the muscle dataset using multiple regression, in a manner similar to the kidney dataset. For each of the 447 genes, we plotted regression slope in kidney against regression slope in muscle, and found an overall weak Pearson correlation of 0.085 (p < 0.004). A Pearson correlation value of 0.085 implies that 0.72% of the variance in the muscle regression coefficients is due to variance in the associated kidney regression coefficients. We note that the muscle dataset had a small sample size (n = 16), which may not be large enough to sufficiently detect similarity in age regulation with the kidney.
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Figure S4 Comparison of Age Regulation of Gene Expression between Humans, Flies, and Worms Reveals No Correlation
We compared patterns of gene expression in the aging time course data from C. elegans (Lund et al. 2002) and D. melanogaster (Pletcher et al. 2002) to those in the data for the human kidney. We identified orthologous genes using the criterion that they exhibit best reciprocal BLAST hits between species. Beginning with the set of 447 age-regulated genes in the human kidney, we identified 119 worm and 142 fly orthologs. From the set of 167 age-regulated genes in the worm, we identified 60 human orthologs. From 1,264 age-regulated genes in the fly, we identified 465 human orthologs.
(A) Regression slopes of age-regulated genes from human kidney and D. melanogaster. Open triangles denote age-regulated genes in humans and their orthologs in flies. Open circles denote age-regulated genes in flies and their orthologs in humans. The scatterplot shows the regression slopes from the human kidney and the fly aging datasets (Pletcher et al. 2002). Specifically, the age-regulated human genes paired with fly orthologs show a Pearson correlation r = −0.05 (p = 0.27) for human and fly, while the age-regulated fly genes paired with human orthologs show a Pearson correlation r = −0.05 (p = 0.12).
(B) Regression slopes of age-regulated genes from human kidney and C. elegans. Open circles denote age-regulated genes in humans and their orthologs in worms. Open triangles denote age-regulated genes in worms and their orthologs in humans. The scatterplot shows the regression slopes from the human kidney and C. elegans aging datasets (Lund et al. 2002). The age-regulated human genes paired with worm orthologs show a Pearson correlation r = 0.05 (p = 0.54). The age-regulated worm genes paired with human orthologs show a Pearson correlation r = −0.01 (p = 0.08).
These results show no evidence for overlap in the aging process between different species.
(509 KB PDF).
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Protocol S1 Affymetrix HG-U133 Set Gene Chip Protocol
(40 KB DOC).
Click here for additional data file.
Table S1 Medical History of Patients
(33 KB XLS).
Click here for additional data file.
Table S2 Patients Recruited by Age Group
(13 KB XLS).
Click here for additional data file.
Table S3 Age-Related Genes (p < 0.001) Arranged by p-Value
(135 KB XLS).
Click here for additional data file.
Table S4 Age-Related Genes (p < 0.001) Excluding Those with Higher Expression Levels in Blood than in Kidney, Arranged by Fold Change
(75 KB XLS).
Click here for additional data file.
Table S5 Age-Related Genes by Location within the Kidney
(53 KB XLS).
Click here for additional data file.
We would like to thank Drs. Jim Brooks, Harcharan Gill, Joseph Presti, Raj Singhal, Jeffrey Reese, Jon Pollack, and Janet Mitchell and the other staff at the Stanford Tissue Bank for providing access to the kidney samples, and to the staff of the Stanford Microarray Database. We are grateful for insightful comments from the reviewers. This work was supported by grants from the National Kidney Foundation of Northern California, the Ellison Medical Foundation (AT-SS-1018-02), and the National Institutes of Health (DK064697).
Conflicts of interest. The authors have declared that no conflicts of interest exist.
Author contributions. SKK conceived and designed the experiments. RS, JW, LW, EC, and JH performed the experiments. SKK, GEJR, JMZ, JL, WX, MM, ES, and ABO analyzed the data. MM, BDM, JDB, RWD and JH contributed reagents/materials/analysis tools. SKK, JMZ, and ABO wrote the paper.
Academic Editor: Thomas Kirkwood, University of Newcastle upon Tyne
Citation: Rodwell GEJ, Sonu R, Zahn JM, Lund J, Wilhelmy J, et al. (2004) A transcriptional profile of aging in the human kidney. PLoS Biol 2(12): e427.
Abbreviations
ANOVAanalysis of variance
cRNAcomplementary RNA
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| 15562319 | PMC532391 | CC BY | 2021-01-05 08:28:08 | no | PLoS Biol. 2004 Dec 30; 2(12):e427 | utf-8 | PLoS Biol | 2,004 | 10.1371/journal.pbio.0020427 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1556232010.1371/journal.pbio.0020429Research ArticleCell BiologyGenetics/Genomics/Gene TherapyMolecular Biology/Structural BiologyNeuroscienceDrosophila
Drosophila Spastin Regulates Synaptic Microtubule Networks and Is Required for Normal Motor Function Functions of Drosophila SpastinSherwood Nina Tang
1
Sun Qi
1
¤1Xue Mingshan
2
¤2Zhang Bing
2
Zinn Kai [email protected]
1
1Broad Center, Division of Biology, California Institute of TechnologyPasadena, CaliforniaUnited States of America2Section of Neurobiology, University of TexasAustin, TexasUnited States of America12 2004 30 11 2004 30 11 2004 2 12 e42920 4 2004 12 10 2004 Copyright: © 2004 Sherwood et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
A Fly Enzyme for Motor Control
The most common form of human autosomal dominant hereditary spastic paraplegia (AD-HSP) is caused by mutations in the SPG4 (spastin) gene, which encodes an AAA ATPase closely related in sequence to the microtubule-severing protein Katanin. Patients with AD-HSP exhibit degeneration of the distal regions of the longest axons in the spinal cord. Loss-of-function mutations in the Drosophila spastin gene produce larval neuromuscular junction (NMJ) phenotypes. NMJ synaptic boutons in spastin mutants are more numerous and more clustered than in wild-type, and transmitter release is impaired. spastin-null adult flies have severe movement defects. They do not fly or jump, they climb poorly, and they have short lifespans. spastin hypomorphs have weaker behavioral phenotypes. Overexpression of Spastin erases the muscle microtubule network. This gain-of-function phenotype is consistent with the hypothesis that Spastin has microtubule-severing activity, and implies that spastin loss-of-function mutants should have an increased number of microtubules. Surprisingly, however, we observed the opposite phenotype: in spastin-null mutants, there are fewer microtubule bundles within the NMJ, especially in its distal boutons. The Drosophila NMJ is a glutamatergic synapse that resembles excitatory synapses in the mammalian spinal cord, so the reduction of organized presynaptic microtubules that we observe in spastin mutants may be relevant to an understanding of human Spastin's role in maintenance of axon terminals in the spinal cord.
Kai Zinn and colleagues use loss- and gain-of function mutations to study the Drosophila homologue of a gene mutated in human autosomal dominant hereditary spastic paraplegia
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Introduction
“Pure” autosomal dominant hereditary spastic paraplegia (AD-HSP) is an inherited disease characterized by bilateral spasticity in the absence of other phenotypes (reviewed in Fink 2003; Reid 2003). Afflicted patients experience difficulty in walking and have a distinctive gait. Degeneration of the lateral corticospinal tracts, which contain the axons of cortical neurons that innervate primary limb motoneurons, is observed in the lumbar regions of the spinal cord in patients with AD-HSP. The distal segments of long dorsal root ganglion axons also display degeneration. No evidence is seen for cell death or for primary myelination defects, and the axons of primary motor neurons do not degenerate (Maia and Behan 1974; Wharton et al. 2003). AD-HSP thus appears to selectively affect the distal regions of the longest axons within the spinal cord.
Because pathology is usually confined to long spinal cord axons, it has been suggested that the primary defect in pure AD-HSP is in axonal transport or some other process required for maintenance of axon terminals. Perturbation of anterograde or retrograde axonal transport might selectively affect the longest axons, because they would be most vulnerable to a reduction in efficiency of transport of material to or from their terminals.
About 40% of cases of pure AD-HSP are caused by mutations in the SPG4 gene, which encodes an AAA ATPase called Spastin (Hazan et al. 1999). AAA ATPases are a large and diverse set of proteins that include an approximately 250–amino acid (aa) conserved domain containing Walker A and B ATP-binding motif sequences (reviewed in Confalonieri and Duguet 1995; Patel and Latterich 1998; Neuwald et al. 1999). They use energy obtained from ATP hydrolysis to catalyze assembly or disassembly of a variety of protein complexes. AAA proteins are involved in many cellular processes, including vesicle trafficking, protein degradation, and microtubule dynamics. Many AAA ATPases form hexameric rings, and it is thought that the ring structures are required for catalytic activity (Vale 2000).
Spastin is a member of the “meiotic” subgroup of AAA ATPases (Frohlich 2001; Frickey and Lupas 2004), which contains proteins involved in vesicle trafficking and microtubule dynamics. The only member of the subgroup whose activities have been biochemically characterized is Katanin-60, which is the catalytic subunit of a microtubule-severing protein (McNally and Vale 1993; Hartman et al. 1998). Katanin-60 and Spastin are homologous only within their AAA domains. However, cell culture studies have provided evidence that Spastin is also involved in microtubule dynamics. Expression of wild-type human Spastin in transfected cell lines and cortical neurons caused disassembly of the microtubule cytoskeleton, while a mutant Spastin lacking catalytic activity colocalized with tubulin (Errico et al. 2002; McDermott et al. 2003).
The mechanisms by which spastin mutations produce dominant spasticity phenotypes in humans are controversial. A wide variety of nonsense and missense mutations, but no complete gene deletions, have been found in families with AD-HSP. It has been suggested that dominance arises from haploinsufficiency (Charvin et al. 2003). This model, however, would require that the processes in which Spastin participates are vulnerable to a 50% decrease in its enzymatic activity. Another possibility is that truncated or missense mutant Spastins function as dominant negatives. Hexameric AAA ATPase ring complexes might be especially vulnerable to the presence of nonfunctional (“poison”) subunits that assemble into rings but lack catalytic activity. Consistent with this idea, expression of a mutant Spastin that associates with microtubules but cannot catalyze severing altered organelle distribution in transfected cells (McDermott et al. 2003). In the dominant negative model, AD-HSP might only occur when Spastin activity is eliminated or greatly reduced.
In this study, we describe the phenotypes arising from mutation of the Drosophila ortholog of human spastin. We initially identified this gene in a gain-of-function screen, in which we found that its overexpression in neurons causes axons in the embryonic central nervous system (CNS) to converge onto the midline (Sun 2000). Overexpression of Spastin in muscles erases their microtubule networks, consistent with the idea that Spastin is a microtubule-severing protein.
We made loss-of-function (LOF) spastin mutations, and found that they produce recessive phenotypes affecting the larval neuromuscular system. The Drosophila neuromuscular junction (NMJ) uses glutamate as its neurotransmitter and employs ionotropic glutamate receptors homologous to vertebrate AMPA receptors (Schuster et al. 1991; Petersen et al. 1997; Marrus et al. 2004). It is organized into presynaptic boutons that are surrounded by a postsynaptic scaffold, and its synapses exhibit plastic behavior during development. These properties make the fly NMJ a useful genetic model system for the study of glutamatergic synapses in the mammalian brain and spinal cord (Keshishian et al. 1996; Koh et al. 2000).
During the period from larval hatching through the third instar stage, the number of boutons at each NMJ increases by up to 10-fold in order to keep pace with the growth of its muscle target. New boutons are added by a process of budding (Zito et al. 1999). As these boutons mature, their microtubule cytoskeleton is thought to progress through a regulated series of alterations (Roos et al. 2000; Pennetta et al. 2002).
In this paper, we show that synaptic growth and function are altered in spastin mutant larval NMJs. Boutons are more numerous than in wild-type larvae, and synaptic transmission is impaired. These changes could result from alterations in synaptic microtubule dynamics, because we find that microtubule bundles are depleted from the distal boutons of NMJs in spastin-null mutants. This is surprising, because the fact that Spastin overexpression destroys microtubule networks might lead one to expect that its removal would increase the number of microtubules. Morphological and microtubule phenotypes are seen only for a total gene deletion, indicating that complete loss of Spastin function is required to alter synaptic microtubules in the fly system. The phenotypes we see are quite different from those described in a recently published study of perturbation of Drosophila
spastin using RNAi methods (Trotta et al. 2004). In particular, the changes in synaptic microtubules that occur in spastin LOF mutants are opposite to those reported in the RNAi perturbation paper.
spastin is not an essential gene, but mutant adults have severely compromised motor behavior. Null mutants cannot fly or jump, they climb slowly, and they often drag their hind legs. While it is intriguing that spastin mutant flies display such movement phenotypes, further work will be required to determine whether Drosophila can provide a useful organismal model system for human AD-HSP. Nevertheless, insights into the cellular functions of Drosophila Spastin obtained from our work should be relevant to an understanding of Spastin's functions in human neurons.
Results
The Drosophila spastin Gene
We identified spastin in an “EP” screen for genes involved in embryonic CNS development. EPs are P element derivatives with a block of “UAS” sites recognized by the yeast transcription factor GAL4 near one end (Rorth 1996). An EP element inserted in the proper orientation upstream of a gene will drive its expression in a cell-specific manner when the insertion line is crossed to the appropriate promoter-GAL4 “driver” line (Brand and Perrimon 1993). We generated approximately 6,000 new EP insertion lines and screened them by crossing to pan-neuronal (ElavC155) and pan-muscle (24B) GAL4 driver lines (Lin and Goodman 1994; Luo et al. 1994). Those lines for which crosses to either driver generated reduced numbers (<20% of expected) of viable adult progeny containing both the EP element and the driver were saved. About 2% of lines (131) exhibited lethality or reduced viability with one of the drivers, and 62 of these were lethal or semilethal with both drivers. The T32 insertion on the third chromosome conferred complete lethality when crossed to either driver, and produced a neuronal-driver-dependent axonal phenotype (see below).
To identify the gene driven by the T32 element, we cloned a genomic DNA fragment adjacent to the insertion site and used it to identify a full-length cDNA encoding a 758-aa protein that is a member of the AAA ATPase family (Figure 1A). The T32
EP element is inserted into the 5′ UTR, 222 nucleotides upstream of the predicted ATG start codon (Figure 1B; Sun 2000).
Figure 1
Drosophila Spastin: Sequence Alignment and Gene Map
(A) Clustal alignment of complete D. melanogaster and H. sapiens Spastin amino acid sequences and the AAA region of Drosophila Katanin-60. Identical and similar residues are highlighted in dark and light gray, respectively.
(B) Map of the Drosophila
spastin gene, including exons (black boxes) and introns, the position of the T32
EP insertion (nucleotide 58 of the 5′ UTR), and the regions deleted by imprecise excision in lines 10-12, 17-7, and 5.75. The 3′ end of the adjacent Rox8 gene is also shown. Arrows indicate direction of transcription.
(C) Unrooted phylogenetic tree generated by the “neighbor” algorithm, showing relationships between the AAA domains of Spastins and their close relatives in human and fly. Dm CG3326 is the counterpart of the human fidgetin/fidlik gene pair, while CG1193 probably encodes a second fly ortholog of human Katanin-60. In the mouse, fidgetin mutations produce inner ear defects that cause head-shaking and circling behaviors (Cox et al. 2000).
The gene driven by T32 is orthologous to the human SPG4
(spastin) gene that is mutated in the most common form of AD-HSP (Hazan et al. 1999). Mammalian Spastins are the only proteins that are homologous to both the C-terminal AAA domain and the N-terminal region of fly Spastin (Figure 1A). Spastin exhibits homology to all other AAA proteins only within its AAA domain (approximately aa 460–754 of fly Spastin). There are about 30 AAA proteins encoded in the Drosophila genome.
The Drosophila Spastin sequence from aa 233 to the C terminus is 49% identical to that of human Spastin (616 aa). The AAA domains of the two proteins are 67% identical. The other region that is conserved between the Spastins (34% identity) corresponds to aa 233–404 of the fly sequence. The same region is also weakly related (26% identity) to human Spartin, the product of the SPG20 gene mutated in Troyer syndrome, a form of “complicated” HSP (Patel et al. 2002; Ciccarelli et al. 2003). Spartin is not an AAA ATPase. The AAA protein with a known biochemical function that is most closely related to Spastin (41% identity in the AAA domain) is Katanin-60 (Figure 1A and 1C; McNally and Vale 1993; Hartman et al. 1998).
Drosophila Spastin Localizes to the Cytoplasm
Human Spastin is thought to be a cytoplasmic protein expressed in many cell types, based on localization of epitope-tagged proteins expressed in transfected cells, and antibody staining of human tissue and neuronal cell lines (Errico et al. 2002, 2004; McDermott et al. 2003; Wharton et al. 2003). However, antibodies generated against Spastin were also reported to stain nuclei in several cell lines and mouse spinal cord neurons (Charvin et al. 2003; Errico et al. 2004). This dual subcellular localization has been proposed to reflect a role for human Spastin in processes involving highly dynamic microtubule states, such as during cell division (reflected in Spastin's nuclear localization) and in axon outgrowth, suggested by the distal cytoplasmic staining of growing axons in culture (Errico et al. 2004).
To investigate the subcellular localization of Drosophila Spastin, we generated a variety of antibodies against different regions of the protein (see Materials and Methods). We evaluated these antibodies by staining stage 16 embryos overexpressing Spastin in the striped Engrailed pattern from an engrailed-GAL4 driver. Two different antibodies revealed Spastin expression in the expected striped pattern, which includes a subset of CNS neurons (Figure 2A). Anti-Spastin antibodies stained both the cell bodies and the axons of these neurons (Figure 2B). In the epithelial portions of the Engrailed stripes, where the cells are flat and spread out, we observed that Spastin is expressed uniformly in the cytoplasm, but did not detect any nuclear staining (Figure 2C; see also Figure 7E, showing cytoplasmic expression in muscles).
Figure 2 Spastin Protein Localizes to the Cytoplasm
(A) In embryo “fillets” in which Spastin overexpression is driven by the engrailed-GAL4 driver, a polyclonal antibody, pAb1239, generated against the C-terminal half of Spastin (aa 380–758) recognizes the characteristic striped pattern of Engrailed cells. Anterior is up; the CNS is the structure in the center, and the lateral epithelial stripes extend to either side.
(B) An enlarged view of the CNS shows Spastin protein in these embryos localizing to neuronal cell bodies (arrow indicates the ventral unpaired midline [VUM] neurons), as well as in commissural and longitudinal axons (arrowheads).
(C) A high-magnification view of the Spastin-positive epithelial cells shows that the protein fills the cytoplasm (arrow), and is excluded from the nucleus (arrowhead).
Scale bar: (A) 25, (B) 10, and (C) 6.5 μm.
Figure 7 Spastin Overexpression in Muscles Erases the Microtubule Network
(A) An antibody against β3-tubulin stains body wall muscles and chordotonal cap cells in stage 16 wild-type embryos. Two abdominal hemisegments are shown; muscle fiber numbers are labeled in one. The cap cells (brackets) are difficult to distinguish in this panel because of high levels of muscle tubulin staining. They extend diagonally from about the middle of muscle 18 to muscle 22. Anterior is to the left, and dorsal is up.
(B) When Spastin is overexpressed in muscles (genotype: G14-GAL4/+; T32/+), β3-tubulin staining is very weak and has a disorganized pattern in most muscle fibers, but an intact microtubule network is still present in the cap cells, which do not express this driver (brackets). The muscle fibers are misshapen and partially (arrowhead) or completely (arrow) detached from their insertion sites.
(C–H) Similarly, the microtubule network (recognized by antibodies to α-tubulin) is almost eliminated by high-level Spastin expression in third instar larval muscles. Larvae of genotype UAS-spastin/MHC-GS-GAL4213-3; spastin5.75/TM3Ser-ActGFP overexpress Spastin protein specifically in muscles to varying degrees. Wild-type larval muscles had undetectable levels of Spastin using pAb 1239 (C) and displayed a dense network of microtubule bundles in the muscle (D and E), as well as in trachea (D, arrow) and neurons (D, arrowhead denotes a terminal arbor). In contrast, larval muscles expressing high levels of Spastin (F) show only faint muscle microtubule staining (G and H), while tracheal (G, arrow) and neuronal (G, arrowhead) staining remain robust.
spastin mRNA is expressed at low levels within the embryonic ventral nerve cord (VNC) in wild-type embryos (Sun 2000; Kammermeier et al. 2003). Endogenous Spastin appears to be a very rare protein, and we have not been able to define a staining pattern in wild-type embryos or larvae that disappears in null mutants. An independently generated anti-Drosophila Spastin antibody was reported to stain the cytoplasm of both neurons and muscles in wild-type larvae, and staining was also detected at NMJ boutons (Trotta et al. 2004). Taken together, these results suggest that fly Spastin, like human Spastin, is likely to be a widely expressed protein that is primarily localized to the cytoplasm.
Spastin Overexpression in Neurons Causes Collapse of the Embryonic CNS
Crossing the T32 insertion line to scabrous
(sca)-GAL4, which is expressed in neuronal precursors and neurons (Klaes et al. 1994), produced very strong CNS phenotypes. In Figure 3, these are visualized by staining with monoclonal antibody (mAb) 1D4 (Van Vactor et al. 1993), which labels a set of three longitudinal axon bundles. At 23 °C, embryos displayed abnormal midline crossing of the inner 1D4 bundle, and the entire VNC was narrowed at these crossing sites (Figure 3B). When crosses were performed at 29 °C, a temperature at which GAL4 transactivation is stronger, the VNC collapsed onto the midline and discrete longitudinal bundles were no longer apparent (Figure 3C). We also made transgenic lines bearing a full-length spastin cDNA driven by a UAS-containing promoter. After crossing to sca-GAL4, such lines produced even stronger phenotypes, in which the VNC collapsed at 23 °C (Figure 3D). Consistent with this observation, we found that more Spastin protein was made in driver × UAS-spastin embryos than in driver × T32 insertion embryos.
Figure 3 Neuronal Overexpression of Spastin Causes Midline Convergence of Embryonic CNS Axons
(A) Anti-Fasciclin II (mAb 1D4) staining of filleted late stage 16 control embryos reveals three longitudinal axon bundles (arrow) on each side of the midline. Anterior is up.
(B) In sca-GAL4/+; T32/+ embryos raised at room temperature, overexpression of Spastin in neurons causes the ladder to constrict toward the midline (e.g., arrow).
(C) Increased Spastin expression at 29 °C causes collapse of the CNS onto the midline (arrow). Longitudinal axon tracts are thin or absent (arrowhead).
(D) A phenotype similar to that in (C) is produced by sca-GAL4-driven expression of the UAS-spastin cDNA insertion at 23 °C. Arrow and arrowhead indicate same as in (C).
spastin LOF Mutations Produce Larval NMJ Phenotypes
To evaluate Spastin's functions during development, we generated several deletion mutations from the T32 insertion by imprecise excision. We mapped their breakpoints by sequencing, and these data are displayed in Figure 1B. In line 10-12, about half of the first exon is deleted. The 17-7 deletion ends within the second intron and thus removes the entire first exon (encoding sequence up to aa 251). In both of these lines, DNA encoding the protein region conserved between human and Drosophila Spastin is still present. Deletion 5.75 removes the entire spastin gene, as well as the intergenic region and 129 bp at the 3′ end of the sequence of the adjacent predicted gene Rox8. Rox8 contains RRM RNA-binding domains. The 5.75 deletion removes the C-terminal 43 aa of the Rox8 protein, but does not delete into the RRM domains. The function of Rox8 is unknown, and there are no existing Rox8 mutations (Brand and Bourbon 1993). Because the only null spastin mutation also affects Rox8, we relied on rescue experiments (see below) to demonstrate that the phenotypes we describe for the null mutant are due to loss of Spastin.
Flies homozygous for spastin10-12 and spastin17-7 have behavioral phenotypes, but they eclose at normal frequencies and are fertile (see below). In contrast, most homozygous spastin5.75 pupae do not eclose. spastin5.75 adults have very severe behavioral phenotypes, and both sexes are sterile. These results suggest that the 10-12 and 17-7 alleles are hypomorphic, and that the spastin5.75 phenotype represents the null condition. RT-PCR analysis of cDNA from spastin10-12 and spastin17.7 animals indicated that low levels of truncated spastin transcripts are still produced (data not shown). These may direct synthesis of proteins initiated from internal ATGs that could retain partial function, since they include the entire conserved AAA domain.
We could not detect anatomical phenotypes in embryos homozygous for any of the spastin mutations. However, we saw striking morphological changes in the NMJs of spastin5.75 third instar larvae. In Figure 4, the two predominant types of glutamatergic boutons at the NMJs, Ib (big) and Is (small), are visualized by double-staining larval fillets with antibodies against Synaptotagmin (Syt, magenta; Menon and Zinn 1998) and Discs-large (Dlg, green; Woods and Bryant 1991). Syt is a presynaptic protein involved in neurotransmitter release that is localized to boutons, while Dlg is a primarily postsynaptic scaffold protein localized to the subsynaptic reticulum that surrounds each bouton (Littleton et al. 1993; Lahey et al. 1994). Figure 4A and 4B show that NMJ boutons are smaller and more numerous at the muscle 6/7 NMJ of spastin5.75 larvae than in Canton S
w− (WCS) control larvae. (WCS was chosen as a control because, like the lines used to generate our EP insertion mutants, it is derived from a Canton S wild-type background, but it is also w−, like the T32 excision derivatives. WCS is also commonly used for behavioral experiments.) Other NMJs are affected in a similar manner (e.g., Figure 4D–4F, showing muscle 4 synapses). The sizes of muscle fibers are normal in spastin mutants.
Figure 4 Synaptic Boutons Are Smaller, More Numerous, and Clustered in spastin LOF Mutants
(A–F) Representative A3 NMJs on muscles 6/7 (A–C) or muscle 4 (D–F) stained with antibodies against Dlg (green) and Syt (magenta) are shown for control larvae (WCS; A and D), spastin5.75 larvae (B and E), and larvae expressing Spastin from the spin-GAL4 driver in a spastin5.75 mutant background (Rescue; C and F). Boutons are arranged in a linear pattern in WCS larvae, whereas in spastin5.75 larvae their distribution is more clustered and individual boutons are smaller. These phenotypes are rescued by Spastin expression via the spin-GAL4 driver. Scale bars, 10 μm.
(G) Quantitation of bouton numbers in spastin mutants relative to wild-type and rescued larvae demonstrates complete rescue of the null phenotype by spin- or Elav-GS-GAL4-driven expression of Spastin. spastin-null mutants have on average 1.6-fold more type Ib boutons on muscle 4 compared to WCS control larvae. Similarly, spastin-null mutants (of genotype spin-GAL4/CyOKr-GFP; spastin5.75) have 1.7-fold more boutons compared to their sibling rescued larvae (genotype spin-GAL4/UAS-spastin; spastin5.75). Boutons are also 1.6-fold more numerous in spastin-null larvae from a neuronal rescue cross (genotype +/CyOKr-GFP; Elav-GS-GAL4,spastin5.75/spastin5.75) compared to their siblings in which UAS-spastin is expressed in neurons postembryonically (genotype UAS-spastin /CyOKr-GFP; Elav-GS-GAL4, spastin5.75/spastin5.75).
To quantify the NMJ phenotype, we counted the numbers of boutons at the muscle 4 NMJs of segments A2 and A3, where boutons typically form on the internal surface of the muscle and are thus easily imaged. Dlg is expressed at much higher levels at Ib compared to Is boutons, allowing the two types of boutons to be distinguished and counted. Because of the greater variability in Is bouton number between NMJs, we focused our quantitative analysis on the type Ib boutons. However, the numbers of both bouton types were similarly affected in spastin5.75 larvae.
The number of Ib boutons per muscle 4 NMJ was increased by 1.6-fold relative to WCS in spastin mutants at room temperature (approximately 23 °C) (Figure 4G), and the boutons often formed dense clusters, particularly at the ends of NMJ branches (Figure 4E). This morphology was rarely observed in wild-type muscle 4 NMJs, where boutons were arranged more linearly (Figure 4D). The clustered boutons resemble the “satellite” boutons described by other investigators (Torroja et al. 1999; Franco et al. 2004; Koh et al. 2004; Marie et al. 2004). Hypomorphic spastin10-12 and spastin17-7 mutants had bouton numbers that did not differ significantly from controls.
To confirm that loss of Spastin produced the observed NMJ alterations, and to determine whether Spastin is required presynaptically or postsynaptically, we needed to evaluate rescue of the phenotype by expression of Spastin from a UAS-spastin cDNA insertion. This was difficult because of the early lethality produced by expression of Spastin from most drivers. UAS-spastin animals bearing pan-neuronal (Elav-GAL4), motoneuronal (OK6-GAL4), or pan-muscle (24B-GAL4 or G14-GAL4) drivers did not survive to larval stages at 23 °C, and few larvae appeared even at 18 °C. However, third instar larvae in which Spastin expression from the cDNA was conferred by spinster
(spin)-GAL4, a weak driver that functions in both neurons and muscles (Sweeney and Davis 2002), did survive at 23 °C. We were also able to obtain larvae in which Spastin expression was induced in neurons postembryonically. This was done by crossing UAS-spastin to Elav-GeneSwitch (GS)-GAL4, a driver line bearing a neuronally expressed GAL4 derivative that is only active in the presence of the progesterone analog RU486 (Osterwalder et al. 2001; McGuire et al. 2004). Newly hatched larvae from this cross were maintained on RU486-containing food until the third instar stage.
To assay rescue, we combined the spin-GAL4 and Elav-GS-GAL4 drivers and UAS-spastin insertions separately with spastin5.75, crossed the driver and UAS-spastin lines together, and assayed NMJ phenotypes in the F1 driver-GAL4/UAS-spastin;
spastin5.75 larvae. In each cross, we compared the rescued larvae to their unrescued spastin mutant siblings (driver-GAL4; spastin5.75), because the presence of the driver chromosome had effects on the absolute number of Ib boutons (see Materials and Methods). We observed that the ratio of muscle 4 Ib bouton numbers in spastin mutant controls versus rescued larvae was 1.7 for spin-GAL4, and 1.6 for Elav-GS-GAL4 (Figure 4G). Since the ratio of bouton numbers for spastin5.75 versusWCS was 1.6, this indicated that rescue was essentially complete in both cases. We also observed that the abnormal bouton clustering was eliminated in rescued larvae using either driver (Figures 4E and S1). These results demonstrate that loss of Spastin from neurons during larval development causes the NMJ bouton phenotypes seen in spastin5.75 mutants.
To examine the consequences of driver-dependent postembryonic neuronal expression of Spastin in a wild-type background, we also counted boutons in UAS-spastin; Elav-GS-GAL4 larvae grown on RU486 food (see Materials and Methods). We observed that these larvae had fewer boutons than their siblings (0.83 ± 0.07 fold change), and some of their boutons appeared larger (Figure S1C). This phenotype is very mild, but it does suggest that loss and increased expression of Spastin can produce opposite effects on the NMJ.
Neurotransmitter Release Is Impaired in spastin Mutants
To evaluate whether spastin mutations cause alterations in the electrophysiological properties of the NMJ, we evaluated synaptic transmission at the muscle 6 NMJ in WCS, mutant, and rescued larvae raised at 18 °C. In spastin5.75 larvae, there was a reduction in the amplitudes of evoked responses (excitatory junction potentials [EJPs]) to depolarization of the innervating nerves. Average EJP amplitudes in the null were reduced to 78% of the levels in control (WCS) larvae (Figure 5A and 5B; p < 0.003). We also examined the average amplitude and frequency of responses to single vesicles of spontaneously released neurotransmitter (“mini” EJPs [mEJPs]). mEJP amplitude was increased slightly, to 117% of WCS levels (Figure 5A and 5C; p < 0.03). There was no significant change in mEJP frequency (Figure 5D; p = 0.3).
Figure 5 NMJs in spastin Mutant Larvae Display Reduced QC
(A) Representative EJP (upper) and mEJP (lower) traces are shown for control (WCS), spastin-null mutant (spastin5.75), and spin-GAL4/UAS-spastin; spastin5.75 (Rescue) larvae. All recordings were from the A3 or A4 muscle 6 NMJ.
(B) The average EJP amplitude is decreased by about 20% in spastin-null mutants (37 ± 2.0 mV, n = 26) relative to control (48 ± 2.4 mV, n = 14) and Rescue (42.4 ± 1.2 mV, n = 28) larvae, and is intermediate between control and null levels in hypomorphic spastin5.75/17-7 transheterozygotes (41.9 ± 1.5 mV, n = 22).
(C) The average amplitude of spontaneous events (mEJPs) is increased slightly in spastin nulls relative to control and Rescue larvae.
(D) The average frequency of spontaneous events is not affected in spastin mutants compared to control. Rescue larvae had a slightly higher mEJP frequency.
(E) Average QC, a measure of the amount of neurotransmitter released per action potential, is significantly lower in transheterozygotes (28 ± 1.3) versus control (35 ± 1.7), and reduced even further in spastin nulls (23 ± 1.2). This decrease is completely rescued by spin-GAL4-driven rescue (30 ± 1.9, p = 0.1 compared to WCS).
(F) Average QC is temperature dependent in spastin5.75/spastin17-7 transheterozygous larvae, but not in homozygous spastin-null or control larvae. QC measured in transheterozygotes raised at 18 °C (light gray bars) is intermediate between that of control and nulls. At room temperature (dark gray) and 29 °C (black bars), similar QC values are measured in transheterozygotes and null mutants.
*, p < 0.05; **, p < 0.005.
Quantal content (QC), a measure of the number of vesicles released per evoked event, was calculated by dividing the EJP amplitude by the average mEJP amplitude. Because the evoked EJP was decreased and the mEJP increased in spastin5.75 mutants, QC was reduced to 67% of WCS levels in these larvae (Figure 5E; p < 3 × 10−6). spastin17-7/spastin5.75 larvae had EJP amplitude, mEJP amplitude, and QC values intermediate between those of spastin-null and control larvae. QC in these transheterozygotes was also decreased significantly, to 78% of WCS levels (Figure 5E; p < 0.002). The changes in EJP amplitude and QC observed in spastin5.75 mutants were completely rescued by spin-GAL4-driven Spastin expression. Average QC in rescued larvae was not significantly different from wild-type (p > 0.1), and was 30% greater than in spastin5.75 mutants (p < 0.005; Figure 5E).
We also observed that the electrophysiological phenotypes of the hypomorphic spastin17-7/spastin5.75 larvae were temperature sensitive. While the average QC in transheterozygotes was reduced to 78% of controls at 18 °C, this effect was exacerbated at higher temperatures. At 29 °C, QC was 54% of wild-type (Figure 5F). However, QC in control or in spastin5.75 null larvae was unaffected by temperature. These results suggest that the N-terminally truncated Spastin protein that is probably made from the spastin17-7 allele is temperature sensitive. At 29 °C, this hypomorphic allele behaves as a null with respect to NMJ electrophysiology. Finally, we examined escaper larvae (Elav-GAL4/+; T32/+) overexpressing Spastin in neurons, and found that synaptic transmission in these larvae was not significantly different from wild-type (data not shown).
spastin Mutant Adults Have Severe Behavioral Phenotypes
We observed that only approximately 20% of homozygous spastin5.75 pupae were able to eclose at room temperature compared to 94% of heterozygotes, and the adults that emerged had severe movement defects (see Video S1). They could not fly at all, and did not even appear to move their wings, although the wings inflated and straightened in a normal manner immediately after eclosion. They also did not jump spontaneously, but would jump if persistently prodded in the abdomen. Their legs were weak: when standing, the metathoracic legs often slipped out from underneath them, and during walking they often dragged these legs (see Video S1). They also had difficulty holding on to surfaces when they were upside down. These phenotypes were temperature dependent. Null mutant flies that developed at 18 °C eclosed at much higher rates (56%) than at higher temperatures and moved more normally. Flies homozygous for the hypomorphic mutations, spastin10-12 and spastin17-7, eclosed at normal frequencies at all temperatures.
To evaluate these movement defects, we assayed flight and climbing ability in spastin-null and hypomorph flies (Figure 6). The flight assay could only be used for hypomorphs since null mutants were flightless. In this assay, flies were released into the top of a vertical cylinder that had been coated on the inside with oil (Benzer 1973; Atkinson et al. 2000). Poor fliers who took longer to fly fell to the bottom or collided with the lower walls of the cylinder, while good fliers who responded rapidly to being dropped collided with the upper walls. A histogram of the distribution of oil-trapped flies along the height of the cylinder showed that more than half of the spastin hypomorphs did not fly in time to avoid falling to the bottom of the cylinder (Figure 6A). In contrast, the majority (approximately 75%) of the controls, including w1118 (another Canton S-derived w− control) flies and flies homozygous for T32 (in the absence of a GAL4 driver; these have orange eyes), flew well enough to distribute themselves along the sides of the column. Interestingly, for those hypomorphs that did fly out to the sides, their distribution paralleled that of the controls, suggesting that flight responses in the column were relatively normal in this subpopulation of the mutants (Figure 6B).
Figure 6
spastin Mutant Flies Have Compromised Motor Behavior and Reduced Lifespans
(A) In a flight test assay, over twice as many adult spastin hypomorphs (spastin10-12 and spastin17-7) fail to fly before falling to the bottom of a cylinder, in comparison to w1118 and T32 homozygous controls.
(B) Although fewer than half of the hypomorphs fly, compared to more than 70% of controls, the distribution of collision sites of the fliers along the height of the cylinder parallels that of the controls, suggesting that these spastin mutations affect flying ability in some animals but not in others.
(C and D) spastin mutants are compromised in their climbing ability. (C) All control (WCS) and nearly all spastin hypomorphs climb to the top of a vial in 30 s, but only 40% of spastin nulls do so. (D) Climbing velocity (measured for those flies that reach the top in 30 s) is 3.8 ± 0.2 cm/s in WCS (n = 45), but only 1.8 ± 0.2 and 1.4 ± 1.1 cm/s in spastin10-12 (n = 28) and spastin17-7 (n = 38) flies, respectively, and 0.3 ± 0.1 cm/s in spastin5.75 null mutants (n = 17; p < 1 × 10−8 for all relative to WCS).
(E) Lifespan curves. The curve inflection point at which WCS and hypomorph flies begin to die off at a rapid rate occurs at 30–35 d after eclosion, and more than 70% of hypomorph flies and 95% of wild-type flies are still alive at 30 d. In contrast, approximately 45% of spastin5.75 null mutant flies die prior to 4 d after eclosion. However, the majority of the remaining null flies survive more than 25 d, so that the curve inflection point for nulls occurs only a few days before that for controls and hypomorphs.
(F) Mean lifespan is 46 ± 2.7 d in WCS controls (n = 39) compared to 35 ± 3.2 and 35 ± 3.4 d, respectively, in spastin10-12 (n = 24, p < 0.02) and spastin17-7 (n = 32, p < 0.006), and 7.6 ± 0.6 d in nulls (n = 62, p < 10−31).
(G) Only about 10% of spastin5.75 flies eclosing at room temperature are males, while 40%–45% are males for controls and hypomorphs.
In the climbing assay, flies were tested for their ability to climb up the side of a vial within a limited time period. All WCS and almost all homozygous spastin10-12 and spastin17-7 flies, but only about 40% of spastin5.75 flies, climbed to the top of the vial within 30 s (Figure 6C). This difference did not reflect a loss of geotactic behavior, since spastin5.75 flies were typically found at the tops of their vials after several minutes. Overall, mean climbing velocity was approximately 9-fold slower for spastin-null mutants than for wild-type flies, while the hypomorphs were about 2-fold slower than controls (Figure 6D).
We also measured the lifespan of the flies. Under our conditions, WCS flies lived an average of 46 d at 25 °C. spastin10-12 and spastin17-7 flies had somewhat shorter lifetimes, surviving an average of 35 d. Lifespan was dramatically reduced in spastin5.75 flies, which lived an average of only 8 d (Figure 6E and 6F). Examination of mortality curves (Figure 6E), however, revealed that, as in the case of flight ability in the hypomorphs, these flies had a bimodal lifespan distribution. Only 55% of flies were still alive at 4 d post-eclosion, but most of these (37% of the total) then remained alive until about 25 d post-eclosion. After this time, they rapidly died off, and no flies remained alive more than 33 d.
Another spastin phenotype observed in adults was male-specific lethality (Figure 6G). For WCS and spastin hypomorphs, more than 40% of eclosed adults were males. However, only approximately 10% of eclosed spastin5.75 flies were male. We do not understand the origins of this phenotype.
In summary, spastin-null adult flies had severely compromised movement behavior and were short-lived, while spastin hypomorphs displayed weaker movement and lifespan phenotypes. We also examined rescue for these behavioral phenotypes. When compared to their non-rescued siblings (spin-GAL4/CyOKr-GFP; spastin5.75) from the same cross, spin-GAL4/UAS-spastin;
spastin5.75 flies climbed better, were more coordinated, and lived longer (Figure S2; Video S1), indicating that partial rescue was achieved. These flies were still very slow, and it is clear that spin-GAL4-driven Spastin expression did not restore behavior to the levels characteristic of control flies such as WCS. However, genetic background effects made the precise efficacy of rescue achieved in this experiment difficult to determine (the non-rescued spastin5.75 sibling flies bearing the driver and balancer chromosomes used in the rescue cross were much more unhealthy and slow-moving than spastin5.75 flies without these chromosomes).
Spastin Overexpression Erases Microtubule Networks In Vivo
To investigate whether Drosophila Spastin affects microtubule networks, we overexpressed it in embryonic muscles using the G14-GAL4 or 24B-GAL4 drivers, and then visualized muscle microtubules in late stage 16 embryos with an anti-β3-tubulin antibody that preferentially stains polymerized tubulin (Buttgereit et al. 1996). In wild-type embryos, a complex network of microtubules aligned along the muscle axes was observed (Figure 7A; two segments are shown). This was clearly seen in both vertically oriented (18 and 21–24) and diagonally oriented (5,11, and 19) muscles.
When Spastin was overexpressed in muscles, the muscle microtubule network completely disappeared (Figure 7B). The muscles themselves appeared rounded, and were partially or totally detached from their insertion sites. This detachment may have been a consequence of the dissolution of the microtubule network. Oriented microtubule networks could still be seen within the cap cells of the chordotonal organs in each segment (Figure 7B, brackets); these cells did not express the GAL4 driver and therefore did not overexpress Spastin. Dissolution of the microtubule network was therefore specific to cells in which Spastin was overexpressed.
We also overexpressed Spastin in larval muscles by crossing the muscle-specific MHC-GS-GAL4 driver line to UAS-spastin flies. MHC-GS-GAL4 is RU486-inducible, but we could not feed the larvae with RU486 as this was lethal. However, MHC-GS-GAL4 also confers late RU486-independent expression in third instar larvae, and we were able to obtain escaper larvae from the cross and double-stain these for Spastin and α-tubulin. As shown in Figure 7C–7E, larvae that lacked detectable Spastin expression had a dense network of muscle microtubules. In contrast, MHC-GS-GAL4/UAS-spastin larvae that had high levels of muscle Spastin displayed a dramatic reduction in microtubules, so that only faint and sparse microtubules were observed in the muscle fibers (Figure 7F–7H). The strongly staining microtubules still visible in these larvae are those of the neurons and tracheae, which do not overexpress Spastin (Figure 7G–7H). These results also show that this α-tubulin antibody preferentially recognizes polymerized tubulin, since the total amount of tubulin dimers would be the same in both sets of muscles (tubulin dimers are very stable proteins and are unlikely to be proteolyzed after severing).
Interestingly, when we overexpressed Spastin in the embryonic or larval CNS, we did not observe an obvious alteration of the axonal microtubule architecture (see also Figure 8). This suggests that Spastin may be unable to disassemble stable axonal microtubule bundles. Nevertheless, the dramatic effects of Spastin overexpression on muscle microtubules suggest that the embryonic CNS collapse phenotype conferred by neuronal overexpression (see Figure 3) may also arise from breakdown of key neuronal microtubules during the axonal growth phase.
Figure 8 The Distribution of Stable NMJ Microtubule Bundles Marked by the MAP1B-like Protein Futsch Is Altered in spastin Mutant Larvae
(A–C) Anti-Futsch labels stable microtubule bundles in axons, NMJ boutons, and interbouton regions. The muscle 4 NMJs in segment A3 of third instar wild-type (A), spastin5.75 (B), and spin-GAL4/UAS-spastin; spastin5.75 (Rescue) (C) larvae were immunostained with anti-HRP (A and B) or anti-Syt antibodies (C) to label presynaptic boutons (magenta), and mAb 22C10 to label Futsch protein (A–C, green). Arrows and arrowheads mark the terminal boutons (those at the ends of synaptic branches); boutons marked by arrows in (A–C) are enlarged in insets in the middle and right panels to show examples of Futsch patterns. (A) In control (WCS) larvae, terminal boutons have both looped (arrowheads) and diffuse, punctate (right panel, arrows and inset) patterns of Futsch staining. (B) In spastin mutants, Futsch staining appears similarly strong in axon bundles (not shown) and along the main branches of the bouton arbor. More distal and terminal boutons, however, have diffuse or no Futsch staining (arrows and arrowheads). Note the absence of green staining in insets. (C) The distribution of Futsch staining is restored to the control pattern by spin-GAL4-driven expression of Spastin in the mutant background (arrows and arrowheads indicate loops). Scale bar, 5 μm.
(D and E) Quantitative assessment of Futsch staining data. Futsch staining at A2 and A3 muscle 4 NMJs was classified as continuous (bundles or splayed bundles), looped, or diffuse or undetectable (none) for each bouton. (D) The percentage of boutons exhibiting continuous or looped Futsch staining (relative to the total number of boutons for each NMJ) is decreased in spastin mutants relative to controls, while the percentage of boutons having diffuse or no staining is increased. In total, 58% ± 4.2% of boutons in controls have a continuous pattern of Futsch staining, while only 42% ± 1.5% do in mutants. Boutons in this class are predominantly along the major (more proximal) branches of the axon arbor. Similarly, 11% ± 1.7% of wild-type boutons have Futsch loops, but only 6.0% ± 1.1% do in mutants. Most mutant boutons show only diffuse or no Futsch staining (52% ± 2.2%, versus 32% ± 5.2% in controls). Futsch distribution is restored to the control pattern by spin-GAL4- or Elav-GS-GAL4-driven expression of Spastin. (E) The difference in Futsch distribution is most pronounced at terminal boutons. There is no detectable Futsch staining in the majority of terminal boutons (65% ± 4.5%) in spastin mutants, compared to only 7.8% ± 5.8% of terminal boutons in wild-type larvae (p < 2 × 10−6). Futsch staining is restored in most terminal boutons of spin-GAL4- or Elav-GS-GAL4-rescued larvae, with only 20% ± 3.7% and 19% ± 5.9% of boutons, respectively, showing no staining (p = 0.09 compared to WCS). Terminal bouton staining in larvae overexpressing Spastin in neurons was unaffected relative to controls (p = 0.12). **, p < 0.005; *, p < 0.03 relative to WCS;
n
>8 NMJs scored in all cases.
Microtubule Bundles Are Depleted in Distal NMJ Boutons of spastin LOF Mutants
The finding that Spastin overexpression erases the microtubule network in muscles suggested that the spastin LOF NMJ phenotypes could arise from alterations in microtubule networks. To investigate this, we first examined the distribution of Futsch, a microtubule-associated protein related to vertebrate MAP1B (Hummel et al. 2000; Roos et al. 2000). Futsch staining is restricted to stable neuronal microtubule bundles. Because Futsch is not expressed in the underlying muscle, Futsch antibody staining provides the optimal method for quantitatively evaluating stable microtubules within NMJ boutons.
At muscle 4 NMJs in wild-type larvae (Figure 8A) we observed continuous microtubule bundles stained by anti-Futsch (mAb 22C10; green) within axons and along the axis of each branch of the NMJ (delineated by anti-HRP, which labels neuronal membranes; magenta). The intensity of Futsch staining weakens in the distal portions of the branches. Consistent with earlier findings, we also observed distinctive “loops” of Futsch staining within some boutons (Roos et al. 2000; Packard et al. 2002; Pennetta et al. 2002). Loops were typically observed in terminal boutons at the ends of branches. In some terminal boutons, however, we detected only punctate staining or no staining at all. This last case may reflect the limits of detection rather than the complete absence of Futsch protein in a bouton.
We quantified Futsch distribution by dividing the patterns of Futsch staining in boutons into three classes: continuous (bundles or splayed bundles), looped, and diffuse or undetectable. In spastin5.75 larvae (Figure 8B), there was a shift in the Futsch pattern toward less organized morphologies (i.e., diffuse/undetectable). At the muscle 4 NMJ of spastin mutant larvae, 74% and 54% as many boutons contained continuous and looped Futsch, respectively, as compared to WCS. In contrast, 63% more boutons in spastin mutants displayed only diffuse or no staining (Figure 8D). These differences were most pronounced at the distal ends of the synaptic branches (Figure 8B, arrows and arrowheads). 65% of terminal boutons in mutants had no detectable Futsch staining, as compared to 8% in WCS (Figure 8E; p < 2 × 10−6).
The Futsch distribution phenotypes were rescued by expression of Spastin from the spin-GAL4 or the RU486-induced, neural-specific Elav-GS-GAL4 drivers. Rescued larvae had Futsch staining patterns very similar to those seen in WCS (Figure 8C–8E). These results show that the reduction in stable synaptic microtubules seen in spastin LOF mutants is due to loss of Spastin from neurons during larval development. We also examined Futsch staining in Elav-GS-GAL4/UAS-spastin larvae grown on RU486 food, but saw no difference from the pattern in wild-type controls. Thus, stable microtubules at the NMJ do not break down when Spastin is overexpressed at the levels induced by this driver.
Having demonstrated statistically significant differences in Futsch localization between control and spastin mutant NMJs, we then directly examined tubulin in muscle 4 NMJ boutons using fixation conditions that reduce muscle microtubule staining (see Materials and Methods). The pattern of NMJ microtubules is complex, and is difficult to quantitatively analyze because of residual signal from microtubules in the underlying muscle. However, using the α-tubulin antibody described above, we were able to clearly visualize looped microtubule structures (Figure 9A, green) within anti-HRP-labeled boutons (magenta). These loops were present both along the branches and in the terminal boutons (inset).
Figure 9 The Microtubule Network in NMJ Boutons Is Altered or Absent in spastin Mutant Larvae
(A) In wild-type (WCS) larvae, an antibody against α-tubulin (green) reveals the distribution of the network of microtubule bundles within the A3 muscle 4 NMJ bouton arbor. Presynaptic bouton membranes are labeled by anti-HRP antibody (magenta). The microtubule network has a complex structure and extends into the terminal boutons (arrowheads and inset). Many proximal boutons have loops (arrows). Microtubules are also observed outside of the boundaries of the NMJ; these are within the muscle fiber, which also expresses α-tubulin. Staining of these muscle microtubules is minimized by the use of Bouin's fix.
(B) In spastin5.75 mutants, the microtubule network is much sparser than in controls, particularly in the distal boutons at the edges of the bouton clumps that are characteristic of spastin mutant NMJs (arrowheads and inset). Many of these distal boutons have little or no detectable α-tubulin staining. More proximal boutons still have tubulin loops, however (arrows).
Scale bar, 5 μm.
spastin5.75 NMJs exhibited weaker α-tubulin staining than wild-type controls, particularly in terminal boutons (Figure 9B). Looped microtubule structures could be seen in some boutons in mutants. However, boutons at the ends of NMJ branches or at the outer edges of the bouton clumps that are characteristic of spastin NMJs often lacked any tubulin staining (inset). Thus, our results indicate that microtubule bundles are selectively depleted from the distal boutons of NMJs in larvae lacking Spastin protein.
Discussion
Mutations in the human spastin gene, which encodes an AAA ATPase, are the most common cause of pure AD-HSP. We identified the Drosophila Spastin ortholog (see Figure 1) in a gain-of-function screen (see Figure 3). Drosophila Spastin is a cytoplasmic protein that can also localize to axons (see Figure 2). spastin-null larvae have altered NMJs in which presynaptic boutons are more numerous and smaller than in wild-type, and are organized in dense clusters (see Figure 4). These changes in bouton number and organization are rescued by expression of Spastin in neurons (see Figures 4 and S1). QC, a measure of the number of vesicles of neurotransmitter released in response to an action potential, is reduced at NMJs in both null and hypomorphic spastin mutants (see Figure 5).
spastin-null flies have severe movement defects. They cannot fly at all, and do not jump. They climb and walk very slowly, often drag their hind legs when walking (see Video S1), and have greatly reduced lifespans. spastin hypomorphs have milder phenotypes, displaying flying defects and a decrease in climbing speed (see Figure 6).
Regulation of Synaptic Microtubule Networks by Spastin
The AAA domain of Spastin is quite similar to that of Katanin-60, which is a microtubule-severing protein. To determine whether Spastin might also sever or otherwise alter microtubules in vivo, we overexpressed the protein in embryonic and larval muscles. Strikingly, this overexpression erases or greatly reduces the microtubule network (see Figure 7). These data are consistent with the finding that overexpression of human Spastin in transfected mammalian cells causes microtubule disassembly (Errico et al. 2002; McDermott et al. 2003).
Having demonstrated that Spastin can cause disassembly of microtubules in vivo, we then examined how its absence affects the synaptic microtubule cytoskeleton. Based on the overexpression phenotype, one might have expected that microtubules would be more stable or more numerous in spastin LOF mutants. However, our observations indicate the opposite: microtubule bundles are depleted in NMJ boutons when Spastin is absent.
At the wild-type muscle 4 NMJ, boutons are arranged along linear axes. Continuous microtubule bundles run along the axes and connect to larger bundles within the innervating axon. Microtubules within boutons are typically arranged in loops and swirls. In spastin-null mutants, boutons are arranged in clumps, and the distal boutons of these clumps often lack any detectable tubulin staining (see Figure 9). Looped microtubule structures are present within some proximal boutons, however, and the bundles connecting the NMJ to the axon are still present. These results suggest that the absence of Spastin selectively affects the construction of the presynaptic microtubule cytoskeleton, and that the severity of the microtubule defects in a bouton are correlated with its distance from the NMJ's axonal branchpoint.
We quantitated these defects using an antibody against the microtubule-associated Futsch protein, which defines a subpopulation of stable neuronal microtubule bundles. In wild-type larvae, Futsch staining forms continuous lines along the main branches of the NMJ. Some individual boutons have Futsch loops, while others display only diffuse staining. A comparison of wild-type and spastin-null larvae shows that the distribution of Futsch within boutons shifts from organized structures (bundles and loops) toward diffuse patterns or the absence of detectable staining. This effect is most pronounced at terminal boutons, and is rescued by neuronal expression of Spastin (see Figure 8).
If Spastin's function in vivo is to disassemble microtubules, as suggested by our overexpression experiments (see Figure 7), why does its absence produce a paradoxical reduction in microtubules within the NMJ (see Figures 8 and 9)? One possibility is that microtubule severing is required for movement of microtubules into or within the presynaptic region. Some evidence for this idea has been published. In one study, injection of function-blocking anti-Katanin-60 antibody into cultured sympathetic neurons reduced process outgrowth, and microtubules were 4- to 5-fold longer in antibody-injected neurons than in control cells (Ahmad et al. 1999). More recent work demonstrated that expression of dominant-negative Katanin-60 reduces axonal outgrowth (Karabay et al. 2004). These results were interpreted as indicating that Katanin is required for severing microtubules to a length that allows their transport along the axon to its growing tip. When Katanin is inhibited, microtubule segments may be too long to be efficiently transported, and this results in a reduction in axon outgrowth.
Based on these findings, we suggest that the depletion of microtubules in the distal boutons of spastin mutant NMJs arises because severing of axonal microtubules by Spastin is necessary to generate microtubule polymers that are short enough to be efficiently moved into and through the presynaptic terminals. Perhaps Spastin normally excises sections of microtubules at branchpoints where NMJ branches leave the axon trunk, and these severed microtubule segments (or individual tubulin dimers) are then moved distally into the boutons of the NMJ as it grows.
Is Spastin also involved in axon outgrowth or guidance, as suggested by its embryonic gain-of-function phenotype (see Figure 3)? Clearly loss of Spastin activity in Drosophila does not strongly affect outgrowth, since the embryonic CNS axon ladder develops in a normal manner and motor axons reach their appropriate targets in spastin mutants. Furthermore, axonal and muscle microtubules are not detectably altered in spastin-null embryos. Severing of microtubules in vivo, however, may usually involve the actions of multiple severing proteins. In addition to Spastin, the Drosophila genome encodes three AAA ATPases whose AAA domains are closely related to that of vertebrate Katanin-60. These are Katanin-60, CG1193, and an ortholog of mammalian Fidgetins, CG3326 (see Figure 1C). None of these proteins have been genetically characterized. Of these four proteins, Spastin is most distant from vertebrate Katanin-60, yet we have shown that Spastin overexpression causes microtubule disassembly in vivo (see Figure 7). Thus, our results suggest that all four fly proteins are microtubule-severing enzymes or proteins that otherwise facilitate disassembly of microtubule networks. Perhaps each is dedicated to severing microtubules in particular cellular and subcellular contexts, and their functions may be partially redundant. If so, generation of severe phenotypes in which microtubule networks are disrupted might require loss of two or more of these AAA ATPases. In mammals, Katanin and Spastin are both expressed in CNS neurons (Wharton et al. 2003; Karabay et al. 2004), consistent with the idea that they could have overlapping functions.
After this manuscript was submitted for initial review, a paper appeared on perturbation of Drosophila
spastin using transgenic RNAi techniques (Trotta et al. 2004). In direct contrast to our results, this paper concluded that (1) spastin is an essential gene (since crossing spastin RNAi flies to a ubiquitous GAL4 driver line was reported to produce lethality), (2) spastin RNAi larvae have reduced NMJs and an increase in synaptic transmission, and (3) loss of Spastin from neurons produces an increase rather than a decrease in stable microtubules in the NMJ.
The conclusions in our paper are based on phenotypic analysis of spastin mutations that delete part or all of the coding region and on rescue of null mutant phenotypes by neuronal expression from a transgene. Our results show that spastin is not an essential gene: even spastin-null flies can eclose and live for several days, and spastin hypomorphs, which would be expected to more closely resemble most RNAi-perturbed flies, eclose at normal rates and have lifespans and behavior that do not greatly differ from wild-type (see Figure 6). We also determined that the 17-7 mutation, which removes more than one-third of the coding region, produces no detectable alterations in bouton number or NMJ microtubules and slightly decreases synaptic transmission, while spastin-null mutants have more boutons than wild-type larvae, a reduction in NMJ microtubule bundles, and more severely reduced transmission (see Figures 4, 5, 8, and 9).
In most transgenic RNAi work in Drosophila, different transgenic lines yield phenotypes that range from hypomorphic to near-null, and transgenic RNAi does not completely eliminate expression of the target protein (e.g., Billuart et al. 2001; Kalidas and Smith 2002). In the Trotta et al. paper, it is unclear whether more than one transgenic RNAi line was analyzed, but RNAi is described as reducing the level of Spastin protein expression by less than 4-fold. Our findings on Spastin hypomorphic phenotypes imply that such RNAi larvae would not have morphological or microtubule bouton phenotypes and that adult flies would be relatively healthy. We do not understand the origin of the discrepancies between the two sets of results.
Implications of Studies of Drosophila Spastin for the Understanding of Human AD-HSP
spastin-null adult flies have severe movement defects. Their hind legs are particularly weak (see Video S1); this is interesting in light of the restriction of symptoms to the legs in most AD-HSP patients. Other aspects of the spastin mutant adult phenotypes also resemble observations made in human AD-HSP patients. The penetrance of the human spasticity phenotype is highly variable, so that some individuals carrying a spastin mutation appear unaffected, while others with the same mutation are confined to wheelchairs. In our experiments, we observed that some spastin hypomorphs exhibit normal flying behavior in a cylinder assay, while most fail to fly and crash into the cylinder base (see Figure 6A and 6B). Half of the spastin-null adults die within 4 d, but most of the survivors then live more than 25 d (see Figure 6E). The selective male lethality we observed (see Figure 6G) is also interesting in light of the discovery of a large SPG4 pedigree in which only males exhibit AD-HSP phenotypes (Starling et al. 2002).
Despite these apparent parallels, there is no evidence at present that the fly behavioral phenotypes arise through mechanisms related to those that cause human AD-HSP. The anatomy of the Drosophila nervous system is quite different from that of the mammalian spinal cord. Furthermore, AD-HSP is thought to be a neurodegenerative disease that progresses over a period of years, and it is unclear whether neurodegeneration as a result of spastin mutations could occur during the short lifespan of Drosophila. Further work will be required to determine to what extent the Drosophila system can provide an organismal model for AD-HSP pathology.
The clearest implications of our work for AD-HSP emerge from the analysis of the cellular phenotypes arising from loss of Spastin. We show that the absence of Spastin alters the microtubule network at nerve terminals. Microtubule bundles are depleted in the distal boutons of the NMJ, which is a glutamatergic synapse that resembles excitatory synapses within the mammalian spinal cord. These results suggest that microtubules within the terminals of neurons in the human spinal cord could also be disordered or absent in patients with AD-HSP. The terminals of these neurons might eventually degenerate as a consequence of these microtubule defects, leading to a selective loss of distal axon segments within the spinal cord.
Materials and Methods
Genetics and molecular biology
For the overexpression screen, approximately 6,000 new EP insertion lines were generated by crossing an X chromosome EP line, EP55, to a SbΔ2-3 transposase line. EP insertions on Chromosome II or III were crossed to the pan-neuronal driver line ElavC155-GAL4 and the muscle driver line 24B-GAL4. Embryos from EP lines that exhibited less than 20% viability in combination with either driver were immunostained with mAb 1D4. Eighteen lines had embryonic CNS and/or motor axon defects when crossed to ElavC155-GAL4, including line T32. The flanking genomic region of T32 was cloned and sequenced by plasmid rescue. This sequence matched three overlapping EST's from BDGP, one of which, GH11184, contained the complete ORF of the gene downstream of the T32 insertion. This cDNA was sequenced in its entirety. Unrooted trees for Spastin and its closest relatives were constructed using six different algorithms (fitch, kitsch, neighbor, upgma, protein maximum likelihood, and parsimony) from the Phylip package, based on alignment to the PFAM AAA consensus.
For the spastin excision lines, alleles 10-12, 17-7, and 5.75 were generated via imprecise excision of EP T32 using SbΔ2-3. All alleles were homozygous viable, and their deletions were mapped by PCR and sequencing of larval or adult genomic DNA. Allele 5.75 causes sterility in both sexes.
For the spastin rescue construct, the UAS-spastin cDNA construct was made by subcloning a 2.9-kb BglII fragment from GH11184 into the BglII site of pUAST (Brand and Perrimon 1993). This fragment contains the spastin cDNA up to 350 bp after the stop codon (excluding 681 bp of the 3′ UTR) and including 28 bp of polylinker sequence from the pOT2 plasmid at the 5′ end. The construct was injected at approximately 300 ng/μl into KiΔ2-3 embryos and several transgenic lines recovered; experiments described here used the Chromosome II insertion line 8-3-5.
Rescue of spastin-null phenotypes by spin-GAL4-driven expression was assayed by crossing UAS-spastin/CyOKr-GFP;
spastin5.75/TM3SerAct-GFP to spin-GAL4/CyOKr-GFP; spastin5.75/TM3SerAct-GFP. The numbers of Ib boutons in rescued larvae (UAS-spastin/spin-GAL4; spastin5.75/spastin5.75) were compared to those in unrescued sibling mutants (spin-GAL4/CyOKr-GFP; spastin5.75/spastin5.75) to calculate the ratios used to determine the efficacy of rescue (see Figure 4G). This was done because we observed that the presence of the driver chromosome in the background increased the absolute number of Ib boutons, so that the appropriate control was to the sibling mutants also bearing this chromosome. Rescue of spastin-null phenotypes by postembryonic Elav-GS-GAL4-driven expression was assayed by crossing UAS-spastin/CyOKr-GFP;
spastin5.75/TM3SerAct-GFP to Elav-GS-GAL4, spastin5.75/TM6B and raising the larvae on RU486-containing food as described (Osterwalder et al. 2001; McGuire et al. 2004). The numbers of Ib boutons in rescued larvae (UAS-spastin/+; Elav-GS-GAL4, spastin5.75/spastin5.75) were compared to those in unrescued sibling mutants (CyOKr-GFP/+; Elav-GS-GAL4, spastin5.75/spastin5.75) as described above for spin-GAL4 rescue. The effect of postembryonic neuronal overexpression of Spastin in a wild-type background was assayed by counting Ib bouton numbers in UAS-spastin/+; Elav-GS-GAL4, spastin5.75/TM3SerAct-GFP larvae from this same cross and comparing them to the numbers for UAS-spastin/+; Elav-GS-GAL4, spastin5.75/spastin5.75. The wild-type control lines used were Oregon R,
w1118, and WCS, a Canton S line backcrossed ten times to white (gift of Anne Simon). For larval overexpression experiments, the MHC-GS-GAL4213-3 driver was used to induce muscle-specific expression of UAS-spastin at 23 °C in the absence of RU486. Expression induced by this driver was confirmed in a separate cross using UAS-EGFP.
Generation of Spastin antibodies
Regions of the spastin cDNA encoding aa 136–416 (pGEX-T32PvuII), 1–167 (pAcG2T-T32BamRIa), and 380–758 (pAcG2T-T32BBA) were subcloned from GH11184 into bacterial (pGEX) or baculovirus (pAcG2T) expression vectors. Expressed protein (in the form of inclusion bodies for T32PvuII) was injected into guinea pigs (Covance, Princeton, New Jersey, United States) and the antiserum tested on en-GAL4/+2/+ embryos. Antisera against T32PvuII (86EX) and T32BBA (1239) both showed strong staining in the Engrailed pattern; thus, both recognized overexpressed Spastin. 86EX was purified by incubation with membrane-bound pGEX-T32PvuII protein and subsequent elution with 100 mM glycine (pH 2.5) followed by neutralization with 3M Tris (pH 8.8). pAb1239 was affinity purified using the immunogen bound to Affi-gel10 beads (Bio-Rad Laboratories, Hercules, California, United States), followed by preabsorption with spastin5.75 larval fillets.
Embryonic and larval immunocytochemistry
Stage 16 embryos were fixed and stained using standard methods (Patel 1994) with mAb 1D4 (1:5) or anti-β3-tubulin (1:500; a gift of R. Renkawitz-Pohl and D. Buttgereit). Third instar larvae were live-dissected in room temperature HL3 (see below) or PBS and fixed in 4% paraformaldehyde or Bouin's fix (for staining of NMJs with anti-tubulin) for 25 min. Primary antibodies used on larvae were mouse anti-Syt at 1:400 (Menon and Zinn 1998), rabbit anti-Dlg (1:2,000; a gift of D. Woods and P. Bryant), mAb 22C10 (anti-Futsch, 1:50; Developmental Studies Hybridoma Bank, Iowa City, Iowa, United States), rabbit anti-HRP (1:250, Cappel, MP Biomedicals, Irvine, California, United States), mouse anti-α-tubulin DM1A (1:500; Sigma, St. Louis, Missouri, United States). Anti-Spastin pAb 1239 was used at 1:300. Staining was visualized with HRP-conjugated goat anti-mouse secondary (1:200; Jackson Laboratory, Bar Harbor, Maine, United States) or Alexa-Fluor 488 and 568 anti-mouse, -rabbit, or -guinea-pig secondaries (1:200; Molecular Probes, Eugene, Oregon, United States). All fluorescently labeled samples were imaged by acquiring z-series projections with a Zeiss (Oberkochen, Germany) 510 inverted confocal microscope and 63×/1.4 n.a. or 100×/1.2 n.a. PlanApo objectives. Only larval segments A2 and A3 were analyzed. Individual boutons were defined as a Syt-positive area encircled by Dlg-positive staining (see Figure 4), or a Syt- or HRP-positive varicosity in the synaptic arbor (see Figure 8). All type Ib boutons were scored for each muscle 4 NMJ. Average bouton numbers per muscle 4 (see Figure 4) were as follows (mean ± s.e.): WCS, 44 ± 2.6 (n = 26 NMJs); spastin5.75 mutant, 68 ± 2.4 (38); spin rescue, 50 ± 4.9 (26); mutant sibling with spin driver chromosome, 86 ± 4.6 (8); Elav-GS rescue, 70 ± 3.0 (19); and mutant sibling with Elav-GS driver chromosome, 110 ± 9.3 (21). p < 0.02 by one-way ANOVA for all paired comparisons.
Electrophysiology
Intracellular recordings were obtained at 18 °C, using sharp microelectrodes (boroscilicate glass, 1.0 mm OD; 18–35 MΩ resistance; World Precision Instruments Sarasota, Florida, United States) filled with 3M KCl, from body wall muscle 6 (segments A3 or A4) of filleted third instar larvae, following standard methods (Jan and Jan 1976). Larvae were bathed in HL3 solution (Stewart et al. 1994), in mM: NaCl, 70 (EM Science, Gibbstown, New Jersey, United States); KCl, 5; MgCl2, 20; NaHCO3, 10; HEPES, 5; Sucrose, 115; Trehalose, 5; and CaCl2, 1 (Sigma). Larvae were visualized with a 5×/0.10 n.a. Olympus (Tokyo, Japan) objective on an Olympus BX50WI microscope. EJPs were evoked by pulling the cut end of the innervating segmental nerve into a heat-polished suction electrode and passing a depolarizing pulse sufficient to depolarize both motoneurons (Grass SD9 stimulator). For each experiment, 10–15 single EJPs evoked at 0.2 Hz were recorded, and then spontaneous mEJPs recorded for 1 min afterwards. Only recordings with resting membrane potential below −60 mV were acquired. The average resting membrane potential for control (WCS) larvae was −72.2 mV, and did not differ significantly from any of the experimental groups. Average muscle input resistance in control larvae was 8.9 MΩ, and differed significantly only from the input resistance determined for spastin 5.75/spastin17-7 transheterozygotes (7.5 MΩ p < 0.04). Recordings were performed using an Axon Instruments (Foster City, California, United States) Axopatch 200B amplifier with CV203BU headstage operating in current clamp mode. The signal was low-pass filtered at 5 kHz, digitized through an Axon Instruments Digidata 1322A 16-bit acquisition system, and recorded using Axon Instruments Clampex 8.2 software. Mean EJP amplitude was determined by averaging all single EJPs with Axon Instruments Clampfit 8.2, and corrected for nonlinear summation according to McLachlan and Martin (1981) and Feeney et al. (1998). mEJPs were measured using Mini Analysis Program (Synaptosoft, Decatur, Georgia, United States). mEJPs with a slow time course arising from neighboring electrically coupled muscle cells were excluded from analysis (Zhang et al. 1998). QC for a given NMJ was estimated by dividing the average EJP amplitude by the average mEJP amplitude. Statistics were calculated using one-way ANOVA.
Adult phenotypes
Eclosion rates were determined by counting numbers of empty versus full (dead) pupae on the sides of bottles in which flies had been allowed to lay for comparable time periods. The flight test assay was performed at room temperature using an opaque cylinder (a 52-cm-tall pipette washer, 18 cm in diameter) coated on the inside with fresh mineral oil. Flies of a given genotype were dumped through a hole in the center of a lid at the top. The cylinder was divided into bins along its height, and the number of flies per bin counted. Flies of different genotypes were age-matched; more than 200 flies were counted for each. The climbing assay was performed on 4–5 d old flies maintained individually in vials. Climbing velocity for each fly was measured by transferring it to an empty vial, banging it to the bottom, and then measuring either the time required to reach the top of the vial or the maximum distance it climbed in 30 s, whichever came first. Three trials were performed, and the best speed was used. For lifespan tests, flies were maintained at 25 °C, transferred every 3 d to fresh food vials, and their lifespan noted.
Supporting Information
Figure S1 Spastin Expression in Neurons Rescues the spastin Mutant Morphology
Representative muscle 4 NMJs stained with antibodies against Dlg (green) and Syt (magenta) are shown for (A) spastin5.75 mutant (genotype +/CyOKr-GFP; Elav-GS-GAL4,spastin5.75/spastin5.75), (B) neuronally rescued (UAS-spastin /CyOKr-GFP; Elav-GS-GAL4,spastin5.75/spastin5.75), and (C) neuronally overexpressing (UAS-spastin /+; Elav-GS-GAL4,spastin5.75/TM3Ser-ActGFP) larvae. The clustered, smaller, and more numerous boutons observed in mutant NMJs (A, arrowhead) are absent in neuronally rescued larvae, which resemble controls (WCS; see Figure 4D). Spastin overexpression in neurons produces an opposite morphological phenotype compared to the loss of function: boutons appear slightly larger than in wild-type, and bouton counts show that they are reduced in number (83% of control; see text). Scale bar, 5 μm.
(1.4 MB TIF).
Click here for additional data file.
Figure S2 Adult Behavior Is Partially Rescued by spin-GAL4-Driven Expression of Spastin in the spastin-Null Background
Behavioral tests were performed on flies from the four genotypes arising from the spin-GAL4 rescue crosses, raised at 18 oC. These genotypes were (1) spin-GAL4/UAS-spastin;
spastin5.75 (spin Rescue), (2) spin-GAL4/CyOKr-GFP;
spastin5.75 (non-rescued spastin mutant, denoted 5.75[R]), (3) spin-GAL4/UAS-spastin;
spastin5.75/TM3SerAct-GFP (Cy+ Ctrl; heterozygous for the spastin mutation), and spin-GAL4/CyOKr-GFP; spastin5.75/TM3SerAct-GFP (Cy Ctrl; heterozygous for the spastin mutation).
(A) Climbing behavior. None of the spastin mutants (0%) from these crosses (5.75[R]; n = 21) reached the top of the vial in the prescribed 30 s time limit, compared to 8% for Rescue flies (n = 75), and 100% for both spastin/+ controls (n = 39 and 21). Twenty-seven percent of mutants (5.75[R]) did not climb at all, compared to only 4% of the Rescue flies and 0% of the spastin/+ controls. Thus, although both genotypes in the mutant background (homozygous for spastin5.75) were much weaker than either spastin5.75 heterozygous control, Rescue flies showed improved climbing ability compared to the mutants.
(B) Similar to the results in (A), mean lifespan in spastin mutants (10 ± 1.3 d, n = 32) was significantly rescued by spin-driven expression of spastin (16 ± 1, n = 95, p < 0.004), although lifespans were much shorter in spastin5.75 homozygotes than in heterozygous spastin/+ controls (43 ± 3.1 and 44 ± 2.7; n = 20 each).
(218 KB PDF).
Click here for additional data file.
Video S1 Motor Behavior in Control, spastin5.75 Mutant, and spin-GAL4-Rescued Flies
Flies are shown moving in a vial.
Segment 1: Wild-type. One female and one male WCS fly are shown. Note the rapid rate at which they walk, as well as exhibiting climbing, jumping and flying behaviors. When still, their legs are controlled, and they are able to walk upside-down (out-of-focus fly near end of segment) for prolonged periods without falling.
Segment 2: Mutant. One spastin5.75 female is shown. Leg weakness is obvious, particularly for the mesothoracic and metathoracic legs, both when walking and standing still. She climbs poorly, and when rotated so that she is upside-down, is unable to maintain a hanging position. No wing movement or jumping is observed.
Segment 3: Rescue. In spin-GAL4/UAS-spastin;
spastin5.75 flies, Spastin expression via the spin-GAL4 driver partially rescues the movement defects seen in spastin5.75 mutants. Two males are shown, followed by one female. Note their improved leg steadiness, velocity, and hanging ability. These flies can also jump spontaneously. The female appears to be less fully rescued; however, she is able to walk upside-down for prolonged periods, and exhibits wing movement.
(7.4 MB MOV).
Click here for additional data file.
We thank Elena Armand, Anna Salazar, Tambrea Ellison, Miguel Lemus, Nora Tu, and Darya Goloub for excellent help on various aspects of this project; Catalina Ruiz-Canada and Detlev Buttgereit for advice on tubulin staining; the Caltech Biological Imaging Center for use of confocal microscopes; David Mathog for generation of phylogenetic trees; Greg Macleod for discussions on electrophysiology; Renate Renkawitz-Pohl, Dan Woods, and Peter Bryant for antibodies; Anne Simon for WCS flies; Henri Bourbon for Rox8 advice and reagents; and David Sherwood and members of the Zinn and Zhang groups for helpful discussions. This work was supported by a National Institutes of Health RO1 grant to KZ and by an American Cancer Society postdoctoral fellowship to NTS. Most of the other driver-dependent lethal EP lines from the screen that produced T32 have been maintained in the Zinn lab, and this collection is available for distribution to other investigators.
Conflicts of interest. The authors have declared that no conflicts of interest exist.
Author contributions. NTS, QS, MX, BZ, and KZ conceived and designed the experiments. NTS, QS, and MX performed the experiments. NTS, QS, MX, and KZ analyzed the data. BZ oversaw the electrophysiology experiments and analysis. NTS and KZ wrote the paper.
Academic Editor: Michael Bate, University of Cambridge
¤1 Current address: Computational Biology Service Unit, Cornell Theory Center, Ithaca, New York, United States of America
¤2 Current address: Division of Neuroscience, Baylor College of Medicine, Houston, Texas, United States of America
Citation: Sherwood NT, Sun Q, Xue M, Zhang B, Zinn K (2004) Drosophila Spastin regulates synaptic microtubule networks and is required for normal motor function. PLoS Biol 2(12): e429.
Abbreviations
aaamino acid
AD-HSPautosomal dominant hereditary spastic paraplegia
CNScentral nervous system
DlgDiscs-large
EJPexcitatory junction potential
GS
GeneSwitch
LOFloss of function
mAbmonoclonal antibody
mEJP“mini” excitatory junction potential
NMJneuromuscular junction
QCquantal content
sca
scabrous
spin
spinster
SytSynaptotagmin
VNCventral nerve cord
WCS
Canton S
w−
==== Refs
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| 15562320 | PMC532392 | CC BY | 2021-01-05 08:21:17 | no | PLoS Biol. 2004 Dec 30; 2(12):e429 | utf-8 | PLoS Biol | 2,004 | 10.1371/journal.pbio.0020429 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 1556232110.1371/journal.pbio.0020432Research ArticleCell BiologyImmunologyInfectious DiseasesVirologyVirusesMus (Mouse)Replication of Norovirus in Cell Culture Reveals a Tropism for Dendritic Cells and Macrophages First In Vitro Replication of a NorovirusWobus Christiane E
1
Karst Stephanie M
1
Thackray Larissa B
1
Chang Kyeong-Ok
2
Sosnovtsev Stanislav V
2
Belliot Gaël
2
Krug Anne
1
¤Mackenzie Jason M
3
Green Kim Y
2
Virgin Herbert W. [email protected]
1
1Department of Pathology and Immunology, Washington University School of MedicineSt. Louis, MissouriUnited States of America2Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human ServicesBethesda, MarylandUnited States of America3Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, and Clinical Medical Virology Centre, University of QueenslandBrisbaneAustralia12 2004 30 11 2004 30 11 2004 2 12 e43226 5 2004 13 10 2004 Copyright: © 2004 Wobus et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Norovirus Cultured for the First Time
Noroviruses are understudied because these important enteric pathogens have not been cultured to date. We found that the norovirus murine norovirus 1 (MNV-1) infects macrophage-like cells in vivo and replicates in cultured primary dendritic cells and macrophages. MNV-1 growth was inhibited by the interferon-αβ receptor and STAT-1, and was associated with extensive rearrangements of intracellular membranes. An amino acid substitution in the capsid protein of serially passaged MNV-1 was associated with virulence attenuation in vivo. This is the first report of replication of a norovirus in cell culture. The capacity of MNV-1 to replicate in a STAT-1-regulated fashion and the unexpected tropism of a norovirus for cells of the hematopoietic lineage provide important insights into norovirus biology.
Noroviruses -- which cause epidemic gastroenteritis -- can now be grown in cells of the innate immune system, providing a tool to examine this pathogen while offering insights into norovirus biology
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Introduction
Viruses within the genus Norovirus (formerly “Norwalk-like viruses”) of the family Caliciviridae are major agents of acute gastroenteritis (Green et al. 2001). Norovirus research, including the development of prevention and control strategies, has been hampered by the failure to grow these viruses in cultured cells despite extensive efforts (Duizer et al. 2004). Most noroviruses identified thus far have been associated with gastrointestinal disease in humans, but members of the genus have been found in other species as well (Green et al. 2001; Karst et al. 2003). Our recent discovery of the first murine norovirus, murine norovirus 1 (MNV-1), and demonstration of its ability to infect the intestinal tract of mice following oral inoculation provided an opportunity to analyze the pathogenesis of this norovirus in mice (Karst et al. 2003). This previous study demonstrated that the cellular transcription factor STAT-1 and interferon (IFN) receptors are critical for resistance to MNV-1 infection in vivo. The availability of MNV-1 and STAT1-deficient (STAT1−/−) mice (Durbin et al. 1996; Meraz et al. 1996) that are highly susceptible to MNV-1 infection allowed us to revisit efforts to develop a cell culture system for noroviruses.
Here we show for the first time that MNV-1 grows in macrophages (MΦ) and dendritic cells (DCs) and provide the first tissue culture model for a norovirus. Using this model we demonstrate that MNV-1 growth in vitro was inhibited by the IFN-αβ receptor and STAT-1. In addition, we isolated the first three-times plaque-purified strain of MNV-1 (MNV-1.CW1) and characterized it in vitro and in vivo. Sequencing of serial passages of MNV-1.CW1 indicated remarkable sequence stability over time and indicated that an amino acid substitution in the capsid protein of serially passaged MNV-1 was associated with a loss of virulence in vivo.
Results/Discussion
MNV-1 Replicates in Murine MΦ and DCs
As part of our ongoing investigation into MNV-1 pathogenesis, STAT1−/− mice were infected with MNV-1 by the oral route and tissue sections analyzed by immunohistochemistry for the presence of MNV-1 protein. MNV-1-specific staining was observed in spleen and liver 2 d postinfection (Figure 1). Interestingly, in the liver, Kupffer cells (resident macrophages of the liver) lining the sinusoids were specifically stained by MNV-1 immune serum (compare Figure 1A and 1B). In the spleen, staining was found primarily in the red pulp and the marginal zone, but also in non-lymphoid cells within the white pulp (Figure 1C and 1D). This pattern is consistent with staining of MΦ and DCs (Metlay et al. 1990; Leenen et al. 1998). Furthermore, in some cases virus-antigen-positive MΦ were detected (Figure 1C).
Figure 1 MNV-1-Specific Staining In Vivo Occurs in Cells of the MΦ Lineage
Immunohistochemistry was performed on liver (A and B) and spleen (C and D) sections from STAT1−/− mice 2 d after oral infection. MNV-1-specific staining was seen in Kupffer cells of infected livers when probed with MNV-1 immune (A) but not preimmune (B) serum. A selected Kupffer cell lining the sinusoid is indicated by an arrowhead. MNV-1-specific staining consistent with MΦ was seen in red pulp (C) and marginal zone (D) in the spleen. The arrow indicates a cell with MΦ morphology. No staining was observed in tissues from mice infected for 1 d, in infected tissues incubated with preimmune serum, or in mock-infected tissues incubated with immune serum. RP, red pulp; WP, white pulp.
Because cells containing viral antigen in infected mice resembled MΦ, we examined whether cells of the hematopoietic lineage such as MΦ and DCs were permissive for MNV-1 replication in vitro. Bone-marrow-derived MΦ (BMMΦ) and bone-marrow-derived DCs (BMDCs) were inoculated with a MNV-1 stock derived from the brain of infected IFNαβγ receptor−/− (IFNαβγR−/−) mice (Karst et al. 2003). Cytopathic effect (CPE) in cell monolayers was visible within 2 d in STAT1−/− BMMΦ and BMDCs, but not STAT1−/− murine embryonic fibroblasts (MEFs) (Figure 2A). While BMDCs showed CPE even when STAT-1 was present, wild-type (wt) BMMΦ exhibited less CPE than their STAT1−/− counterparts. These data showed that MNV-1 had a marked tropism for MΦ and DCs but not fibroblasts.
Figure 2 MNV-1 from Brain Homogenate Replicates in Cells of the DC and MΦ Lineage In Vitro
BMDCs and BMMΦ, as well as MEFs from wt or STAT1−/− mice, and RAW 264.7 cells were infected with a MOI of 0.05.
(A) MNV-1 causes CPE in permissive cells. MNV-1- or mock-infected cells were observed by light microscopy 2 d postinfection. The boxed area is magnified further to show the border of the plaque.
(B) Infected cell lysates were analyzed in two to four independent experiments by plaque assay at various timepoints postinfection to calculate standard deviations. For wt BMMΦ, MNV-1 growth was detected in two out of four experiments.
We used this information to screen available MΦ cell lines for growth of MNV-1, including the murine lines RAW 264.7 (Raschke et al. 1978) and J774A.1 (Ralph et al. 1975), and the human/murine hybrid line WBC264-9C (Aksamit 1986). These cells also showed visible CPE when inoculated with the MNV-1 stock (Figure 2, data not shown). Plaques were observed when infected RAW 264.7 monolayers were maintained under agarose (Figure 2A), allowing us to develop a plaque assay and quantitate virus titers.
STAT1−/− BMMΦ, STAT1−/− and wt BMDCs, and RAW 264.7 cells consistently supported the growth of MNV-1, while wt BMMΦ varied in their ability to support virus growth (Figure 2B). BMMΦ and BMDCs cells lacking STAT-1 always yielded higher MNV-1 titers than their wt counterparts. Furthermore, a low level of virus replication was observed in STAT1−/− MEFs, but as reported previously, no virus growth was observed in wt MEFs (Karst et al. 2003). MNV-1 replication proceeded rapidly in permissive cells, with newly synthesized infectious virions first detected in cell lysates 9 to 12 hours postinfection (h.p.i.). Taken together, these data indicated that MNV-1 could productively infect MΦ and DCs.
Verification of Viral Growth In Vitro
Several approaches were used to verify that the observed CPE and plaques were caused by MNV-1. We first performed a clonal selection from the MNV-1 stock (from infected brain tissue) with three rounds of plaque purification in RAW 264.7 cells to generate the MNV-1.CW1 strain. This strain was amplified in RAW 264.7 cells, after which virus particles were concentrated and subjected to purification by isopycnic centrifugation in CsCl. A distinct band was observed in CsCl gradients at a density of 1.35 ± 0.01 g/cm3, consistent with that described for noroviruses (Kapikian et al. 1996). Examination of the material in this fraction by negative staining electron microscopy showed the presence of virus particles with calicivirus morphology (Figure 3A). Furthermore, SDS-PAGE analysis of this material revealed a major protein of approximately 59 kDa, consistent with the calculated mass of the MNV-1 capsid protein (Figure 3B,C). Western blot analysis with antibodies generated against bacterially expressed MNV-1 capsid protein (Figure 3B) and mass spectrometry (data not shown) confirmed its identity as the MNV-1 capsid protein. A genomic-sized RNA molecule of approximately 7.4 kb was detected in nucleic acid isolated from the purified virions with a probe specific for the MNV-1 genome in Northern blots (data not shown). Finally, a neutralization assay was performed with the monoclonal antibody (mAb) A6.2 specific for the MNV-1 capsid protein (see Materials and Methods). MAb A6.2 specifically bound to CsCl-purified MNV-1 virions in an immunoassay, while the isotype-matched mAb 10H2, an anti-reovirus μ1c mAb (Virgin et al. 1991), did not bind (Figure 3D). MAb A6.2, but not the isotype control antibody 10H2, showed neutralization activity in a plaque reduction assay for both the virus in the original brain homogenate (MNV-1), and the three-times plaque-purified strain MNV-1.CW1 (Figure 3E). Together these data confirmed that MNV-1 was the infectious agent associated with viral growth observed in the infected cell cultures.
Figure 3 Characterization of the Triple Plaque-Purified Strain MNV-1.CW1
(A–C) MNV-1.CW1 purified on CsCl density gradients was visualized by (A) negative staining electron microscopy, (C) Coomassie staining, and (B) Western blot analysis with a polyclonal anti-MNV-1-capsid antibody. Molecular weight markers are indicated in kiloDaltons.
(D) Specific binding of mAb A6.2 to two different concentrations of CsCl-purified MNV-1 particles in an enzyme-linked immunosorbent assay.
(E) Neutralization of MNV-1 from brain homogenate and MNV-1.CW1 by mAb A6.2 but not the isotype control (10H2) mAb in a plaque neutralization assay. The assay was repeated three times to calculate standard deviations. The limit of detection is indicated by the dashed line.
(F) Timecourse of viral RNA synthesis in RAW 264.7 cells. Northern blot analysis of viral RNA from cells infected with MNV-1.CW1 (MOI of 2.0) or mock-infected cells. The size of RNA markers in kilobases is shown on the left. The positions of subgenomic- and genomic-length RNA are indicated on the right. This timecourse is a representative of two independent experiments.
(G) Timecourse of viral protein synthesis in infected RAW 264.7 cells. MNV-1-specific proteins were precipitated from radiolabeled cell lysates of MNV-1.CW1-infected RAW 264.7 cells (MOI of 2.0) at indicated times after infection. The size of the proteins in kiloDaltons is indicated.
MNV-1 RNA and Protein Production in Permissive Cells
To compare MNV-1 replication in cells with that of other caliciviruses, we analyzed viral RNA and protein synthesis in MNV-1.CW1-infected RAW 264.7 cells. Northern blot analysis using a probe specific for the positive strand of the MNV-1 genome showed an increase in the accumulation of full-length (7.4 kb) and subgenomic-length (2.3 kb) MNV-1 genome over time (Figure 3F). Radiolabeled MNV-1-infected RAW 264.7 cell lysates were analyzed by immunoprecipitation with serum from a MNV-1 infected mouse, and a 59-kDa protein consistent with the capsid protein was detected as early as 6 h.p.i. (Figure 3G). Additional proteins accumulated over time that corresponded in size to expected calicivirus nonstructural proteins such as the 76-kDa proteinase-polymerase precursor and an approximately 40-kDa NTPase protein (Sosnovtsev et al. 2002). These data showed that the viral RNA and proteins synthesized in infected cells were consistent with calicivirus replication (Green et al. 2001).
Ultrastructural Examination of MNV-1-Infected RAW 264.7 Cells
Positive-strand RNA viruses (Dales et al. 1965; Mackenzie et al. 1999; Pedersen et al. 1999), including caliciviruses (Love et al. 1975; Studdert and O'Shea 1975; Green et al. 2002), are known to replicate in association with intracellular membranes. Therefore, we examined the ultrastructural morphology of MNV-1.CW1-infected RAW 264.7 cells (Figure 4). Over time, virus-infected cells showed a striking change in overall morphology and intracellular organization (Figure 4D–4L) compared to mock-infected cells (Figure 4A–4C). Structures resembling virus particles were observed within or next to single- or double-membraned vesicles in the cytoplasm by 12 h.p.i. (Figure 4D). The vesiculated areas increased in size with time (Figure 4G–4I), and by 24 h.p.i., large numbers of these vesicles and viral particles occupied most of the cytoplasm, displacing the nucleus (Figure 4J–4L). In addition, a complete rearrangement of intracellular membranes with some confronting membranes occurred (Figure 4J), leading to a rearrangement of the endoplasmic reticulum and loss of an intact Golgi apparatus (Figure 4E; data not shown). Interestingly, these smooth-membraned vesicles were often surrounded by mitochondria. A small proportion of cells also showed crystalline arrays of cytoplasmic virus particles (data not shown). These observations indicate that like other positive-strand RNA viruses, norovirus RNA replication likely occurs in association with intracellular membranes.
Figure 4 Ultrastructural Studies of MNV-1.CW1-Infected RAW 264.7 Cells
Cells were infected with MNV-1.CW1 (P3) (MOI of 2.0) (D–L) or mock-infected (A–C) and processed for electron microscopy 12 (D–F), 18 (G–I), or 24 (A–C; J–L) h.p.i. MNV-1 particles are indicated by arrows and confronting membranes by arrowheads. VA, vesiculated areas; Nuc, nucleus; rER, rough endoplasmic reticulum. Scale bars, 200 nm for (A), (D), (G), and (J); 500 nm for (B), (E), (H), and (K); 2 μm for (C), (F), (I), and (L).
Characterization of the Plaque-Purified Strain MNV-1.CW1 In Vitro
To determine whether the plaque purification and sequential amplification of MNV-1 in RAW 264.7 cells had altered its growth characteristics, different cell types were infected with passage (P) 3 of MNV-1.CW1. In general, the growth of MNV-1.CW1 (P3) in wt or STAT1−/− MΦ and MEFs (Figure 5A) as well as RAW 264.7 cells (data not shown) was similar to that observed for the original parental MNV-1 virus stock (compare Figure 2B and 5A). Virus titers were reproducibly higher in STAT1−/− cells compared to wt cells, and MNV-1.CW1 (P3) growth was consistently observed in wt BMMΦ. These data demonstrated that our plaque purification and serial passage in RAW 264.7 cells had not changed the tropism of the virus for primary DCs and MΦ and confirmed the importance of STAT-1 in controlling MNV-1 growth at the cellular level.
Figure 5 Critical Role for STAT-1 in Limiting MNV-1 Growth In Vitro
(A) MNV-1.CW1 has no defect in viral growth in vitro. Growth curves (MOI of 0.05) were performed two or three times with MNV-1.CW1 (P3) on indicated cells to calculate standard deviations.
(B) MNV-1 growth in MΦ is controlled by STAT-1 and Type I IFNs. BMMΦ of the indicated genotype were infected with MNV-1.CW1 (P3) at the indicated MOI. The experiment was performed twice to calculate standard deviations. The p-values for PKR versus wt infection at MOI 0.05 and 2.0, 0.8867 and 0.1616, respectively, are not significant. Statistical analysis was performed using the paired t-test (GraphPad Prism, version 3.03).
Cellular Factors Controlling MNV-1 Growth In Vitro
Previous studies demonstrated that a lack of STAT-1 or both IFNαβR and IFNγR increase susceptability to MNV-1 infection. Mice lacking individual IFNR, inducible nitric oxide (iNOS)−/−, or protein kinase R (PKR)−/− are not susceptible (Karst at al. 2003). Therefore, we determined whether molecules other than STAT-1 exhibited antiviral effects at the level of the infected cell. Primary BMMΦ from wt mice or mouse strains deficient in STAT-1, IFNαβR, IFNγR, IFNαβγR, iNOS, or PKR were directly compared for their ability to support virus replication at two different multiplicities of infection (MOIs) (Figure 5B). Again, BMMΦ cells from both wt and STAT1−/− mice supported MNV-1 virus replication, with higher titers observed in cells deficient in STAT-1. Cells obtained from mice lacking both Type I and II IFNR (IFNαβγR−/−) or Type I IFNR alone (IFNαβR−/−) supported replication of virus as efficiently as STAT1−/− cells. In addition, wt BMMΦ and wt BMDCs secrete IFNα after MNV-1-infection, as determined by IFNα enzyme-linked immunosorbent assay (ELISA) (data not shown). This is consistent with a direct role for IFN signaling in MNV-1 growth but does not rule out the possibility that effects of STAT-1 and IFNαβR occur in vivo prior to explantation of the bone marrow. Absence of IFNγR, iNOS, or PKR did not have a statistically significant effect on MNV-1 growth in BMMΦ. Together, these data demonstrate that the antiviral molecules STAT-1 and IFNαβ are part of a cellular response that limits norovirus growth.
Characterization of the Plaque-Purified Strain MNV-1.CW1 In Vivo
To address the effects of cell culture adaptation on virulence, STAT1−/− mice were infected orally with MNV-1.CW1 from three successive passages (P1, P2, and P3) (Figure 6A). Oral administration of MNV-1.CW1 (P1) resulted in lethal infection, similar to that previously reported for the parental MNV-1 brain tissue stock (Karst et al. 2003). These data fulfill a Koch's postulate with regard to MNV-1 infection and are consistent with the identification of MNV-1 as the infectious agent that was passaged in animals in our initial studies (Karst et al. 2003). In contrast, MNV-1.CW1 (P3) failed to cause a lethal infection in STAT1−/− mice after oral inoculation, even when administered a dose of 1.5 × 106 plaque-forming units (pfu), 5,000 times greater than the lethal dose for P1. In addition, immunohistochemical analysis of sectioned spleen and liver from STAT1−/− mice infected orally with 1.5 × 106 pfu of MNV-1.CW1 (P3) did not reveal any MNV-1-specific staining, unlike the parental virus (see Figure 1, data not shown). This striking difference in virulence and decrease of viral antigen in infected mice, coupled with an intermediate lethality phenotype of the MNV-1.CW1 (P2) virus, showed that serial passage of the virus in cell culture could attenuate MNV-1 virulence in vivo.
Figure 6 Changes in Virulence of Plaque-Purified MNV-1 over Multiple Passages Are Associated with Limited Amino Acid Changes
(A) Serial passage of MNV-1.CW1 in cell culture causes attenuation. STAT1−/− mice were infected orally with the indicated virus dose. The number of mice analyzed is indicated in parentheses.
(B) Summary of sequence analysis of MNV-1 over several passages. The nucleotide and amino acid differences between the indicated viruses are shown (for detail see Table 1).
Table 1 Sequence Analysis of MNV-1 over Several Passages
Nucleotides are numbered according to consensus sequence of the parental MNV-1 virus genome (in brain tissue stock) as follows: ORF1 (nt 6–5,069), ORF2 (nt 5,056–6,681), and ORF3 (nt 6,681–7,307), encoding a large polyprotein (viral nonstructural proteins), VP1 (major capsid structural protein), and VP2 (minor capsid structural protein), respectively. Amino acid residues are numbered according to location in the corresponding ORF. The nucleotide position of interest is underlined and its location in the codon of the translated ORF is shown. Sequence heterogeneity at a particular residue was determined from the sequence chromatogram, and the data shown represent direct sequence analysis of PCR-amplified cDNA products. A change in deduced amino acid sequence from the previous passage is indicated in parentheses
Molecular Analysis of Serially Passaged MNV-1.CW1
To examine the molecular basis for this attenuation, consensus sequence analysis was performed on the RNA genome of MNV-1 present in the original brain tissue stock (parental virus), and in viruses from each subsequent cell culture passage of MNV-1.CW1 (P1 through P3) (Figure 6B; Table 1). Three nucleotide changes occurred between the parental virus and P1, with one of these resulting in an amino acid substitution (histidine to arginine) at residue 845, located within the predicted “3A-like” region of the nonstructural polyprotein. In the P2 virus, which retained virulence but at a reduced level compared to the parental and P1 viruses, a second nucleotide substitution within the predicted “3A-like” coding region was observed that caused an amino acid change (valine to isoleucine) at residue 716. The partial attenuation of virulence of the P2 virus in vivo is of interest since the homologous protein in poliovirus, the 3A protein, alters the amount of cytokines secreted from cells, with likely effects on viral pathogenesis (Dodd et al. 2001). Of note, a mixed population of A and G nucleotides was detected at position 5,941 of the P2 viral genome that could potentially yield two populations of virus with either amino acid lysine or glutamic acid at residue 296 of the capsid protein. In the P3 virus, which was avirulent in mice, the G nucleotide sequence at position 5,941 emerged as the predominant sequence. The resulting amino acid substitution was of interest because of its location within the hypervariable P2 domain, which contains the putative receptor-binding site (Prasad et al. 1994, 1999; White et al. 1996). However, altered virus binding to permissive cells cannot explain the attenuated phenotype since the parental virus and MNV-1.CW1 (P3) replicate to similar levels in BMDCs and BMMΦ in vitro. Similar to our findings, the P2 domain was also implicated in attenuation of porcine enteric calicivirus virulence (Guo et al. 2001). This study suggests that the norovirus capsid protein, especially the hypervariable P2 domain, and possibly the 3A-like protein, may be important sites for the development of virulence-attenuating mutations.
Conclusion
Detection of MNV-1-positive cells of the MΦ and DC lineage in infected organs of STAT1−/− mice led to our finding that MNV-1 grows in these cell types in vitro. This provides the first tissue culture model for a norovirus. In addition, the antiviral Type I IFN response with signaling through STAT-1 is crucial for resistance to murine norovirus infection in vivo and in vitro. Taken together the previous in vivo data (Karst et al. 2003) and the tropism of this norovirus for cells of the innate immune system, underscore the importance of the innate immune response, specifically STAT-1 and Type I IFNs, in resistance against norovirus infection. These data may aid the development of a culture system for human noroviruses since neither cells of the MΦ/DC lineage nor cells with defects in the Type I IFN/STAT-1 antiviral pathway have likely been investigated (Duizer et al. 2004). Furthermore, this MNV-1 tissue culture model will help elucidate stages of the viral life cycle and cellular factors essential for norovirus replication that may provide targets for prevention or control of an important human disease.
The demonstration of a tropism of MNV-1 for DCs was unexpected, as a calicivirus tropism for DCs has not been previously described. However, like MNV-1, other caliciviruses do interact with MΦ. Viral RNA from rabbit hemorrhagic disease virus, a lagovirus, was detected in splenic and alveolar MΦ by in situ hybridization (Kimura et al. 2001). In addition, feline calicivirus, a vesivirus, showed a small, transient increase in viral titers in alveolar MΦ cultures, indicative of abortive infections (Langloss et al. 1978). It is possible that MΦ contribute to the spread of the virus through the host, but it must be noted that MΦ supported MNV-1 growth to a lower extent than DCs unless they lacked specific immune defense molecules such as STAT-1. This argues that MΦ may be the cell through which STAT1-dependent innate immunity limits MNV-1 virulence (Karst et al. 2003).
In contrast to MΦ, DCs were permissive even when STAT-1 was present (see Figure 2). DCs are sentinels of the immune system whose function is to acquire antigens and stimulate lymphocytes. Intestinal DCs are found in the gut in specialized lymphoid tissues where they can sample enteric antigens by extending their dendrites into the gut lumen (Stagg et al. 2003; Kraehenbuhl and Corbett 2004). We therefore speculate that DCs in humans and mice provide noroviruses access to subepithelial regions of the intestine, thereby contributing to norovirus disease pathogenesis. Further studies are in progress to address the role of MΦ and DCs in the intestine and the physiologic relevance of these cells for MNV-1 pathogenesis in general.
Material and Methods
Cell cultures and mice
MEFs were generated and cultured as described previously (Pollock et al. 1997). RAW 264.7 cells were purchased from
ATCC (Manassas, Virginia, United States) and maintained in DMEM (Cellgro, Mediatech, Herndon, Virginia, United States) supplemented with 10% low-endotoxin fetal calf serum (SH30070.03, HyClone, Logan, Utah, United States), 100 U penicillin/ml, 100 μg/ml streptomycin, 10 mM HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), and 2 mM L-glutamine (Biosource, Camarillo, California, United States). Bone marrow was harvested and MΦ were cultured as described previously (Heise et al. 1998). To culture DCs, bone marrow cells were resuspended in RPMI1640 containing 10% low endotoxin fetal calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate (Biosource), 100 U penicillin/ml, 100 μg/ml streptomycin, 1% nonessential amino acids (Biosource), and 20 ng/ml recombinant mouse GM-CSF (BD Biosciences, San Jose, California, United States) and plated at a concentration of 3 × 105 cells/ml in six-well plates in a total volume of 3 ml per well. The percentage of CD11c-positive DCs was determined by FACS staining after culturing cells for 7 d at 37 °C and 5% CO2. Approximately 70% of the cells were CD11c positive.
Wt 129 and STAT1−/− mice were purchased from Taconic (Germantown, New York, United States). IFNαβR−/−, IFNγR−/−, and IFNαβγR−/− (Muller et al. 1994), PKR−/− (Yang et al. 1995), and iNOS−/− (MacMicking et al. 1995) mice were bred and housed at Washington University in accordance with all federal and university policies.
Preparation of rabbit anti-MNV-1 serum
Rabbits were immunized subcutaneously with 140 μg of MNV-1 VLPs in complete Freunds adjuvant and boosted 4 or 8 wk later with 70 μg of MNV-1 VLPs or 50 μg of UV-inactivated CsCl-purified MNV-1 in incomplete Freunds adjuvant. Serum was collected two weeks after the last boost, heat inactivated, and filtered before use.
Immunohistochemistry
Seven-week-old STAT1−/− mice were infected orally with 25μl of brain homogenate containing MNV-1 (6 × 105 pfu) or brain homogenate from uninfected mice. Organs were collected into 10% buffered formalin and embedded in paraffin for sectioning by standard methods. Immunohistochemistry was performed as described previously (Weck et al. 1997) using tyramide signal amplification (NEN Life Science Products, Boston, Massachusetts, United States). Slides were blocked in tyramide signal amplification blocking reagent (NEN Life Science Products) containing 10% mouse serum (IHC blocking buffer) for 30 min before adding antibodies. Serum was diluted 1:20,000 (spleen) or 1:100,000 (liver) in IHC blocking buffer, and tissue sections were incubated overnight at 4 °C. Horseradish peroxidase–conjugated donkey anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, United States) was diluted 1:250 in IHC blocking buffer and applied to tissue sections for 1 h at room temperature. Biotin-tyramide was added at a dilution of 1:50 in 1× amplification diluent (NEN Life Science Products) for 10 min, slides were washed, and horseradish peroxidase–conjugated streptavidin (NEN Life Science Products) was added at a 1:100 dilution in tyramide signal amplification blocking reagent and incubated for 30 min at room temperature before washing. Antigen was visualized by a 3-min staining with a solution of 3, 3′-diaminobenzidine (Vector Laboratories, Burlingame, California, United States). Slides were washed and lightly counterstained with hematoxylin, dehydrated, and covered with Cytoseal XYL (Richard Allan Scientific, Kalamazoo, Michigan, United States) coverslips. No staining was observed in infected tissues incubated with preimmune serum or mock-infected tissues incubated with immune serum.
Infection of cells
Adherent cells were plated in 12-well plates and allowed to attach for several hours. Infections were carried out at an MOI of 0.05 or 2.0 for 30 min on ice in a volume of 0.5 ml per well. DCs were infected in bulk in the same volume. Cells were then washed twice with 2 ml of ice-cold PBS per well. To allow viral entry, 1 ml of medium was added to each well, and cells were incubated at 37 °C and 5% CO2 for different time periods. For growth curve samples, infected cells and media were subjected to two or three cycles of freezing and thawing before plaque titration.
Generation of mAb A6.2
A MNV-1-seropositive 129 mouse was injected intraperitoneally with 100 μl of a brain homogenate containing MNV-1, and the spleen was harvested 3 d later. Hybridoma fusions were performed as described previously (Virgin et al. 1991) with the following modifications. Hybridoma supernatants were screened for binding to recombinant MNV-1 capsids by ELISA as described (Karst et al. 2003). Stable hybridomas were characterized by Western blotting and ELISA after two rounds of subcloning by limiting dilution. A6.2 was unable to detect MNV-1 capsid protein by Western blot analysis but specifically bound to recombinant MNV-1 capsids by ELISA. The A6.2 isotype is IgG2a and was determined using the mouse mAb isotyping kit (Amersham Biosciences, Amersham, United Kingdom) and following manufacturer's protocol.
MNV-1 plaque assay and plaque neutralization assay
RAW 264.7 cells were seeded into six-well plates at a density of 2 × 106 viable cells per well. On the following day, 10-fold dilutions of virus inoculum were prepared in complete DMEM medium and plated in duplicate wells. Plates were incubated for 1 h at room temperature on a rocking apparatus before aspirating the inoculum and overlaying the cells with 2 ml of 37–40 °C 1.5% SeaPlaque agarose in MEM supplemented with 10% low-endotoxin fet al.calf serum, 1% HEPES, 1% penicillin/streptomycin, and 2% glutamine (complete MEM) per well. Plates were incubated at 37 °C and 5% CO2 for 2 d. To visualize plaques, cells were stained with 2 ml of 56 °C 1.5% SeaKem agarose in complete MEM containing 1% neutral red per well for 6–8 h.
For plaque neutralization assays, differing concentrations of purified mAb (A6.2, anti-MNV-1 capsid; isotype control, 10H2, anti-reovirus μ1c) were incubated with equal plaque-forming units of either MNV-1.CW1 or MNV-1 brain homogenate for 30 min at 37 °C prior to performing the MNV-1 plaque assay.
Purification of virus particles
RAW 264.7 cells were infected with MNV-1.CW1 for 2 d at an MOI of 0.05. Cellular debris was removed from freeze/thaw lysates by low-speed centrifugation for 20 min at 3,000 rpm. Supernatants were layered on top of a 5-ml 30% sucrose cushion and centrifuged at 4 °C for 2.5 h at 27,000 rpm (90,000 g) in a SW32 rotor. Cell pellets were then resuspended in PBS and mixed with CsCl to a final density of 1.335 g/cm3 and centrifuged for at least 18 h at 35,000 rpm (115,000 g) in a SW55 rotor. A wide lower band (1.35 ± 0.01g/cm3) and narrow upper band (1.31 ± 0.01g/cm3) were typically seen in the gradient. Each band was collected by puncturing the side of the tube with a needle before overnight dialysis against PBS at 4 °C.
Protein analysis
CsCl-purified virions were separated by SDS-PAGE gel electrophoresis using standard protocols (Sambrook et al. 1989). Proteins were visualized by Coomassie blue staining using the Simply Blue safe stain (Invitrogen, Carlsbad, California, United States) according to manufacturer's instructions. For Western blot analysis, proteins were transferred to nitrocellulose membrane and incubated with an anti-MNV-1-capsid rabbit polyclonal antibody, followed by a peroxidase-labeled secondary antibody, and visualized by ECL (Amersham Biosciences) according to manufacturer's instructions. Immunoprecipitation of radiolabeled infected cell lysates was performed as described previously (Sosnovtsev et al. 2002) with serum obtained from a 129 wt mouse infected orally with MNV-1.
Northern blotting
The region of the MNV-1 genome from nt 5,617 to 7,039 was amplified by RT-PCR and cloned into the pGEM-T Easy (Promega, Madison, Wisconsin, United States) vector between the T7 and SP6 promoters. The resulting plasmid was linearized with Bsu361 and in vitro transcribed with SP6 RNA polymerase (Roche, Indianapolis, Indiana, United States) to generate RNA transcript probes for detection of positive-sense viral RNA, or with T7 polymerase (Roche) to generate transcripts for detection of negative-sense viral RNA. To label probes, the transcription reaction was carried out in the presence of [P32]-UTP according to manufacturer's recommendations. Total RNA from virus-infected or mock-infected cells was isolated using Trizol (Invitrogen) according to the manufacturer's recommendations. One microgram of total RNA from MNV-1- or mock-infected cells was subjected to electrophoresis on a 1% formaldehyde gel. RNA Millennium Size Markers (Ambion, Austin, Texas, United States) were used as size markers. Northern blotting was performed using standard protocols (Sambrook et al. 1989). Probes were hybridized overnight at 68 °C in 50% formamide containing 6× SSC, 5× Denhardt's, 0.5% SDS, and 100 μg/ml ssDNA.
MNV-1 ELISA
The ELISA was performed as described previously (Karst et al. 2003) with the following modifications. ELISA plates were coated overnight at 4 °C with CsCl-purified MNV-1 particles at 0.2 or 1.0 μg/well. Diluted purified anti-MNV-1-capsid (A6.2) and isotype control (reovirus 10H2) mAbs, as well as the peroxidase-labeled secondary antibodies, were incubated for 60 min at 37 °C.
Electron microscopy
Negative staining electron microscopy of CsCl-purified virions was performed as described previously (Karst et al. 2003). For thin-section electron microscopy, RAW cells were infected with MNV-1.CW1 at an MOI of 2.0, as described above. At various times postinfection cells were washed with PBS and fixed with 3% glutaraldehyde diluted in PBS at room temperature for 2 h. Cells were pelleted and washed with buffer prior to incubation with 1% osmium tetroxide (in 0.1 M cacodylate buffer) for 40 min at room temperature. After washing, the cells were incubated overnight at 4 °C in 2% uranyl acetate/80% acetone. The pellets were then dehydrated with an acetone series and embedded in Epon before polymerization at 65 °C for 72 h. Ultrathin sections (60 nm) were cut with a Micro Star (Huntsville, Texas, United States) diamond knife, and the sections were stained and contrasted with uranyl acetate and lead citrate before viewing on a JOEL 1010 electron microscope at 80 kV. Images were captured on a MegaView III side-mounted CCD camera (Soft Imaging System, Lakewood, Colorado, United States), and figures were processed using Adobe Photoshop software (Adobe Systems, San Jose, California, United States).
Consensus sequence analysis of viral RNA
RNA was extracted from brain tissue or cell culture material with Trizol (Invitrogen) and reverse transcribed with Superscript II enzyme (Invitrogen). Genome-specific sequences were PCR-amplified with Elongase enzyme (Invitrogen) to produce seven overlapping fragments. The DNA fragments were gel-purifed and sequenced directly with reagents in the BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, California, United States) on a 3100 DNA sequencer (Applied Biosystems). Data were analyzed with the Sequencher software package (Gene Codes Corporation, Ann Arbor, Michigan, United States). Oligonucleotide primer sequences are available upon request.
This work was supported by National Institutes of Health grant RO1 AI54483 to HWV. CEW was supported by training grant T32-CA09547 and SMK by training grants T32-CA09547 and T32-AI07163. We thank W. Beatty for negative staining electron microscopy. LBT was supported by training grant AI007163
Conflicts of interest. The authors have declared that no conflicts of interest exist.
Author contributions. CEW, SMK, LBT, JMM, KYG, and HWV conceived and designed the experiments. CEW, SMK, LBT, KOC, SSS, GB, JMM, and KYG performed the experiments. CEW, SMK, LBT, KOC, SSS, GB, AK, JMM, KYG, HWV analyzed the data. AK contributed reagents/materials/analysis tools. CEW, KYG, and HWV wrote the paper.
Academic Editor: Michael Emerman, Fred Hutchinson Cancer Research Center
¤Current address: Department of Medicine, Technical University Munich, Germany
Citation: Wobus CE, Karst SM, Thackray LB, Chang KO, Sosnovtsev SV, et al. (2004) Replication of a Norovirus in cell culture reveals a tropism for dendritic cells and macrophages. PLoS Biol 2(12): e432.
Abbreviations
BMDCbone-marrow-derived dendritic cell
BMMΦbone-marrow-derived macrophage(s)
CPEcytopathic effect
DCdendritic cell
ELISAenzyme-linked immunosorbent assay
h.p.i.hours postinfection
IFNinterferon
IFN[αβγ]Rinterferon[αβ] receptor and interferon[γ] receptor
iNOSinducible nitric oxide
mAbmonoclonal antibody
MΦmacrophage(s)
MEFmurine embryo fibroblast
MNV-1murine norovirus 1
MOImultiplicity of infection
ORFopen reading frame
Ppassage
pfuplaque-forming units
PKRprotein kinase R
wtwild-type
==== Refs
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| 15562321 | PMC532393 | CC BY | 2021-01-05 08:21:17 | no | PLoS Biol. 2004 Dec 30; 2(12):e432 | utf-8 | PLoS Biol | 2,004 | 10.1371/journal.pbio.0020432 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020436SynopsisBioinformatics/Computational BiologyBiophysicsCell BiologyEubacteriaComputer Simulation of the Movement of Listeria Bacteria Synopsis12 2004 30 11 2004 30 11 2004 2 12 e436Copyright: © 2004 Public Library of Science.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
In Silico Reconstitution of Listeria Propulsion Exhibits Nano-Saltation
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Each of the cells in our body is a complex machine. Within each cell, thousands of proteins and other molecules interact to produce the highly organized cellular events that are needed for life. However, there is no cellular line manager telling the individual proteins where to go and what to do. It's rather like an ant colony. No one tells the individual ants how to build a nest. They just do it. Similarly, no one tells the individual actin molecules, for example, to get together and polymerize to form the cytoskeleton that is essential for cell movement and other cellular processes. It just happens.
Ant colonies and actin polymerization are both examples of emergent behavior. Broadly defined, emergent behavior is when a collection of individuals interact without central control to produce results that are not explicitly programmed. Scientists are only just beginning to understand how complex behaviors of this type can emerge from a myriad of individual interactions, many of which are well-defined experimentally.
Jonathan Alberts and Garrett Odell have turned to an unlikely ally—the bacterium Listeria monocytogenes—in their study of the complexities of cellular actin polymerization. This rod-shaped microbe, which lurks in well-ripened brie and other unpasteurized cheeses, can cause the sometimes fatal disease listeriosis, particularly in young babies or people with weakened immune systems.
When Listeria microbes in food reach a person's gut, they penetrate the cells lining the gut, reproduce, and then spread from cell to cell without ever exposing themselves to the extracellular environment. In this way, they avoid the host's immune system. One particular protein produced by Listeria is central to this sneaky intracellular lifestyle: ActA. Expression of ActA allows the microbes to hijack the machinery in the host cell that controls the growth of actin networks. Some of the cellular actin, instead of forming the cytoskeleton of the cell, polymerizes around the bacterium, forming a dense “comet tail” of actin that generates a force to move the bacterium around the cell and push it into neighboring cells. This clandestine use of our cellular machinery for actin polymerization is far simpler to understand than the elaborate use our cells normally make of it, so the study of Listeria propulsion provides a scientific stepping-stone to understanding the involvement of the actin cytoskeleton in normal cell movements.
Alberts and Odell have used established data on the biochemical and mechanical details of actin polymerization and the physiological concentrations of the proteins involved in the process to build a computer simulation of how an actin network assembles around a moving, rod-shaped bacterium. Their “in silico” reconstitution, which considers the behavior of individual actin filaments and requires a cluster of 80 computer processors to be run for several days at a time, produces realistic bacterial motion in terms of speed and persistence, and models the actin tail shape. The model also reproduces smaller scale emergent behavior. Real Listeria cells do not move smoothly. Instead, the microbes move jerkily, with runs interspersed with pauses. The simulation faithfully reproduces this type of movement and offers a mechanistic explanation for it.
The approach described by Alberts and Odell can now be used to investigate many other, more complex cell mechanics problems, such as how the cellular movements involved in cell division are achieved. With the availability of detailed biological information and powerful computers, we may at last start to solve the mystery of how interactions among maybe 100,000 gene products can produce the organized cellular processes that cell biologists have been watching under the microscope for years.
| 0 | PMC532394 | CC BY | 2021-01-05 08:28:08 | no | PLoS Biol. 2004 Dec 30; 2(12):e436 | utf-8 | PLoS Biol | 2,004 | 10.1371/journal.pbio.0020436 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020445SynopsisCell BiologyImmunologyInfectious DiseasesVirologyVirusesMus (Mouse)Norovirus Cultured for the First Time Synopsis12 2004 30 11 2004 30 11 2004 2 12 e445Copyright: © 2004 Public Library of Science.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Replication of Norovirus in Cell Culture Reveals a Tropism for Dendritic Cells and Macrophages
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Norwalk virus and related noroviruses cause a short but unpleasant illness known variously as stomach flu, viral gastroenteritis, and winter-vomiting disease. They are the major causative agent of epidemic gastroenteritis worldwide. Norwalk virus was first described in 1968 when teachers and pupils at a school in Norwalk, Ohio, succumbed to acute gastroenteritis. The causative agent was identified in 1972, and since then, many other Norwalk-like viruses have been recognized. Noroviruses are usually picked up from contaminated food or water but can also be spread by person-to-person contact. There are no cures for norovirus infections and no vaccines. Worse still, noroviruses survive freezing, heating to 60 °C, and the amounts of chlorine added to public water supplies. Little surprise, then, that around 23 million Americans get a norovirus infection annually.
To reduce this disease burden, better prevention and control strategies for noroviruses are urgently needed. However, the development of such strategies has been hampered by the inability of scientists to find a way to grow noroviruses in cultured cells. Like all viruses, noroviruses replicate inside host cells, and they are choosy about which cells they will grow in. The discovery by Herbert Virgin and colleagues that a mouse norovirus can be grown inside dendritic cells and macrophages, two types of immune system cells, opens the door to a better understanding of norovirus biology and better disease control strategies.
The first norovirus cell culture system
In 2003, Virgin's team discovered MNV-1, a mouse norovirus, and reported that the innate arm of the immune system (as opposed to the adaptive arm) was important in combating MNV-1 infection. The adaptive immune system involves cells that respond to a disease-causing bacterium or virus by making “adaptations” to their genomes that result in specific anti-pathogen responses. The innate immune system, on the other hand, contains cells that recognize general features of pathogens and rapidly attack them when they enter our bodies. It is our first line of defense against bacteria and viruses, and Virgin and coworkers found that mice with defective innate immune systems were particularly sensitive to MNV-1.
Now, by examining tissues taken from mice infected with MNV-1 infection, the researchers show that in live animals the virus infects macrophages (cells that engulf and kill pathogens) and dendritic cells (cells that display pathogen proteins to the adaptive immune system). This observation provided the clue needed to grow a norovirus in cultured cells for the first time: when the researchers took uninfected dendritic cells or macrophages out of animals with defective innate immune systems and grew them in the laboratory, they found that MNV-1 could replicate within these cells. The researchers then used physical and biochemical techniques to verify that what they were growing in culture was indeed MNV-1 and also determined the cellular factors that control MNV-1 growth in culture, thereby confirming that the innate immune system is important for combating norovirus infection. Analysis of the sequence of a virus attenuated by growth in vitro identified an important part of the viral capsid that plays a role in MNV-1 virulence, potentially opening up an avenue to vaccination with attenuated viruses.
The researchers speculate that dendritic cells in the gut may provide the route by which noroviruses gain access to cells deep within the lining of our guts and thus cause disease. The next step will be to see whether human noroviruses can also grow in macrophages and dendritic cells. If they do, researchers should at last be able to elucidate the lifecycle of human noroviruses and identify the cellular factors needed for their replication. Hopefully, this new knowledge will swiftly suggest ways to prevent and control the human diseases caused by noroviruses.
| 0 | PMC532395 | CC BY | 2021-01-05 08:21:21 | no | PLoS Biol. 2004 Dec 30; 2(12):e445 | utf-8 | PLoS Biol | 2,004 | 10.1371/journal.pbio.0020445 | oa_comm |
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PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020446SynopsisCell BiologyImmunologyInfectious DiseasesMicrobiologyEubacteriaDrosophilaWhen Immune Defenses Turn Traitor Synopsis12 2004 30 11 2004 30 11 2004 2 12 e446Copyright: © 2004 Public Library of Science.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Secreted Bacterial Effectors and Host- Produced Eiger/TNF Drive Death in a Salmonella-Infected Fruit Fly
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When pathogens enter your body, most wind up engulfed (by phagocytes), poisoned (by stomach acids), or flushed out of your system. These defenses kick in when a bacterium's toxic secretions bind to cellular receptors that trigger a chain of events ending in an innate response. A wide variety of cells and chemicals, including phagocytes and cytokines, initiate the inflammatory reaction that redirects innate defenses in the bloodstream to the infected site.
If all goes well, the innate system successfully contains and eliminates a pathogen at the site of infection. But if infection persists, so will the inflammatory response, and these first responders turn traitor. It's thought that the immune response rather than the bacteria, for example, causes diarrhea in food poisoning. And when infection leads to death—as happens in septic shock—the immune response may be just as culpable as the infectious microbe.
To better understand the interplay between pathogen and host in the onset of infection and disease, David Schneider and colleagues turned to the genetically compliant fruitfly Drosophila and the food-borne pathogen Salmonella. Working with mutant strains of both fly and bacteria, the authors identified genes important to the development of infection and disease and showed that the host's reaction can indeed be lethal.
Pathogens possess various means to infect their host, unleashing toxins and secreting molecules that enhance virulence by breaching cell membranes and altering the intracellular environment. Fruitflies combat these intrusions with various innate responses, including phagocytosis. In this study, the authors investigated how Drosophila phagocytes find, engulf, and kill invading microbes and then alert the rest of the immune system—and how Salmonella circumvents these defenses to initiate disease.
Adult fruit fly infected with flourescent Salmonella
Schneider and colleagues used a Salmonella strain (S. tyhphimurium) that produces two pathogenicity complexes, called type III secretion apparatuses, which shuttle virulence molecules through the host's cell membrane and into its cytoplasm. One complex, SPI1, facilitates cell entry while the other, SPI2, retools the intracellular environment to suit bacterial growth. The authors created a series of less virulent Salmonella strains and examined their effects on wild-type (nonmutant) flies. In addition, they looked at infections of wild-type Salmonella in flies carrying mutations in two critical immune response pathways (called Toll and imd, for immune deficiency).
Imd mutants infected with nonmutant Salmonella died much faster than the Toll mutants and had far more bacteria in their blood. When Schneider and colleagues correlated Salmonella population numbers with fly fate, they discovered something surprising. In other bacteria models, flies die when bacterial pathogen numbers reach a critical mass. Here, Salmonella populations hit a ceiling and the flies died with comparatively few bacteria. In flies infected with Salmonella strains lacking either one or the other virulence complex, the flies survived despite increased bacterial growth.
The authors explain this surprising finding with a model in which the fly's immune response produces substances that ultimately engineer its own destruction. Their model is supported by experiments showing that flies carrying a mutation in the eiger gene—a homolog of the human TNF gene—live longer with Salmonella infections. This is the same phenomenon seen in TNF-induced septic shock, when patients die as much from their immune system's response to infection as from the bacteria itself. Since many of the proteins involved in the Drosophila imd pathway have counterparts in the mammalian antibacterial immune response, the model described here can help identify the genetic agents of metabolic collapse associated with bacterial infections in humans.
| 0 | PMC532396 | CC BY | 2021-01-05 08:21:18 | no | PLoS Biol. 2004 Dec 30; 2(12):e446 | utf-8 | PLoS Biol | 2,004 | 10.1371/journal.pbio.0020446 | oa_comm |
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