The PaO2 and peripheral oxygen saturation (SpO2) are closely related and both are used to monitor oxygenation status. However, SpO2 values cannot be used as an exact substitute for PaO2. The aim of this study in acutely ill and stable patients was to determine at which SpO2 level PaO2 is more or less certain to be in the physiological range.In the context of an ongoing debate on the potential risks of hypoxemia and hyperoxemia, it seems prudent to maintain the partial arterial oxygen pressure . A second cohort of retrospective data of patients who underwent pulmonary function testing was also included . Arterial hypoxemia was defined as PaO2 < 60 mmHg and hyperoxemia as PaO2 > 125 mmHg. The SpO2 cut-off values with the lowest risk of hypoxemia and hyperoxemia were determined as the 95th percentile of the observed SpO2 values corresponding with the observed hypoxemic and hyperoxemic PaO2 values.This is an observational study prospectively collecting data pairs of PaO2 measurements occurred in patients with an SpO2 below 94%, and 95% of hyperoxemic PaO2 measurements occurred in patients with an SpO2 above 96%. Additionally in the 1379 data pairs of the ROP cohort, 95% of hypoxemic PaO2 measurements occurred in patients with an SpO2 below 93%.220 data pairs were collected in the PIA cohort. 95% of hypoxemic PaO2 level marking an increased risk of arterial hypoxemia is not substantially different in acutely ill versus stable patients. In acutely ill patients receiving supplemental oxygen an SpO2 target of 95% maximizes the likelihood of maintaining PaO2 in the physiological range.The SpO PaO2 on the other hand is the parameter of primary interest, since it reflects the balance between oxygen delivery and consumption [2 and PaO2 are closely related, the nature of which is reflected by the oxyhemoglobin dissociation curve [2 or SpO2 cannot be interpreted as an exact substitute of PaO2 [2 and PaO2 [2 measurements [2 is close to or at its maximum value of 100%. Indeed, corresponding PaO2 values for any given SpO2 value may range widely [Non-invasive measurement of SpOsumption , 14. SaOon curve . However of PaO2 , 17. Lowand PaO2  and alteurements –24. Alsoe widely .2 of 94–98% in adults in healthcare and emergency settings [2 above 96% and advocate a lower SpO2 range [2 was 96% or lower [In order to prevent hypoxemia and hyperoxemia and its possible associated risks, the British Thoracic Society Guideline recommends a target SpOsettings . Others O2 range , 30. A ror lower .2–PaO2 relationship, particularly in acutely ill patients receiving oxygen. In this study, we aim to identify a target SpO2 value, with relative safety margins, resulting in a high likelihood of maintaining PaO2 within a physiological range while administering oxygen [In light of the debate on the trade-off between the risks of arterial hypoxemia and hyperoxemia, it is important to increase our understanding of the SpOg oxygen .2 and SpO2 values in two different patient cohorts. The study was registered at clinicaltrials.gov (NCT02666937). This cohort consists of acutely ill patients admitted to the Emergency Room (ER) and the ICU and is referred to as the Prospective Inpatient Acutely ill cohort (PIA cohort).We performed a prospective observational study collecting simultaneously measured PaOThe medical research ethics committee of the Amsterdam University Medical Centers (Amsterdam UMC) approved the study protocol. The patients in the PIA cohort were informed about the use of their data and could object to their data being used through an opt-out procedure. The medical research ethics committee specifically approved the use of opt-out consent for these patients.2 and SpO2 values. This was a retrospective dataset of patients undergoing pulmonary function testing. This dataset was used to gain more insight into the lower range of SpO2 and PaO2 values in a larger population. This cohort of patients is referred to as the Retrospective Outpatients Pulmonary cohort (ROP cohort). The medical research ethics committee granted a waiver of consent for the use of this data.We also included a second cohort of patients with simultaneously measured PaO2 and PaO2 value.For the PIA cohort, data was collected prospectively from December 2015 to August 2017 in mechanically ventilated ICU-patients and acutely ill patients visiting the ER at the Amsterdam UMC. This cohort consisted of a convenience sample, based on the availability of researchers. Adult patients were eligible for inclusion if they required arterial blood gas analysis (ABGA) as part of routine care. Up to 10 data pairs could be collected per patient with a time interval of at least two hours between samples. A data pair is one single measurement of a combined SpO2 was measured with the use of a Nellcor OxiMax DS-100A  pulse oximeter. Reliability of the SpO2 curve was assessed and defined as a curve showing complete pulsatile tracing of the beat-to-beat arterial pulse wave on the pulse oximeter. On the ICU, PaO2 was measured using a combined blood gas analyzer and CO-oximeter . The samples were analyzed immediately after sampling.Simultaneous with the ABGA the SpO2/min when at risk for insufficient oxygenation, according to local guidelines.Patients with methemoglobinemia (> 1.5%), carboxyhemoglobinemia (> 1.5%) or known hyperbilirubinemia (> 20 μmol/L) were excluded. Additionally, ICU patients were only included if they had an arterial cannula and required mechanical ventilation, and were excluded when undergoing extracorporeal membrane oxygenation or therapeutic hypothermia. Oxygen was titrated to achieve a stable arterial oxygen saturation of ≥ 92% in the ICU during routine clinical care. ER patients were included upon presentation when they required ABGA. Most ER patients were stabilized with a non-rebreather mask with 15 liter O2 measurement. Per patient between one and four ABGA’s were collected during each pulmonary function test. The pulmonary function test consisted of a cycling protocol without supplemental oxygen. The arterial puncture was performed by a pulmonary function technician in the radial artery. SpO2 was measured with a NONIN Avant 9600  pulse oximeter at the Amsterdam UMC and a NONIN 8000S  pulse oximeter at the Northwest Clinics.For the ROP cohort, a dataset was used containing adult patients performing a pulmonary function test at the Amsterdam UMC between 1995 and 2014 and at the Northwest Clinics between 2016 and 2017. A researcher accessed the database to obtain the retrospective data in July 2017. All patients having performed a test were screened for eligibility and were included if they had had an ABGA and a simultaneous SpO2 of 60 mmHg or lower in a clinical trial of oxygenation targets [2 < 60 mmHg [2 > 125 mmHg [The exact cut-offs for hypoxemia and hyperoxemia are relatively arbitrary and not well defined in literature. For example hyperoxemia is defined in a range from > 120 mmHg  up to >  targets . We ther 60 mmHg , 34, 36 125 mmHg , 37, 38.Data were analyzed using SPSS  and RStudio . Values are presented as mean with standard deviation (SD) unless otherwise stated.2 and PaO2 data were first visually assessed with a scatterplot. We determined a hypoxemic and hyperoxemic SpO2 limit for the PIA cohort. The hypoxemic SpO2 limit was determined as the 95th percentile of the observed SpO2 values corresponding with the observed hypoxemic PaO2. The hyperoxemic SpO2 limit was determined as the 5th percentile of the observed SpO2 values corresponding with the hyperoxemic PaO2 values. For the ROP cohort we only defined a hypoxemic SpO2 limit, since the patients did not receive supplementary oxygen.SpO2 and PaO2 values were transformed such that relation between the SpO2 and PaO2 became linear (ln(1—SpO2/100) and ln(PaO2)). The transformed SpO2 was the dependent variable and the transformed PaO2 was included as a covariate with fixed effect. A two-sided 95% prediction interval was constructed for each specific value of PaO2. The upper and lower limit of the prediction interval and the predicted mean value for SpO2 were then transformed back to the original scale.Since multiple measurements were available for most patients, we constructed a mixed linear model in both cohorts. This included a random effect for each patient. First SpO2 curves, resulting in the inclusion of 220 data pairs in 146 patients. 139 data pairs were collected at the ICU and 81 at the ER. We screened 693 patients in the retrospective database for eligibility in the ROP cohort. Thirteen patients were excluded because of no or unreliable SpO2 measurements, resulting in the inclusion of 1379 data pairs in 680 patients. 1003 data pairs were collected at the Amsterdam UMC, and 376 pairs at the North West Clinic. We screened 152 patients in the PIA cohort for eligibility. One patient was excluded because of hyperbilirubinemia and five were excluded because of unreliable SpO2 < 60 mmHg, and 20.5% had a PaO2 > 125 mmHg. In the ROP cohort 10.7% of the data pairs had a PaO2 value < 60 mmHg.Demographics of the included patients and characteristics of the collected data pairs are shown in 2 and SpO2 measurements for both cohorts. In the PIA cohort 95% of data pairs corresponding to hypoxemia had SpO2 values below 94%, and 95% of data pairs corresponding to hyperoxemia had SpO2 values exceeding 96%. The horizontal red dotted lines in 2 limits. The patients in the ROP cohort have more hypoxemic PaO2 values and did not receive supplemental oxygen. In the ROP cohort, 95% of data pairs corresponding to a hypoxemic PaO2 had SpO2 values below 93%.2 >125 mmHg in the PIA cohort and more measurements with a PaO2 < 60 mmHg in the ROP cohortA multilevel analysis was performed for each cohort, taking into account that multiple measurements could be included per patients see . The cur2 measurements within a certain PaO2 range for each SpO2 level in both cohorts. In the PIA cohort at an SpO2 value of 95%, 94,4% of the data pairs had a PaO2 value between 60 and 125 mmHg. For an SpO2 value of 96% and 98%, the corresponding rates of hyperoxemic values were 3.8% and 17.4%, respectively. In the ROP cohort at an SpO2 value of 93%, 4% of the data pairs had a hypoxemic PaO2 value. For an SpO2 value of 92%, the corresponding rate was 11.5%.2 values of 94–96% both hypoxemia and hyperoxemia can be effectively excluded. In the ROP cohort hypoxemia can be excluded with a lower SpO2 target of 93%. The lower PIA and ROP targets excluding hypoxemia are not substantially different. Combining these cut-offs, we recommend an SpO2 target of 95%, which clearly has margins of relative safety. This target SpO2 results in the highest likelihood of physiological PaO2 values for the average patient.In the PIA cohort with SpO2 range between 92–95% that indeed resulted in a significant decrease of the number of hyperoxemic episodes, while hypoxemic episodes remained unchanged [2 level excluding hyperoxia of 96% [2 levels above 96% [2 of 98% for acutely ill patients [2 target of 98% may result in more hyperoxemia, without being associated with a meaningful lower risk of hypoxemia.Our findings are supported by data from an uncontrolled study, aiming at a target SpOnchanged . Moreovea of 96% . As for bove 96% . The Bripatients . Our dat2 value below 93% results in a higher risk of hypoxemia, for the PIA cohort this is the case below 94%. The lower limits are quite similar in these different cohorts of acutely ill and stable patients. These lower safe limits are in line with recent clinical guidelines recommending a lower SpO2 limit of 92–94% [2 values increases the risk for hypoxemia.In the ROP cohort an SpOf 92–94% , 29. Aim2 [2 [2 is not heavily influenced by specific pulmonary morbidity.This observational study has some limitations. Different oximeters were used for the two cohorts and potential differences between measurements can depend on the oximeter used. The Nellcor oximeters, used in the PIA cohort, slightly overestimate SaO2 , 41, whi2 < 60 mmHg [2 > 125 mmHg [2 in a physiological range, defined as between 60 and 125 mmHg [It is important to note, that there is no clear definition of hypoxemia and hyperoxemia available. We therefore pragmatically defined hypoxemia in line with literature and clinical practice as PaO 60 mmHg , 34, 36 125 mmHg , 37, 38.125 mmHg .2 values, since stable patients without pulmonary pathology will hardly ever be hypoxemic. Thus, this limitation is intrinsic to the study question of whether the safe SpO2 margin is different in acutely ill versus stable patients.The PIA cohort consisted of a convenience sample. The patient profiles in the PIA cohort varied, with mechanically ventilated patients at the ICU and patients requiring blood gas analysis at the ER. In both cohorts, the number of patients with hypoxemia was relatively small, because of oxygen administration. The ROP cohort consisted of retrospective data of patients performing a pulmonary function test with a cycling protocol. There are very few alternatives to the ROP cohort to obtain more insight in hypoxemic PaO2 and SaO2 or the accuracy of SpO2 measurements, so we cannot provide separate recommendations about specific conditions, such as sepsis, hypotension, heart failure, or acute respiratory failure. The generalizability of the results of this study should be confirmed with sufficiently powered studies in patients with specific conditions with different oximeters to define how clinical variables might change SpO2 targets. Also the different definitions of hypoxemia and hyperoxemia and clinical outcome variables should be taken into account in future studies.This study did not explore the clinical variables potentially modifying the relation between PaO2-target of 95%, or as close to this level as possible, to avoid the occurrence of either hypoxemia or hyperoxemia in acutely ill patients receiving supplementary oxygen.In conclusion, we recommend an SpOS1 File(XLSX)Click here for additional data file.


Scientific Reports 10.1038/srep21336, published online 22 February 2016Correction to: This Article contains an error in Figure 5 where the data is incorrect in panel (c). The correct Figure 5 appears below as Figure


The 5T33 multiple myeloma is one of a series of transplantable murine myelomas arising spontaneously in C57BL/KaLwRij mice. This study describes the establishment and characterisation of the 5T33 murine myeloma in vitro as a cultured cell line in terms of its morphology, growth rate, expression of paraprotein (IgG2b) and tumorigenicity in syngeneic animals. The 5T33 cell line has been in continuous culture for over 10 months and has achieved more than passage 34. In culture, 5T33 myeloma grows as single cells or in small clusters of loosely adherent cells on an adherent stromal cell layer. Maximum doubling time is approximately 25 h, and over 90% of the cells express cytoplasmic IgG2b paraprotein. The cultured 5T33 myeloma cells are highly tumorigenic in C57BL/KaLwRij mice with as few as 500 cells inducing paralysis and death as early as day 36 post-tumour inoculation. Kinetics of tumour development and detection of IgG2b paraprotein are dose dependent. Two weeks following intravenous inoculation of 5 x 10(5) cultured 5T33 myeloma cells, tumour cells were readily identified in the bone marrow. By 3 weeks post-tumour inoculation, 5T33 myeloma cells were found in various tissues throughout the animal. Studies are now underway to determine the sensitivity of this cell line to various therapeutic modalities.


The pharmacokinetics of 5-fluorouracil (5FU) following its administration via the hepatic artery in conjunction with biodegradable albumin microspheres and angiotensin II have been studied. Peripheral venous concentrations of 5FU are lower and plasma clearance values higher following intrahepatic arterial administration compared with a similar dose administered by intravenous infusion over both 2 h and 24 h. For the 2 h drug infusions, plasma 5FU concentrations following co-treatment with angiotensin II and microspheres via the hepatic artery were intermediate between those of arterial and venous infusions of 5FU alone. There was a trend towards the peak plasma drug concentrations and the area under the plasma concentration-time curve (AUC) being significantly lower following co-treatment with angiotensin II and microspheres compared with intra-arterial and intravenous infusions of 5FU over 24 h. Co-administration of 5FU, angiotensin II and microspheres via the hepatic artery may reduce drug exposure in the systemic compartment and therefore may increase the therapeutic ratio of 5FU administration via the hepatic artery.


Twelve boys and 10 girls on similar long term remission maintenance treatment for lymphoblastic leukaemia had 79 random assays of their red cell 6 thioguanine nucleotide ( 6TGN ) concentrations performed as an index of cytotoxic activity generated by oral 6-mercaptopurine ( 6MP ). Correlation between the dose of 6MP and 6TGN was statistically significant in the girls  but not in the boys (r = 0.15). Additionally, as a group the boys tolerated more 6MP (P less than 0.05), despite similar prescribing criteria, but this did not result in a higher mean 6TGN concentration or increased myelotoxicity. It appears that girls develop 6MP cytotoxicity at lower doses and more predictably than boys. If so, this may be relevant to the as yet unexplained but marked sex difference in prognosis apparent in some studies.


EXPB1, the most abundant isoform of Zea m 1) on the production of Zea m 1 protein, pollen viability, and pollen tube growth, both in vitro and in vivo. We also examined the effect of the insertional mutation on the competitive ability of the pollen by experimentally varying the sizes of the pollen load deposited onto stigmas using pollen from heterozygous plants and then screening the progeny for the presence of the transposon using PCR. We found that the insertional mutation reduced the levels of Zea m 1 in maize pollen, but had no effect on pollen viability, in vitro pollen tube growth or the proportion of progeny sired when small pollen loads are deposited onto stigmas. However, when large pollen loads are deposited onto the stigmas, the transposon mutation is vastly underrepresented in the progeny, indicating that this major pollen allergen has a large effect on pollen tube growth rates in vivo, and plays an important role in determining the outcome of the pollen-pollen competition for access to the ovules. We propose that the extraordinary abundance (4% of the extractable protein in maize pollen) of this major pollen allergen is the result of selection for a trait that functions primarily in providing differential access to ovules.Worldwide, 400 million people suffer from hay fever and seasonal asthma. The major causative agents of these allergies are pollen specific proteins called the group-1 grass pollen allergens. Although details of their antigenicity have been studied for 40 years with an eye towards immunotherapy, their function in the plant has drawn scant attention. Zea m 1 constitutes a class of abundant grass pollen allergens coded for by several genes that loosen the walls of grass cells, including the maize stigma and style. We have examined the impact of a transposon insertion into one of these genes ( Considering that only the first pollen tube to reach the micropyle passes its genes to the next generation, it is not surprising that the entire process is very fast. In maize, rehydration and germination of the pollen grain occur within 5 min of deposition on the silk, and pollen tubes grow at rates exceeding 1 cm hMu transposon insertion in EXPB1 (GenBank Accession AY197353), a gene that codes for Zea m 1d, the most abundantly expressed of four Zea m 1 isoforms in vivo and the ability of pollen to achieve fertilization under conditions of pollen competition.The group-1 grass pollen allergens are pollen-specific proteins originally identified by immunologists 40 years ago as the main causative agents of hay fever and seasonal asthma induced by grass pollen Mu) insertions  and wild type plants (EXPB1/EXPB1), as well as heterozygous (EXPB1/expb1) plants by crossing the true breeding plants. Analysis of pollen protein extracts by two-dimensional gel electrophoresis and immunoblotting from EXPB1/EXPB1 and expb1/expb1 plants revealed that overall Zea m 1 production was reduced by 31% in expb1 pollen compared with the overall production of Zea m 1 in EXPB1 pollen  also code for the same isoform From a large library of maize lines bearing Robertson's Mutator  revealed no significant effect of plant genotype on pollen viability . For each of the three maize genotypes  75–78% of the pollen stained a deep purple . Tukey pairwise comparisons with adjusted probabilities for multiple comparisons revealed that there was no significant difference in the in vitro growth of pollen tubes from expb1/expb1 and EXPB1/EXPB1 plants but that the pollen from the EXPB1/expb1 plants grew faster in vitro .When pollen from 20–22 plants from each of the 3 genotypes were germinated and grown on a medium in Petri plates in vitro  most likEXPB1 and expb1 pollen to achieve fertilization under conditions of pollen competition, we varied the volume (number) of pollen grains from EXPB1/expb1 plants deposited onto the silks of EXPB1/EXPB1 plants. We found that the transmission of expb1 depended upon the volume of pollen grains used in the pollination  , indicating that as the intensity of pollen competition increases, the proportion of seeds sired by expb1-bearing pollen decreases.To determine the ability of <0.0001) . When thin vivo pollen tube growth rates indicate that these differences in the ability to achieve fertilization under competitive conditions are due to differences in pollen tube growth rates. When we examined the silks at 8 h after pollination, we found that silks pollinated with EXPB1 pollen contained a significantly greater number of pollen tubes  at 8 cm below the site of pollen deposition than silks pollinated with expb1 pollen (0.98±0.06) . At 22 h after pollination, we found pollen tubes in 37.5% of the ovaries following pollinations by EXPB1 pollen  and higher plants  (see EXPB1 gene (the most abundant isoform of Zea m 1) has no significant effects on pollen viability or in vitro pollen tube growth rates but has a large effect on in vivo pollen tube growth rates and the ability to achieve fertilization under conditions of pollen competition. Because the pollen competition experiment varied the size of the pollen load produced by heterozygous (EXPB1/expb1) plants rather than using the pollen produced by the true breeding (homozygous) plants, the results are not an artifact of differences in vigor between the true breeding lines. This experimental design, however, does not exclude the possibility that loci distinct from EXPB1 could account for our results. After backcrossing the original line with the Mu insertion to the non-mutator parental line for 3 generations, the resulting plants still contain, on average, 12.5% of the genes from the original Mu insertion line. The vast majority of these genes would randomly segregate into the EXPB1 and expb1 pollen produced by the heterozygous plants used in the pollen competition experiment. Only those genes that consistently cosegregate with expb1 could potentially influence performance of expb1 pollen. Because only 3% of the seeds produced by our largest pollen load contained expb1, another mutation that is responsible for the large effect on pollen performance that we observed would have to lie no more than 3 cM away from the EXPB1 locus to cause this skewed ratio. The EXPB1 locus is located on chromosome 9 which has a genetic distance of approx. 150 cM EXPB1 locus represents <4% of the genetic distance of chromosome 9 in maize (chromosome n = 10). It would be extremely unlikely to find in this small region a second mutation with the precise phenotype described in this paper.Our findings reveal that a 31% reduction in Zea m 1 caused by a transposon insertion into the EXPB1 does not perform a vital role in pollen development or in the internal growth processes of the pollen tube per se. Previous in vitro studies demonstrated that Zea m 1 loosens the cell walls of silks and other studies showed that Zea m 1 is secreted by pollen upon hydration and tube growth EXPB1 is gametophytically expressed because the performance of pollen from heterozygous plants depended upon whether the pollen carried the EXPB1 or the expb1 allele. If the protein (or the mRNA) from EXPB1 was sporophytically produced  and then moved into the pollen, there would be no difference in the performance of pollen bearing different alleles. A few pollen expressed genes have already been shown to affect pollen tube growth rates in vivo  access to the stylar resources necessary for their growth Group-1 pollen allergens (β-expansins) have been detected in every grass [Because of the association that we observed between Zea m 1 production and breeding success under conditions of pollen competition, it would be difficult for plant breeders to develop and maintain low allergenic cultivars of turf, pasture, and agricultural grasses. This does not bode well for the 400 million people worldwide who suffer from hay fever and seasonal asthma due to this abundantly expressed β-expansin  Mu insertion in EXPB1  in the progeny of plants that were both self pollinated and crossed to wild type (EXPB1/EXPB1) silks. Ears were allowed to set seed and progeny of both crosses were screened for the presence of Mu in EXPB1 , cleaned by passing through a series of sieves, and stored separately at −80°C. Approximately 25 mg of maize pollen was extracted in 4 volumes (0.1 mL) of 50 mM sodium acetate, pH 4.5, for 1 h at 4°C. The extract was centrifuged at 20,800 g for 10 min. Proteins in the supernatant were quantified colorimetrically with the Coomassie Plus® Protein Assay Reagent  according to the manufacturer's instructions.In order to assess the impact of the These proteins were then subjected to 2-dimensional gel electrophoresis. For the first dimension – isoelectric focusing (IEF) – Immobiline DryStrip gels  and IPG buffer (pH 6–11) were obtained from GE Healthcare Bio-Sciences Corp. . The gels were rehydrated for 16 h with the rehydration buffer  containing the protein extracts and then were focused in a PROTEAN IEF cell apparatus  at the following program: running temperature: 20°C; maximum current: 50 µA/gel; Step 1: 200 V for 30 min (linear ramp); Step 2: 300 V for 30 min (rapid ramp); Step 3: 8,000 V for 150 min (linear ramp); Step 4: 8,000 V for 55,000 Vh (linear ramp). After the completion of IEF, the gels were incubated for 15 min in SDS equilibration buffer  with 10 mg/mL dithiothreitol and then switched into the same buffer containing 25 mg/mL iodoacetamide for another 15 min. For the second dimension, proteins were separated by discontinuous SDS-PAGE in a Criterion Dodeca Cell apparatus  using 12.5% precast gels. 2-D gels were stained for protein with SYPRO Ruby  according to the manufacturer's instructions and quantified using a laser scanner (Molecular Imager FX Pro PLUS from Bio-Rad) and 2-D image analysis software (PDQuest Version 7.3 from Bio-Rad). The protein marker, Mark12 Unstained Standard, for SDS-PAGE, was from Invitrogen Inc. .To identify the β-expansins, the resulting 2-D gels were then subjected to immunoblot analysis. This analysis was performed in a Bio-Rad Criterion blotter as described by Li et al. Mu insertion into EXPB1 on pollen viability, we collected pollen at anthesis from 20–22 plants from each of the three genotypes (EXPB1/EXPB1 (N = 20), EXPB1/expb1 (N = 22), expb1/expb1 (N = 20) plants), stained it with thiazolyl blue to assess membrane integrity—a trait that is highly correlated to germinability in vitro growth of pollen tubes from each of the three genotypes by sprinkling the pollen from 20–22 plants of each genotype onto Petri plates containing a maize pollen germination and pollen tube growth media To assess the impact of the Mu insertion into EXPB1 on the competitive ability of pollen in vivo, we experimentally manipulated the intensity of competition between mutant and wild type pollen. Pollen from field grown, heterozygous (EXPB1/expb1) plants was collected, cleaned, aliquoted into 50, 100, 250 and 500 µL volumes, and sprinkled over virgin silks of true breeding wild type plants . We reasoned that under conditions of intense pollen competition  only the fastest growing pollen tubes would achieve fertilization, whereas under conditions of little or no pollen competition  both the fast and slowly growing pollen would achieve fertilization. A random sample of 30 seeds from each of the 16 ears  was assessed for the presence of the mutant allele (expb1) by PCR.To determine the effect of the EXPB1 and expb1 pollen tubes through maize silks. We pollinated the silks of 16 wild type plants (EXPB1/EXPB1) with pollen from either EXPB1/EXPB1 or expb1/expb1 plants. On eight plants (four from each type of pollination), we removed the silks after 8 h; stained the silks with 0.1% aniline blue for 30 min and examined 10–14 silks from the central region of each ear for the presence of pollen tubes within the region from 7.5 to 8.5 cm from the site of pollen deposition using fluorescence microscopy We also directly examined the growth of


A study was made of the incidence of p53 mutations in Japanese males with prostate cancer or benign prostatic hyperplasia. Polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) was used as a primary screening technique with gene sequencing being carried out in positive cases. Two out of 21 prostate cancers (9.5%) were found to have p53 mutations. These were stage B2 and D2 prostate cancers. No abnormalities were found in the remaining cases or benign prostatic hyperplasia. Mutations of the p53 gene would thus appear infrequent in the tumourigenesis of primary prostate cancer.


The reactivation of U.V.-irradiated adenovirus 2 in HeLa cells is enhanced 8-9 fold if the cells are given a brief hyperthermic shock before infection. Maximum reactivation is achieved by heating for 10 min at 45.5 degrees C and with a delay of 36 h between heating and infection. The induction process requires protein synthesis only during the 3 h period immediately following heating; cycloheximide does not prevent the expression of enhanced reactivation if added to the cells after this time. Heat-enhanced reactivation exhibits properties similar in some respects to radiation-enhanced reactivation and indicates an increased capacity of the heated cells to tolerate DNA damage.


Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) appears to play an key role as both a virulence factor and as a target of naturally acquired immunity var genes of which there are 60 variants in every genome var sequences is now available from clinical isolates around the world var genes contain a DBL1α region var genes to create DBL1α sequence tags var genes and the fact that they undergo intra-genic recombination A rapidly growing collection of var genes that supports the use of information present in DBL1α sequence tags in making comparisons between the expression levels in different isolates. Analysis of the fully sequenced genome of a single P. falciparum isolate 3D7 suggests that the genomic location of the 60 var genes promotes genetic structuring and the maintenance of genetically distinct sequence types var tag grouping system, though it is based on portion of the DBL1α domain .Further support for the cysteine/PoLV groupings comes from recent publications. Trimnell et al. found a good correspondence between cysteine/PoLV groupings of cys2 sequences and groups defined phylogenetically within a globally sampled subset of var genes from 3D7, HB3 and IT4 var gene classification. With the exception of group 6 sequences which were not found in HB3 var genes all sequence groups were represented. In all three genomes cysteine/PoLV group 1 sequences are exclusively found in group A var gene and long genes with >5 domains whereas cysteine/PoLV group 5 are found only in non-group A genes and those with 4–5 domains. Cys2 sequence tags (groups 1–3) were never found in group C var genes.Kraemer et al. have recently performed an analysis and re-classification of whole Kyriacou et al. used a phylogenetic approach to compare DBL1α sequence tags from Mali var genes from South American isolates in support a recent study of Amazonian isolates At a higher level of resolution, the distinct sequence identifier (DSID) see A is a poSince the cysteine/PoLV system of classification is based on commonly occurring sequence features it is hoped that it will useful for initial analysis and annotation, comparison of different geographical regions over time and identification of unusual sequences.


SLC11A1 is known to link infections, autoimmunity and cancers. A review is presented of the mechanisms by which a balance is maintained between infections caused by pathogens  and disorders resulting from (acute or chronic) inflammation, and of the interactions that determine how the initial innate immune system directs subsequent acquired immune responses in human populations Slc11a1 in mice encodes a polytopic integral 10–12 transmembrane protein, which is expressed exclusively in macrophages and polymorphonuclear leukocytes and neurons (This review discusses the role of the solute carrier family 11 member 1 protein (SLC11A1), formerly NRAMP1 , in linking infections, autoimmunity and cancer. Chronic inflammation is caused by a variety of factors including bacterial, viral and parasitic infections, chemical irritants and nondigestible particles. Chronic inflammation is associated with a predisposition to cancer and autoimmunity because the longer the inflammation persists, the higher the risk of associated autoimmunity and carcinogenesis. Immune cells involved in inflammation include leucocytes such as neutrophils, monocytes, macrophages and eosinophils. These cells provide the soluble factors that are thought to mediate the development of inflammation. When inflammation goes unchecked it often leads to chronic inflammation-associated diseases. 2+) levels  levels . Thus, sMycobacterim bovis infection are significantly more acidic than those formed in slc11a1-negative ones .SLC11A1 should lead to apoptosis, in accordance with the notion that genes that deplete the iron content of the cell cytosol antagonize cell growth. By contrast, transcriptional repression of SLC11A1 could lead to cell survival. SLC11A1 might influence immune responses to viral vector delivery systems 5 ac (gt)5 ac (gt)9 g  drives high gene expression, while the allele t(gt)5 ac (gt)5 ac (gt)10 g  drives low gene expression , at 0.1%, is similar to that in the African populations  and within intronic regions, thus suggesting that this gene might be differentially regulated in different species. One major difference at this locus between species includes the type and locations of transcription factor binding sites in the 5′UTR of SLC11A1. Of particular importance is the binding site for the activating transcription factor-3 (ATF-3; also known as an AP-1-like element, Homo sapiens, Pan troglodytes and Macaca mulatta, this sequence is absent in Mus musculus. In Rattus norvegicus and Pongo pygmaeus, the second cytosine is replaced by a thymine. ATF-3 is a negative regulator of TLR4 (SLC11A1 but it is unclear whether it is an active ATF-3 binding site in SLC11A1. The formation of Z-DNA structure is known to play an active role in modulating inhibitory chromatin structure (SLC11A1 it should repress transcriptional activation of SLC11A1; conversely, heterodimerization of ATF3 with other transacting factors should promote transcriptional activation of SLC11A1.Agents that activate macrophage together with host SLC11A1 alleles will exert different effects on SLC11A1 function, but how SLC11A1 functions help determine the outcome of infection by modulating the production of these cytokines in not known. Comparative analysis of the  of TLR4 , where iIt could be that transcriptional activation of SLC11A1 leads to apoptosis and cell death while transcriptional repression of SLC11A1 leads to cell proliferation and survival if unchecked could result in cancer and autoimmunity. The ATF-3 putative site in the promoter of SLC11A1 along with other transacting factors might have a regulatory role in modulating the effects of SLC11A1. This review has thus highlighted some of the mechanisms by which SLC11A1 might be mediating, influencing or modulating the outcome of these diseases and disorders in human populations.Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation.


Recombination between hepatitis C single stranded RNA viruses is a rare event. Natural viable intragenotypic and intergenotypic recombinants between 1b-1a, 1a-1c and 2k-1b, 2i-6p, respectively, have been reported. Diagnostically recombinants represent an intriguing challenge. Hepatitis C genotype is defined by interrogation of the sequence composition of the 5' untranslated region [5'UTR]. Occasionally, ambiguous specimens require further investigation of the genome, usually by interrogation of the NS5B region. The original purpose of this study was to confirm the existence of a suspected mixed genotype infection of genotypes 2 and 4 by clonal analysis at the NS5B region of the genome in two specimens from two separate individuals. This initial identification of genotype was based on analysis of the 5'UTR of the genome by reverse line probe hybridisation [RLPH].The original diagnosis of a mixed genotype infection was not confirmed by clonal analysis of the NS5B region of the genome. The phylogenetic analysis indicated that both specimens were natural intergenotypic recombinant forms of HCV. The recombination was between genotypes 2k and 1b for both specimens. The recombination break point was identified as occurring within the NS2 region of the genome.The viral recombinants identified here resemble the recombinant form originally identified in Russia. The RLPH pattern observed in this study may be a signature indicative of this particular type of intergenotype recombinant of hepatitis C meriting clonal analysis of NS2. Hepatitis C virus infects approximately 170 million individuals' world wide ,3. HepatDQ417427], with no definable subtype or indication of a mixed genotype infection with a strain of genotype 4. In light of this fact, clonal analysis of the NS5B region from HC9A98987 was undertaken. 14 clones were sequenced [DQ417439–DQ417452]. A search for similarities with previously published sequences was performed with the program Blast.  and not the expected genotype 2 or 4. The corresponding amino acid sequences analysis indicated that there were only 7 unique isolates, GenBank .Following routine RLPH of specimen HC9A98987 it was noticed that an unusual banding pattern indicative of a putative genotype 2 and 4 mixed infection was evident Figure . This inm Blast.  The resuDQ417428–DQ417433] and (ii) the HVR1 sequences [DQ417434–DQ417438] indicated that these regions of the genome were in fact homologous to viruses within the 2k subtype . Two clones from the core region had synonymous mutations . The HCV 2k  isolate has a relatively high prevalence in the countries of the former USSR. Kalinina et al reported in 2002 the identification of a natural intergenotypic recombinant form of HCV which had homology to 2k at the 5' end of the genome and to 1b at the 3' end of the genome  and a phylogenetic analysis was carried out. Phylogenetic trees were constructed with the NEIGHBOR program in the PHYLIP package and genetic distances were calculated with PRODIST program in the PHYLIP package based on Kimura's distance.  (2k/1b)] was recovered by nested RT-PCR [Table DQ839386–DQ839389] with a minor quasispecies [DQ839390]. Sequence analysis indicated that this region of specimen HC9A98987 is homologous with the reference strain [AY587845]  2k/b] was re] Figure . This isAY587845 (2k/1b), the laboratory investigated whether an additional specimen  from another individual with the same RLPH pattern was also a candidate natural intergenotypic recombinant form. Clonal analysis of the NS2 region was performed on this specimen from the second patient (HC9A99966). The sequences of six clones were determined. All six clones were of identical nucleotide sequence [DQ904446] and were found by sequence analysis to be homologous to the natural intergenotypic recombinant related to HC9A98987, i.e., AY587845 (2k/1b) . Nucleotide sequences reported in this study have been assigned the following accession numbers which were derived from HC9A98987 Genbank: [DQ417427] (5'UTR). [DQ417428 to DQ417433] (Core sequences), [DQ417434 to DQ417438] (HVR1 sequences), [DQ417439 to DQ417452] (NS5B sequences), [DQ839386 to DQ839390] (NS2 sequences). [DQ904446] was derived from the NS2 sequence from sera HC9A99966.Reference strains used in the present study were all obtained from GenBank [Hepatitis C Virus HCVUntranslated Region UTRReverse Line Probe Hybridisation RLPHHuman Immunodeficiency Virus HIVThe author(s) declare that they have no competing interests.IM Carried out NS2 clonal analysis, sequence alignment and phylogenetic analysis. Preparation of manuscriptSH Performed core, HVR1 and NS5B sequence/cloningJL Determined qualitative, quantitative and initial genotype of specimens described herePS Assisted SH in RT-PCR and amplicons cloningOC & EKW Clinicians who manage HCV at Cork University HospitalLJF Supervised project and assisted with analysis and preparation of manuscript.


The tumour suppressor gene p53 has been found to be mutated or inactivated at high frequency in several common human tumours. We have examined a series of exocrine pancreatic carcinomas for over-expression of mutant forms of p53 by immunohistochemistry with a panel of specific antibodies. We found immunodetectable p53 in 13 of 22 (60%) frozen pancreatic cancers and seven of 13 pancreatic cell lines. One of the antibodies, CM1, recognises p53 in formalin-fixed, paraffin-embedded archival material and using this reagent we found immunodetectable p53 in 28 of 124 (23%) pancreatic cancers. We have successfully demonstrated the presence of point mutations by direct sequencing of genomic DNA extracted from archival tissue showing CM1 immunoreactivity. We conclude that p53 activation is an important event in human pancreatic tumorigenesis and that the CM1 antibody can detect a proportion of cases of overexpression of mutant p53 in archival pathological material.


A Phase 2 trial was conducted using intramuscular lymphoblastoid interferon , 4 MU per day, in 10 patients with chemotherapy-resistant teratomas. There was stabilisation of disease in 2 patients both of whom were in retrospect considered to have had differentiated teratoma at the time of IFN administration. There was progression of presumed active anaplastic germ cell tumour in 8 patients. One of these patients, a 15-year-old boy with biopsy proven differentiated teratoma has received 2 courses of lymphoblastoid IFN and 1 course of recombinant leukocyte A IFN (Roche Products Ltd.) lasting 5 1/2, 8 and 8+ months respectively. He has had a mixed response in his differentiated tumour which on each occasion has been maintained for the duration that he received IFN. Rising HCG levels during his second course of interferon required additional cytotoxic chemotherapy. Lymphoblastoid IFN does not appear to be active against anaplastic germ cell tumours but both lymphoblastoid and recombinant leukocyte A IFN may be useful in the treatment of unresectable differentiated teratoma.


The pattern of TGF beta expression and in vitro response to TGF beta has been defined in three ovarian carcinoma cell lines . Marked differences in both mRNA expression and growth responses were detected between the cell lines. All expressed mRNA for TGF beta 3, PEO1 and PEO4 but not PEO14 expressed mRNA for TGF beta 1, whereas PEO14 but not PEO1 and PEO4 expressed TGF beta 2. Growth of PEO14 cells in culture was markedly inhibited by both TGF beta 1 and beta 2. PEO1 cells were inhibited by TGF beta 1, but not TGF beta 2 whilst growth of PEO4 cells were not affected by exposure to either of these peptides. These data indicate that several elements of potential autocrine loops involving TGF beta's are present within ovarian cancer cells.


Gli3 gene encodes a zinc finger transcription factor and homozygous loss-of-function mutations of Gli3 are lethal. Humans heterozygous for mutations in this gene suffer Greig cephalopolysyndactyly or Pallister-Hall syndromes, in which limb defects are prominent, and mice heterozygous for similar mutations have extra digits. Here we examined whether eye development, which is abnormal in mice lacking functional Gli3, is defective in Gli3+/- mice.Knowledge of the consequences of heterozygous mutations of developmentally important genes is important for understanding human genetic disorders. The Gli3 is expressed in the developing eye but that Gli3+/- mice have only very subtle eye defects. We then generated mice compound heterozygous for mutations in both Gli3 and Pax6, which encodes another developmentally important transcription factor known to be crucial for eye development. Pax6+/-; Gli3+/- eyes were compared to the eyes of wild-type, Pax6+/- or Gli3+/- siblings. They exhibited a range of abnormalities of the retina, iris, lens and cornea that was more extensive than in single Gli3+/- or Pax6+/- mutants or than would be predicted by addition of their phenotypes.We showed that Gli3 can impact on eye development. The importance of a normal Gli3 gene dosage becomes greater in the absence of a normal Pax6 gene dosage, suggesting that the two genes co-operate during eye morphogenesis.These findings indicate that heterozygous mutations of  GLI3 allele can cause Greig cephalopolysyndactyly (GCPS) or Pallister-Hall Syndrome (PHS) . Mut and man ,17. Miceorm eyes ,18,19 wh defects . There i defects  and losspression -26. ThusAs shown in Fig. Gli3+/-, Pax6+/- and Gli3+/-;Pax6+/- eyes, immunostained for Pax6 expression, are shown in Fig. The appearances of E14.5 +/- eyes appeared slightly larger than those of wild-type siblings, although their appearances were otherwise normal  was significantly less than that of wild-type eyes     was significantly less than that of wild-type eyes , but not significantly different to that of Pax6+/- eyes. There was much greater variation than among eyes of the other genotypes  showed no retinal dysplasia  Some retinae exhibited no dysplasia but were thinner than those of other genotypes (15/28) 28) Fig. . 2) Some28) Fig.  with arePax6+/-, Gli3+/- and Pax6+/-;Gli3+/- animals. Compound heterozygotes that had retinal dysplasia in the regions measured were excluded. Whole retinal thickness was significantly reduced in Pax6+/-;Gli3+/- animals compared to their wild-type, Pax6+/- and Gli3+/- siblings, all of which had similar thicknesses , the outer plexiform layer (OPL), the inner nuclear layer (INL), the inner plexiform layer (IPL) and the ganglion cell layer (GCL). We found that the thicknesses of both the ONL and INL of Pax6+/-; Gli3+/- retinae were significantly reduced compared to those of the other genotypes , the OPL was not discernible; where this layer was present, its average thickness was not significantly different to that of wild-type animals   showed highly dysmorphic and dysgenic lenses, which were not observed in any of the other genotype groups  Fig. , which w28) Fig.  in one Pups Fig. .Pax6+/- eye has been reported to be thinner than normal and the corneal stroma is hypercellular [Gli3+/- animals  was significantly thinner than that of wild-type (n = 16) and Gli3+/- (n = 27) eyes, both centrally and peripherally . A similar result was observed in Pax6+/-; Gli3+/- eyes (n = 25) . Overall, these data suggest that loss of one copy of both Pax6 and Gli3 together does not create a corneal epithelium thinner than that resulting from loss of Pax6 alone.Thicknesses of the corneal epithelium and corneal stroma, centrally and peripherally, were measured for the four genotype groups. There was no difference in both central or peripheral corneal epithelium thickness between wild-type and yes Fig. . As prevPax6+/- or Gli3+/- eyes compared to wild-type eyes . Thus, the mean stromal thickness  of Pax6+/-; Gli3+/- eyes was significantly less than that of wild-type , Pax6+/-  and Gli3+/-  eyes.There was no difference in either central or peripheral corneal stromal thickness in yes Fig. . The perPax6 [Gli3[Pax6 results in various eye defects [Gli3 results in eye defects. Thus, in this study, we set out to characterise the morphology of the Gli3+/- eye. Furthermore, because both Pax6 and Gli3 are expressed in the optic cup/retina, lens and iris during development [Pax6 and Gli3 play mutually co-operative roles in eye formation by studying animals compound heterozygous for mutations in both Pax6 and Gli3.The importance of the transcription factors Pax6 ,18,19 anax6 [Gli3,9,10 in  defects -22, littelopment ,18,27-29Gli3+/- eyes that we examined. Where abnormalities were seen, they were very mild. Gli3+/- eyes had a slightly higher mass than wild-type eyes as well as a thicker OPL compared to wild-type retinae. Retinal folding has been described previously in some neonatal Gli3+/- animals [Gli3+/- eyes are either not detectable or minor.No abnormality was observed in the majority of the  animals . OverallPax6 and Gli3 exhibited unique and more severe eye defects than either Pax6+/- or Gli3+/- animals or than would be predicted by addition of their phenotypes. As previously described [Pax6+/- adult eyes was significantly smaller than that of wild-type eyes. The additional loss of one copy of Gli3 resulted in eyes with a wide range of eye mass, from some eyes with smaller mass than Pax6+/- eyes to some eyes which fell within the wild-type range of eye mass.Animals compound heterozygous for mutations in both escribed , the masPax6+/-; Gli3+/- eyes. Some Pax6+/-; Gli3+/- retinae were thinner than normal but exhibited normal lamination while others were severely dysgenic with considerable laminar dysmorphology. The severe retinal defects found in Pax6+/-; Gli3+/- animals were never seen in either Pax6+/- or Gli3+/- animals. One mechanism by which Pax6 and Gli3 might cooperate during retinal development is through their actions in the Shh signalling pathway. This pathway is critical for normal eye development. Shh from the ventral midline of the early neural plate stage embryo is required for early patterning of the visual system [Gli3 can partially rescue the Shh-null phenotype [Gli3+/- mice) mimics the effects of raising Shh levels. There is evidence that Shh suppresses Pax6 expression [Gli3  might not lower Pax6 levels enough to create morphologically detectable defects if both alleles of Pax6 are functional, levels might fall below a critical threshold in Pax6+/-;Gli3+/- heterozygotes. Furthermore, there is evidence that Pax6 suppresses Shh expression [Pax6+/-;Gli3+/- heterozygotes might allow increased production of Shh, thereby further exacerbating the overstimulation of the Shh pathway. The mechanism by which reduced levels of the repressor form of Gli3 result in reduced Pax6 levels might involve loss of repression of intermediate transcription factors such as Pax2 and Vax1, that themselves repress Pax6 [A range of retinal phenotypes was observed in l system . Later, l system  and has l system ,31. Gli3l system . Lowerinhenotype , suggestpression ,33, and pression -26 and sess Pax6 . These pess Pax6 ,34Pax6+/-; Gli3+/- animals were more severe than those of either Pax6+/- or Gli3+/- animals. Pax6+/-; Gli3+/- animals had highly dysgenic lenses which were never seen in Pax6+/- or Gli3+/- animals. There were particularly high incidences of abnormal contacts involving the iris in Pax6+/-; Gli3+/- animals and, in contrast to the iris hypoplasia observed in Pax6+/- animals, many Pax6+/-; Gli3+/- animals exhibited hyperplastic irises. Together these data suggest not only that the iris is sensitive to levels of Pax6 expression but that normal Gli3 expression may also be required, in conjunction with Pax6, for normal iris development to occur. Pax6+/-; Gli3+/- animals also showed thinning of the corneal epithelium and disorganisation of the corneal stroma, similar to that observed in Pax6+/- animals. Unlike Pax6+/- and Gli3+/- animals, however, the peripheral corneal stroma was significantly thinner than in wild-types. As Gli3+/- animals showed no difference in the thickness of the corneal stroma, the reduction of Gli3 dosage alone does not affect stromal thickness but does so only in combination with reduced Pax6 dosage.Like the defects in the retina, abnormalities in the anterior segment of the Pax6 and Gli3 have eye abnormalities that are either not present in, or are more severe than, those present in animals heterozygous for mutations in either gene. Addition of the phenotypes of Pax6+/- and Gli3+/- animals is insufficient to explain the severe phenotypes of the compound mutants. Our findings suggest that Gli3 and Pax6 cooperate during eye morphogenesis.We have found that animals compound heterozygous for mutations in both Pax6+/-  strain was maintained on a mixed  background as described previously [Gli3+/- strain  was maintained on an inbred CBA/Ca background. Animals wild-type at the Pax6 and Gli3 loci, heterozygous for either mutation (Pax6+/- or Gli3+/-) and compound heterozygous for mutations in both genes  were obtained by crosses between Pax6+/- and Gli3+/- animals. 65 animals from 17 litters were analyzed. All comparisons were between wild type, Pax6+/-, Gli3+/- and Pax6+/-; Gli3+/- siblings. Animals were killed by cervical dislocation; eyes were removed and some were weighed before fixing for histological preparation. All animal procedures were performed in accordance with Home Office (UK) legislation.The eviously . The GliEyes were fixed in either 4% paraformaldehyde or Bouin's fluid overnight before dehydrating and processing to paraffin wax. Bouin's fluid was selected for tissue for morphometric analysis as it causes few artefacts of tissue fixation. Sections were cut at 10 μm and stained with haematoxylin and eosin. Midsections through the eye containing the optic nerve were identified for morphometric retinal and corneal analysis.Pax6 gene were assessed as described previously [Gli3XtJ allele has revealed a 51.5-kb deletion [Gli3 and mutant Gli3XtJ alleles were identified using the following primers: 580For 5' TACCCCAGCAGGAGACTCAGATTAG-3' and 580Rev 5'-AAACCCGTGGCTCAGGACAAG-3'; C3For 5'-GGCCCAAACATCTACCAACACATAG-3' and C3Rev 5'-GTTGGCTGCTGCATGAAGACTGAC-3' producing products of 193 bp and 580 bp for the wild-type and mutant alleles respectively.Mutations in the eviously . Mappingdeletion . Using adeletion , wild-tyMorphometric analysis of the retinal layers was performed only in eye sections where normal laminar morphology of the retina was maintained. Animals with eyes which were severely dysplastic were not included in this analysis. Transverse eye sections at the level of the optic nerve  were photographed with a digital camera at ×40. The thicknesses of the whole retina as well as the thicknesses of the inner nuclear layer (INL), outer nuclear layer (ONL), inner plexiform layer (IPL) and outer plexiform layer (OPL) were measured using ImageTool™. Two separate measurements were taken on either side of the optic nerve Fig.  and averAnalysis of corneal epithelial and stromal thickness was performed essentially as described previously . Serial Slides were microwaved in 10 mM sodium citrate to achieve maximal antigen retrieval before addition of primary antibody. Mouse monoclonal anti-Pax6 antibody (1:100) was obtained from the Developmental Studies Hybridoma Bank . Signal was enhanced using the Dako ABC Kit and visualised with diaminobenzidine (DAB).A 611 base pair fragment comprising nucleotides 560–1170 of the mouse Gli3 cDNA (a gift from T. Theil) was PCR amplified and subcloned into a pGEM-Teasy vector (Promega). The plasmid was linearized with SpeI and transcribed with T7 RNA polymerase. Non-radioactive RNA in-situ hybridisation on paraffin wax-embedded sections was carried out using a protocol described previously .GCPS Greig cephalopolysyndactylyDAB diaminobenzidineFGF8 fibroblast growth factor 8GCL ganglion cell layerINL inner nuclear layerIPL inner plexiform layerONL outer nuclear layerOPL outer plexiform layerPHS Pallister-Hall SyndromeShh Sonic hedgehogXt extra-toesPZ and JQ designed and carried out most of the experiments, MC participated in the quantitative analysis, JT carried out in situ hybridizations, IS participated in the design and carrying out of genotyping, DP participated in the analysis and writing of the manuscript.


We have investigated X-ray survival parameters and repair of potentially lethal damage ( PLDR ) in ten early passage squamous cell carcinoma cell lines derived from patients who were biopsied before initiation of radiotherapy or after radiation therapy failure. Radiosensitivity (D0) ranged from 1.07 to 1.93 (Gy), extrapolation numbers (-n) from 1.17 to 2.14 and PLD recovery at 24 h from 1.4 to 20.3. Despite significant differences in these parameters amongst the cell lines, a firm correlation between radiocurability and any individual radiobiological parameter could not be established. Our data suggest that the mechanisms associated with radioresistance are complex and that any single radiobiological parameter may not predict clinical success or failure.


The distribution of labelled cells through 5 different mouse tumours was measured after a single injection of [3H]-thymidine [( 3H]-TdR) or [3H]-deoxyuridine [( 3H]-UdR). All the tumours had areas where the percentage of labelled cells  was high and areas where the LI was very low. The total area with a low LI was greater after [3H]-TdR than after [3H]-UdR injection in all 5 tumours. In one of the tumours, carcinoma NT, repeated injections of [3H]-UdR at 2 h intervals caused the areas of high LI to spread, eliminating all areas of low LI in many specimens. When 5-fluorodeoxyuridine (FUdR) was injected, to block de novo DNA synthesis in carcinoma NT, [3H]-TdR was incorporated by many more cells. The LI was increased throughout the tumour and no area had a LI below 20% after FUdR plus [3H]-TdR. After flash-labelling with [3H]-TdR alone, nearly half the tumour had a LI below 20%. We conclude that the labelling seen after FUdR plus [3H]-TdR represented the true distribution of S phase cells in carcinoma NT. Routine flash-labelling with [3H]-TdR or [3H]-UdR left nearly half the S phase cells unlabelled and gave an erroneously low value for the proportion of DNA synthesising cells in the tumour. The results suggest that many tumour cells have very large endogenous nucleotide pools which cannot be flooded by a single injection, even of [3H]-UdR.


Administration of adenosine triphosphate (ATP) in sinus rhythm identifies dual atrioventricular node physiology (DAVNP) in 75% of patients with inducible slow / fast AV nodal reentrant tachycardia (AVNRT). The incidence of DAVNP following termination of AVNRT with ATP is unknown. Incremental doses of ATP (10-60mg) were administered, first in sinus rhythm and then during tachycardia induced at electrophysiologic study, to 84 patients with inducible AVNRT and to 18 control patients with inducible AV reentrant tachycardia (AVRT) and no electrophysiologic evidence of DAVNP. Study end-points were the occurrence of DAVNP or ≥ 2nd degree AV block following administration of ATP in sinus rhythm and tachycardia termination following administration of ATP during tachycardia. Of the 82 patients with AVNRT who completed the study, 62 (75.6%) exhibited DAVNP following administration of 17.1 ± 9.4 mg ATP in sinus rhythm, while 30 (36.5%) exhibited DAVNP at the termination of AVNRT following administration of 10.6 ± 2.4 mg ATP. The occurrence of DAVNP following the administration of 10 mg ATP in sinus rhythm.was a good predictor (62%) of its occurrence after termination of AVNRT with ATP. The dose of ATP had a strong correlation between the presence of DAVNP following AVNRT termination and the ATP doses needed for tachycardia termination. Of the 18 control patients, none had DAVNP at ATP test during sinus rhythm but 1 (5.5%) showed slight (60 msec) PR jump after termination of AVRT with ATP. In conclusion, DAVNP is present in a relatively high proportion (36.5%) of patients following termination of AVNRT with ATP but is much less frequent (5.5%) in control patients. Thus, findings at termination of tachycardia by ATP may be useful in the noninvasive diagnosis of the mechanism of a paroxysmal supraventricular tachycardia. Atrioventricular nodal reentrant tachycardia (AVNRT) is the most frequently encountered type of regular, paroxysmal supraventricular tachycardia in clinical practice. The typical form, observed in >95% of cases of AVNRT, is due to a reentrant mechanism that involves a slow pathway in the antegrade direction and a fast pathway in the retrograde direction. Adenosine-5'-triphosphate (ATP) is almost always effective for terminating AVNRT usually by blocking the antegrade slow pathway . BesidesThe study group consisted of consecutive patients in whom the effects of the intravenous administration of ATP were tested both in sinus rhythm and during sustained (≥ 1 min) slow / fast AVNRT induced at electrophysiologic study. All patients were evaluated in the absence of antiarrhythmic drug therapy and patients in whom isoproterenol was required to induce AVNRT were excluded. Most patients were included in another study dealing with the use of a simplified ATP test for the noninvasive diagnosis of dual AV nodal physiology and the assessment of the results of slow pathway ablation in patients with AVNRT .The control group consisted of patients who had inducible sustained AVRT involving a concealed accessory pathway and in whom the effects of the intravenous administration of ATP were tested both in sinus rhythm and during sustained (≥ 1 min) AVRT. Patients were excluded if they had electrophysiologic evidence of DAVNP defined as sudden prolongation of AH by ≥ 50 msec when shortening the cycle length of atrial pacing or the coupling interval of the atrial extrastimulus by 10 msec. Most of these patients were part of a larger study that assessed the use of ATP in the noninvasive diagnosis of concealed accessory pathway .None of the patients participating in the study had a history of asthma (a contraindication to ATP administration), or was treated with drugs known to interfere with ATP metabolism .This protocol was approved by our Ethical Committee and all patients gave informed consent. The effects of ATP on AV nodal conduction in sinus rhythm were evaluated before the introduction of any electrode catheter into the cardiac chambers in order to avoid any catheter-induced arrhythmia or trauma to the fast or slow pathways . After pDAVNP was considered to be present when at least one of the following events occurred following ATP injection: 1) PR interval increased or decreased by ≥50 ms in 2 consecutive sinus beats; 2) an AV nodal echo beat was observed; 3) AVNRT developed. All 3 criteria were solely based on the analysis of simultaneous 12-lead surface electrocardiogram. Single AV nodal echoes are often difficult to discern without the aid of intracardiac recordings. However, AV nodal echoes would be expected to reset the sinus activity. Therefore, AV nodal echos were considered to be present when, following a sinus complex conducted with an increased PR interval: a) a >70% increment in P-P interval was observed or b) retrograde P waves were seen at the end of the QRS complex. We previously found a good correlation between these electrocardiographic criteria and the diagnosis of DAVNP using intracardiac recordings  reached a study end-point following administration of ATP in sinus rhythm and AVNRT and constituted the study group.Of the 82 study patients, 62 (75.6%) patients exhibited signs of DAVNP following administration of 17.1 ± 9.4 (range 10 - 40) mg ATP. DAVNP occurred after administration of 10 mg ATP in 34 (55%) patients, after 20 mg in 22 (35%) patients and after ≥ 30 mg in 6 (10%) patients . This ocAVNRT was terminated by ATP in all 82 (100%) study patients. This occurred 6 to 25 (mean 16.3 ± 4.9) sec following the ATP bolus. The doses of ATP that terminated the tachycardia were 10 mg and 20 mg in 77 and 5 patients, respectively (mean 10.6 ± 2.4 mg). In all patients, tachycardia terminated due to antegrade block in the slow pathway. DAVNP at the termination of AVNRT was observed in 30 (36.5%) patients  . This oDAVNP after termination of AVNRT was more frequently observed in patients who exhibited DAVNP in sinus rhythm  than in those who did not  but the difference did not reach statistical significance (p=0.08).In the whole group of patients, the dose of ATP necessary for demonstrating DAVNP in sinus rhythm  was greater than that required to show DAVNP after conversion of AVNRT  (p <0.001). However, the time of onset of DAVNP in these 2 groups was similar . Also, the number of conducted beats over the slow pathway following ATP administration was not statistically different in the 2 settings . Among the 26 patients who exhibited DAVNP both in sinus rhythm and after termination of AVNRT, the doses of ATP required were 12.7 ± 6 mg and 10.4 ± 2 mg, respectively (p<0.05). The time of occurrence of DAVNP was shorter after ATP injection in sinus rhythm (14.7 ± 3.3 sec vs 19.3 ± 6.9 sec p<0.05); however, the number of conducted beats over the slow pathway following ATP administration was not statistically different in the 2 settings .There was a strong correlation between the ATP dose needed to demonstrate DAVNP in sinus rhythm and the odds for demonstrating DAVNP following AVNRT termination: 62% (21/34) of the patients who exhibited DAVNP following administration of 10 mg ATP in sinus rhythm, exhibited DAVNP following termination of AVNRT. In contrast, only 18% (5/28) of the patients who required ≥ 20 mg ATP for demonstrating DAVNP in sinus rhythm, subsequently exhibited DAVNP upon termination of AVNRT with any ATP dose (p<0.0005).This group included 18 patients [13 females and 5 males (age 30 ± 14 years)] who had AVRT and no electrophysiologic evidence for DAVNP. Administration of ATP (15.6 ± 7.4 mg) resulted in ≥ second degree AV block without DAVNP in all patients. A mean dose of ATP (10.7 ± 2.7 mg) terminated AVRT in all patients after 16 ± 5 sec. A slight jump (+ 60msec) in PR interval was noted on 1 beat after tachycardia termination in only 1(5.5%) of the 18 patients.The present study shows that administration of ATP in sinus rhythm reveals the presence of DAVNP in 75.6% of patients with inducible sustained slow / fast AVNRT and in none of the control patients with no electrophysiologic evidence of DAVNP. These results are consistent with those found in our pilot study using ATP (2) and by others using adenosine [This incidence rate of DAVNP upon termination of AVNRT with ATP seems even more remarkable if one takes into account that the study end-point following administration of ATP during AVNRT was tachycardia termination rather than exposure of DAVNP. Since doses of ATP required to achieve DAVNP after drug administration in sinus rhythm were higher than those required for termination of AVNRT, it is tempting to speculate that higher ATP doses during AVNRT might have resulted in a higher incidence of DAVNP after tachycardia termination.As explained elsewhere  administThe occurrence of DAVNP following administration of ATP during AVNRT was more frequently observed in those patients who exhibited DAVNP following ATP administration in sinus rhythm than in those who did not. This difference, however, did not reach statistical significance. In contrast, the occurrence of DAVNP after termination of AVNRT with ATP best correlated (in 62% of patients) with its occurrence following the administration of 10 mg ATP in sinus rhythm.The present study was performed during the course of an electrophysiologic procedure and therefore its results cannot be extrapolated to the clinical setting. When adenosine compounds are administered in the acute management of spontaneously occurring AVNRT, this is generally after several hours of sustained tachycardia compared to 1 min of tachycardia as in the present study. Sympathetic activation resulting from hypotension and stress may be more marked during spontaneous, long-lasting tachycardias than during short-lasting laboratory-induced arrhythmias. Taking in account that adenosine's negative chronotropic effect is enhanced in the setting of adrenergic stimulation , one mayAlthough the diagnosis of AVNRT is usually easy when a 12-lead ECG is available, the actual diagnosis may remain difficult in some instances. In addition, single strip electrocardiographic leads are frequently the only available data, especially when the patient is treated in a mobile care unit. According to our study, the presence of DAVNP following tachycardia termination by ATP would suggest that the mechanism of the tachycardia is actually AVNRT. Caution should prevail, however, since DAVNP can also be present in other types of PSVT. For example, we recently found that DAVNP could be suspected with ATP test in sinus rhythm in 7 (21%) of 33 patients with AVRT ; howeverTermination of induced slow / fast AVNRT with ATP is followed by electrocardiographic evidence of DAVNP in a relatively high proportion of patients. Such a finding may be useful in the noninvasive diagnosis of the mechanism of paroxysmal supraventricular tachycardia.


A biochemical response index comprising ESR, CEA and CA 15.3 was evaluated in 67 patients with systemic breast cancer treated by chemotherapy; 55 were assessable by UICC criteria and the response index . Marker changes at 2 and 4 months showed a highly significant correlation with the UICC assessed response at 3 and 6 months (P < 0.001); sensitivity 100%, specificity 87%; positive predictive value 85%; negative predictive value 100%. This index was then used to select out truly responsive patients and to prospectively direct their chemotherapy. Twenty-six responding  patients were randomised to discontinue cytotoxics after 6 months and move to maintenance hormones (n = 13) or continue chemotherapy whilst the biochemical markers kept falling or remained within the normal range. Biochemical progression prompted a change of chemotherapy. Continuous chemotherapy in biochemically defined responders was associated with a significant lengthening of remission duration and an improved quality of life and survival. We are now using the index to routinely direct chemotherapy and select out true responders for maintenance chemotherapy.


Effects of recombinant tumour necrosis factor (TNF) on functional and structural vascular volumes in solid murine Meth A tumours were investigated by injection of Hoechst 33342 and staining for the vascular basement membrane component laminin, respectively. Systemic injection of 3 x 10(4) U TNF caused an initial increase in functional volume in the tumour, but a strong decrease from 1 to 48 h after treatment. Early effects of intralesional treatment were more moderate. Systemic injection of 10(4) U TNF or 0.3 or 3 micrograms lipid A caused a fall in functional volume at 4 h, but a recovery was seen at 24 h. This recovery did not occur after treatment with a combination of 10(4) U TNF and 0.3 micrograms lipid A. Structural vascular volume was not markedly reduced until 24 h after treatment with the high doses of the separate agents and the combination. All effects appeared generally more prominent in the tumour centre than in the borders. Data suggest that TNF induces initially an active hyperaemia that rapidly converts to passive hyperaemia. A prolonged disturbance of tumour blood supply is probably necessary for therapeutic activity. Breakdown of laminin in the vascular basement membrane may be a cause of loss of vascular integrity.


Epidermal growth factor receptor (EGFR) was studied in ovarian tumours with immunohistochemical (IH) and ligand-binding assay (LBA). Two different monoclonal antibodies  with respect to detecting EGFR with different ligand-binding affinities  were used. When comparing the IH data of MoAbs, 2E9 and EGFR1 a significant correlation was found (2P < 0.0001). Both antibodies stained 77% of the adenocarcinoma samples. The incidence of positivity as well as the mean percentage of stained cells was increased in metastases when compared with primary lesions. In 12.5% overexpression of EGFR (score 3) was noticed in some of the tumour cells. This was not due to amplification of the EGFR gene in any of the 25 ovarian tumours studied (including 6 which showed high expression of EGFR in IH). EGFR was detected in 66% of the adenocarcinomas analysed with LBA. A statistically significant correlation was found between the maximum binding capacities of EGFR obtained from Scatchard plots and the percentage of positive tumour cells determined by MoAb EGFR1 (2P < 0.0001). A weaker correlation was found between the reactivity of MoAb 2E9 and LBA (2P < 0.1). Clinical studies are necessary to determine the possible prognostic impact of EGFR determined with either method, or whether a combination of both will give a better discrimination between high- and low-risk patients.


CHEK2 1100delC allele has been identified at a particularly high frequency in families with hereditary breast and colorectal cancer.Development of multiple primary tumors is a hallmark of hereditary cancer. At least 1/10 of breast cancers and colorectal cancers occur because of heredity and recently the cell cycle kinase 2, CHEK2 1100delC to the development of such metachronous tumors.We utilized the Southern Sweden population-based cancer registry to identify women with double primary breast and colorectal cancer and sequenced tumor material in order to assess the contribution of the CHEK2 1100delC allele. which was not significantly different (p = 0.26) from the 1% (3/300) carriers identified in the control group.Among the 75 patients successfully analyzed, 2 (2.5%) carried the CHEK2 1100delC is not a major cause of double primary breast and colorectal cancer in Sweden, which suggests that this patient group should not routinely be screened for the CHEK2 1100delC variant.In summary, our data suggest that the  BRCA1 and BRCA2 genes are the major causes of hereditary breast and ovarian cancer, but the underlying genetic defect remains unresolved in the majority of the families with familial or hereditary breast cancer , through ATM phosphorylation [CHEK2 1100delC mutation leads to premature termination and abrogates kinase activity. This variant allele occurs in about 1% of normal populations, but has been identified at increased frequencies (1.2–1.9%) in individuals with breast cancer [CHEK2 1100delC mutation [CHEK2 1100delC allele have by some investigators been reported in non-BRCA1/2 families with hereditary breast cancer [CHEK2 1100delC variant does not show clear co-segregation with disease it is considered to be a modifier, and may act in cooperation with yet unidentified high-penetrance genes or together with multiple low-penetrant genes as part of polygenic inheritance.Double-strand DNA breaks lead to activation of the cell cycle checkpoint kinase 2, CHEK2 [GenBank:rylation . CHEK2 pt cancer ,8. An apt cancer . These dCHEK2 1100delC has been reported at frequencies of 1.6–2.6% among individuals with colorectal cancer, which is not significantly higher than in unaffected individuals from the population, but these studies may be compatible with a risk of 1.5–2.0 for development of colorectal cancer among carriers [CHEK2 1100delC allele has been reported in families with hereditary breast and colorectal cancer where 10/55 Dutch families (18%) carried the mutation [In studies from Finland and the Netherlands the carriers -16. A paCHEK2 1100delC mutation to the development of metachronous cancers of these types.Since development of multiple primary tumors in an individual is a sign of hereditary cancer we utilized a population-based cancer registry to identify women who had developed breast cancer and colorectal cancer in order to assess the contribution of the Ethical approval was obtained from the ethics committee at the Lund University. We utilized the population-based cancer registry of the Southern swedish health care region (which currently has approximately 1.7 million inhabitants). The registry was established in 1958 and because of mandatory cancer registration for clinicians as well as for pathologists the registry is estimated to contain 98% of all cancers diagnosed in this area. All females diagnosed with at least one breast cancer and one colorectal cancer during the time period 1958–2000 were identified. Totally 96 patients were identified in whom breast cancer was the first tumor in 64 and colorectal cancer was the first tumor in 25, whereas the remaining 7 patients developed synchronous breast and colorectal cancer. The mean age at diagnosis of the first of tumor was 68 (range 40–95) years, breast cancer developed at mean age 70 (range 40–95) and colorectal cancer at age 74 (range 47–98). After omission of patients who were misclassified or from whom the tumor blocks could not be retrieved, 84 patients remained for analysis.Tumor-containing paraffin-embedded tissue and normal tissue (e.g. resection borders or benign lymph nodes) were retrieved. Fresh sections were obtained and stained with Hematoxylin & Erythrosin in order to verify that tumor tissue was present in the samples. DNA was extracted from 3 × 10 μm paraffin sections using treatment with Proteinase K in 65°C for at least 2 hours and boiled for 10 minutes for enzyme inactivation, whereafter the samples were centrifuged and the aqueous phase was removed for use.CHEK2 exon 10. The primers used were 5'-TGGCAAGTTCAACATTATTCCC-3' (forward) [Mutation analysis was carried out using dHPLC  and samples with aberrant patterns were further analyzed by direct sequencing . Due to homologous sequences primer design is complex for forward)  and 5'-ACHEK2 1100delC mutation was detected in 2 patients (2.5%)  were successfully analyzed. In total, 36 breast cancers and 67 colorectal cancers were analyzed and from 34 patients normal tissue was also analyzed. The majority of the patients developed breast cancer as their first tumor table  and 68 oCHEK2 1100delC in the Southern swedish population dHPLC analysis and direct sequencing was performed from 300 healthy individuals with identification of the variant allele in 3/300 individuals, thus at a population frequency of 1% [In order to determine the expected frequency of the cy of 1% .et al. already in 1972 [CHEK2 1100delC mutation was proposed to represent a low-penetrant breast cancer susceptibility allele [CHEK2 1100delC had been described in hereditary breast and colorectal families, we assessed the contribution of this variant to the development of double primary breast and colorectal cancer in a population-based patient material. Among the 75 patients with metachronous tumors of the breast and the colorectum successfully studied, 2 (2.5%) carried the CHEK2 1100delC mutation compared to 1% in the control group [per se not alone to identify individuals with a high likelihood of being carriers of the CHEK2 1100delC mutation. Although development of double primary tumors may be a sign of heredity it is not enough to recommend genetic analysis, though the family history of cancer should be carefully reviewed. Our findings are in line with previous studies that exclude CHEK2 1100delC as a major contributor to the breast and colorectal cancer phenotype [et al. [CHEK2 1100delC mutation among 24 patients from the US with breast cancer and colorectal cancer. Also, in the study by Meijers-Heijboer et al. that primarily identified the link between CHEK2 1100delC and familial breast and colorectal cancer in Dutch families, the vast majority, 45/55 families, who fulfilled these criteria did indeed not carry this variant [CHEK 1100delC mutation.A subtype of familial breast cancer that includes colorectal cancer was recognized by Lynch y allele . Since dol group . These fhenotype . Huang e [et al.  did not  variant . HoweverCHEK2 1100delC allele in our study, one presented with two separate, synchronous, breast cancers. An increased risk of multiple primary breast cancers (OR 5.7–6.5) has been reported in individuals carrying this CHEK2 1100delC allele [CHEK2 1100delC mutation has not only been suggested to act as a low-penetrant susceptibility gene in breast cancer families, but the CHEK2 I157T alteration has been proposed to act as a multiorgan cancer susceptibility allele based on observations of an increased risk for development of breast cancer, colon cancer, prostate cancer, thyroid cancer, and renal cancer in Polish families [et al. who identified an increased risk of renal cancer associated with CHEK2, albeit in carriers of the CHEK2 I157T variant [Among the two women with the C allele ,22. Howefamilies . Both pa variant . HoweverCHEK2 1100delC occurs at a low frequency in Swedish women with double primary breast cancer and colorectal cancer, and thus suggests that development of these two tumor types is not sufficient to recommend mutation analysis of CHEK2.In summary, our findings demonstrate that the The author(s) declare that they have no competing interests.AI conceived of the study, performed the sequencing analysis, interpret data and drafted the manuscript. MB also carried out the sequencing. AB participated in its design and helped to draft the manuscript. MN conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the manuscript.The pre-publication history for this paper can be accessed here:


Thirty-four primary, untreated sporadic breast cancers were examined for loss of heterozygosity (LOH) at tumour suppressor loci involved in colorectal cancer: APC/MCC at 5q21 and DCC at 18q21. LOH was identified in 28% informative patients at 5q21 and 31% at 18q21. LOH at 5q21 and 18q21 was compared with allele loss at 17p13 and concurrent LOH at two or more of the loci was noted in 24% of tumours. Expression of a 12 kb DCC mRNA was demonstrated in 14/34 (42%) of the cancers and in all five tumours with LOH at the DCC locus there was an additional 11 kb DCC mRNA. Abnormalities of three loci involved in colorectal cancer  therefore also occur in sporadic breast cancer. The accumulation of such genetic abnormalities may confer a growth advantage important in the development of breast cancer.


The value of chemotherapy in advanced non-small cell lung cancer (NSCLC) remains contentious. Because of this two separate but very similar trials were set up in Australia and Southampton (UK). Two hundred and one patients with stage IIIb or IV NSCLC were randomly assigned to cisplatin 120 mg m-2 on days 1 and 29 and vindesine 3 mg m-2 weekly x 6 or to no chemotherapy. Both groups were eligible to receive radiotherapy or other palliative treatment as required. Of 188 evaluable patients, 97 received chemotherapy and 91 were in the control arm. Response was assessed between days 42 and 49. Responders continued chemotherapy at the same doses though cisplatin being given 6 weekly x 4 and the vindesine 2 weekly x 12. The overall response rate to chemotherapy was 28%; there were no significant differences according to major prognostic criteria. Although the overall survival of the chemotherapy group (median 27 weeks) was longer than that of the no chemotherapy group (median 17 weeks) this was not statistically significant (log rank P = 0.33). For patients without dissemination (IIIb), median survival was 45 weeks in the chemotherapy arm and 26 weeks in the non-chemotherapy (log rank P = 0.075). Toxicity was universal and frequently severe: of 17 patients discontinuing chemotherapy after one cycle, 13 did so because of unacceptable toxicity. This chemotherapy cannot be recommended as routine treatment. Further phase III studies of chemotherapy in advanced NSCLC should continue to use a no chemotherapy control and should also attempt to measure quality of life, an issue not addressed effectively in this or other recent trials.


Patients with T2 grade 3 and T3 bladder cancer were randomised to be treated with radiation alone (NO MISO) or with radiation and misonidazole (PLUS MISO). Patients in both groups initially received 40 Gy in 2 Gy fractions (5/week). Patients in the NO MISO arm received a further 20 Gy in 2 Gy fractions (5/week). Patients in the PLUS MISO arm received a further 12 Gy in 6 Gy fractions (1/week). MISO was administered orally (3.0 g m-2) and intravesically (1.0 g in 35 ml of solvent) 4 h and 2 h respectively prior to each fraction of 6 Gy. Fifty-eight patients were randomized of whom 53 are evaluable. There is a minimum follow-up of 5 years in the surviving patients. In the NO MISO and PLUS MISO arms, the complete response rate at cystoscopy at 6 months was 63% and 69%, the 5-year survival rate was 41% and 48% and the 5-year local control rate with bladder preservation was 46% and 36% respectively . These differences are not statistically significant. Two patients had grade 3 RTOG late bowel complications. Both patients were in the PLUS MISO arm, had undergone salvage cystectomy and subsequently required colostomies for bowel obstruction for a 5-year late complication rate (RTOG grade 3) of 9%. In addition, two patients in the PLUS MISO arm developed wound sepsis post cystectomy. We were not able to demonstrate improved results from the use of oral and intravesical MISO in this study. The number of patients entered are relatively low and large differences would have been required to be detected with a power of 0.80. The use of an unconventional radiation fractionation schedule may have resulted in increased bowel morbidity in patients in the PLUS MISO arm who subsequently underwent cystectomy.