DXZ4 transcripts could be detected originiating from both the active and inactive X chromosome. Expression levels of DXZ4 varied significantly between males, but did not relate to the size of the array, nor did inheritance of the same array result in similar expression levels. Collectively, these studies provide considerable insight into the polymorphic nature of DXZ4, further highlighting the instability and variation potential of macrosatellites in the human genome.Macrosatellites are some of the most polymorphic regions of the human genome, yet many remain uncharacterized despite the association of some arrays with disease susceptibility. This study sought to explore the polymorphic nature of the X-linked macrosatellite DXZ4. Four aspects of DXZ4 were explored in detail, including tandem repeat copy number variation, array instability, monomer sequence polymorphism and array expression. DXZ4 arrays contained between 12 and 100 3.0 kb repeat units with an average array containing 57. Monomers were confirmed to be arranged in uninterrupted tandem arrays by restriction digest analysis and extended fiber FISH, and therefore DXZ4 encompasses 36–288 kb of Xq23. Transmission of DXZ4 through three generations in three families displayed a high degree of meiotic instability (8.3%), consistent with other macrosatellite arrays, further highlighting the unstable nature of these sequences in the human genome. Subcloning and sequencing of complete DXZ4 monomers identified numerous single nucleotide polymorphisms and alleles for the three microsatellite repeats located within each monomer. Pairwise comparisons of DXZ4 monomer sequences revealed that repeat units from an array are more similar to one another than those originating from different arrays. RNA fluorescence  At least half of the human genome is composed of repetitive DNA Macrosatellites consist of repeat units ranging from 1–12 kb that are arranged in tandem. The number of repeat units is polymorphic in the general population, and an array can be composed of only a few to over one hundred repeat units, and therefore can encompass large genomic intervals. Most macrosatellite arrays are specific to one or two chromosomal locations in the genome, and only a small number have been confirmed and characterized to some extent PLS3), and 296.5 kb proximal to the angiotensin II receptor (AGTR2)(Our interest in macrosatellite repeats came about through examination of chromatin organization on the human inactive X chromosome (Xi). X chromosome inactivation (XCI) is the mammalian form of dosage compensation r (AGTR2). DXZ4 isr (AGTR2), that acHere we report our findings on four aspects of DXZ4 variation: tandem repeat copy number variation, array instability, monomer sequence polymorphism and differences in array expression.in vivo, but would be aligned on top of one another in silico. A comparison of a single DXZ4 monomer sequence (Accession Number HQ659112) against the assembled human genome sequence (hg19) using the UCSC genome browser (http://genome.ucsc.edu/), reveals approximately fourteen ∼3 kb DXZ4 monomers covering 50 kb, arranged in tandem centered at 115 Mb on the X chromosome. In addition, two partial and one complete monomer reside in an inverted orientation relative to the main array within the immediate distal 70 kb sequence  revealed hybridizing DXZ4 EcoRI fragments of between 150–300 kb by Southern analysis. This translates into arrays composed of between 50 to 100 monomers. We extended this analysis to an additional 22 unrelated individuals of diverse ethnicity. Agarose embedded genomic DNA was digested with XbaI (a restriction endonuclease for which there are no recognition sites within DXZ4) and were then separated by PFGE before transfer to nylon membrane by Southern blotting. As expected, hybridization of the blots with a DXZ4 probe identified two hybridizing signals in female samples and one in males .Next we sought to confirm that DXZ4 is indeed a tandem array of individual 3 kb monomers arranged in a head-to-tail orientation. In order to do this we used two complementary approaches; restriction endonuclease digest analysis of a DXZ4 bacterial artificial chromosome (BAC) clone, and extended DNA fiber fluorescence http://blast.ncbi.nlm.nih.gov/Blast.cgi). Several clones were identified including clone 2272M5 from the human genomic sperm CITB BAC library D (Accession number AQ745776). The BAC clone was obtained and DNA isolated. Given that the BAC clone insert matches DXZ4 sequence at both ends, uninterrupted tandem arrangement of DXZ4 monomers should result in the generation of a predictable pattern of restriction fragments (BamHI cuts twice per monomer and once in the BAC vector (pBeloBAC11). HindIII cuts once per monomer and is the cloning site used for generation of this library BamHI and 3.0 kb HindIII fragment relative to the vector backbone fragment indicated that the BAC contained several DXZ4 monomers. To confirm this, the BAC clone was digested with four different restriction endonucleases that have recognition sites within the vector backbone but not within DXZ4, and the cut DNA was separated by PFGE. For all four digests the resulting fragment was greater than 100 kb . The two cloned fragments were then labeled with different fluorophores and used for FISH. A tandem arrangement of DXZ4 would result in an alternating red-green signal, as was observed . Most variation was accounted for by polymorphism in copy number of repeat units in the three internal microsatellite repeats. The [GGGCC] repeat ranged from 2 to 5 tandem copies, the [CT] repeat ranged from 9 to 16 tandem copies, and the [TAAA] repeat ranged from 8 to 13 tandem copies  The 18 unique monomers shared 99% or greater sequence identity according to pairwise alignments using BLAST  were identified in monomers from 2272M5 , as wellPvuII, separated by PFGE and Southern blotted, showed the same pattern of bands when hybridized with a DXZ4 probe , two diploid female primary fibroblast cultures and one EBV transformed diploid female lymphoblast cell line. Representative examples of the results are shown in Next we sought to investigate the expression of DXZ4 from the Xi and Xa in 10 different female lymphoblast cell lines by RNA FISH with direct labeled probes; five from CEPH family 1331 and five from family 1345. DXZ4 expression was readily detected in all females . The patPreviously, we had shown that all regions of DXZ4 could be detected by RT-PCR Finally, we examined expression of DXZ4 in a panel of complementary DNA (cDNA) samples prepared from 20 different human tissues. RT-PCR analysis showed robust expression of DXZ4 from all sources  indicatiLike DXZ4 Next we sought to examine if inherited DXZ4 alleles showed comparable expression levels. Once again, to ensure expression originated from a single allele we restricted our analysis to males. Due to X-linkage we also restricted our analysis to sons inheriting DXZ4 from their maternal grandfather. As with the size versus expression analysis described above, DXZ4 expression was variable , and inhExploring variation in the human genome is essential to begin to understand how polymorphism impacts gene expression, phenotypic variance and disease susceptibility. Here we report on variation of the X-linked macrosatellite DXZ4.In this study, we confirm that DXZ4 is a polymorphic uninterrupted tandem array, and extend the range of observed alleles to between 12 and 100 head-to-tail 3 kb repeat units. This variability is comparable to that described for other macrosatellites In addition to monomer copy number variation, we report variation in both the internal microsatellite repeats, and numerous SNPs within the unique regions of a monomer. Our analyses indicate that DXZ4 monomers within one array are more similar to one another than they are to monomers in an array from a different individual. This suggests that mutations acquired in a monomer can spread through an array, most likely via complex gene conversion mechanisms as has been described for other satellite DNA Previously we have described several different RNA species originating from DXZ4, with expression from both the Xa and Xi As with expression of DXZ4 from the Xa, DXZ4 expression from the Xi is variable, differing both between cell lines and from one cell to the next within the same line. Using direct RNA FISH, it is also clear that the level of expression of DXZ4 differs from cell to cell within the same cell line; with some cells showing little to no expression whereas others may show DXZ4 localized transcript patterns comparable in size to that of XIST RNA. It is important to note that large diffuse DXZ4 signals were not common, and were only observed in the hTERT and EBV transformed cell lines. However, primary cells were not extensively examined in this study and therefore no significance can be placed on the extent of the DXZ4 signal and cell immortalization. DXZ4 monomers on the Xi are packaged into both euchromatin and heterochromatin CT). The exception was the SST1 array that is expressed in all tissues examined. Here we show that DXZ4 is also expressed at comparable levels in all tissues, and therefore DXZ4 is not a CT loci, distinguishing DXZ4 from two other X-linked macrosatellite repeats CT47 CT family. The significance of DXZ4 expression is unclear. Like SST1, DXZ4 shows no obvious protein coding function. It is possible that expression of DXZ4 is associated with chromatin organization of the array Recently we described the characterization of four autosomal macrosatellite arrays The data we present here extends our knowledge base of DXZ4 and the biology of macrosatellite arrays, providing the basis for formulation of new hypotheses to explore the role of DXZ4 on the X chromosome.Human tissue total RNA was obtained from Clontech (636643). Residual genomic DNA was removed by pre-treating the RNA with DNaseI (Invitrogen) for 20 minutes at room temperature, before heat inactivating the DNaseI at 70°C in the presence of 2.5 mM EDTA for 15 minutes. cDNA was prepared using 1 ug of total RNA with or without M-MLV reverse transcriptase (Invitrogen) according to the manufacturers instructions.cDNA was amplified using Taq polymerase (NEB) with the following cycle: 95°C for 2 minutes, followed by 35 cycles of 95°C 20 seconds, 58°C 20 seconds, 72°C 30 seconds. The sequence of oligonucleotides used to amplify DXZ4 cDNA and the anticipated product size are given in the Supporting Information .QRT-PCR was performed on a CFX96 thermocycler and analyzed using the CFX Manager Software (Biorad). The sequence of oligonucleotides used to amplify DXZ4 cDNA are given in the Supporting Information . Amplifiwww.coriell.org/). Cells were maintained according to Coriell recommendations. Culture media (RPMI), fetal bovine serum and supplements were all obtained from Invitrogen corp. Telomerase immortalized cell lines hTERT-RPE1 , hTERT-HME1  and hTERT-BJ1  were all obtained from Clontech and maintained as recommended by the supplier. Fetal lung fibroblast primary cells IMR-90  and WI-38  were obtained from the American Type Tissue Culture Collection (ATCC), and maintained according to supplied instructions.Lymphoblastoid cell lines of CEPH family members and the individuals used in the variation panels were obtained from the Coriell Institute for Medical Research . Cells were pelleted three more times, resuspending each time in 3∶1 fix. Fixed cells were either stored at −20°C or used immediately for fiber preparation. Cells in 3∶1 fix were applied to the raised end of a poly-L lysine coated microscope slide propped up on a paper towel. Using a cover glass, the cells were gently drawn down the length of the slide, dragging only the liquid, not touching the glass slide. Cells were air dried for 5 minutes before immersing in 3∶1 fix for 5 minutes. Fibers were dehydrated through 70% and 100% ethanol for 2 minutes each before air-drying. Fibers were denatured for 5 minutes in 70% formamide, 2× SSC at 75°C before dehydrating for 3 minutes each in cold 70% and 100% ethanol.FISH probes consisted of 449 bp or 550 bp pCR2.1 cloned PCR fragments of DXZ4 that are approximately 900–1100 bp apart in a single monomer . Probes Images were collected using a Zeiss Axiovert 200 M fitted with an AxioCam MRm and were managed using AxioVision 4.4 software (Carl Zeiss microimaging). Image files were exported to Adobe Photoshop CS (Ver.8.0) for preparation of figures.A direct labeled Spectrum Green probe of BAC clone 2272M5 was prepared by Nick Translation according to the manufacturers instructions (Abbot Molecular). The probe (1 microgram) was ethanol precipitated along with 25 micrograms of human Cot-1 DNA before resuspending in 0.1 ml of Hybrisol VII . A direct labeled Spectrum Red probe of XIST exon 1 was prepared as described previously DXZ4 sub-region direct-labeled FISH probes were prepared from sub-fragment clones of DXZ4. Sub-fragment clones were prepared by PCR amplifying unique regions of DXZ4  and TA cImaging was performed as described as above.7 cells were resuspended in 1 ml of L-buffer , before mixing 1∶1 with 1.0% (w/v) molten low-melt agarose (Biorad). The cell mixture was transferred to plug molds (Biorad) with ∼80 ul of the cell suspension per plug (approximately 1.6×106 cells/plug). Plugs were allowed to set at 4°C for 10 minutes before transfer to 10 volumes of L-buffer containing 1% (w/v) sarkosyl and 1 mg/ml Proteinase-K (Roche) and incubating overnight at 50°C. Plugs were rinsed with water before three washes of one hour each with 50 volumes of TE [8.0]. Plugs were incubated at 50°C for 30 minutes in 10 volumes of TE [8.0] supplemented with 80 ug/ml PMSF (Roche). Plugs were rinsed once more with water before three additional hour-long washes in 50 volumes of TE [8.0] at room temperature before storage at 4°C.Approximately 4×10Agarose embedded DNA was digested with the restriction enzymes given in the legend for the appropriate data figures. All enzymes were obtained from NEB. Each plug was first equilibrated in 300 ul of 1× digest buffer at room temperature for 20 minutes, before replacement of buffer with 100 ul of 1× digest buffer containing 200 units of restriction enzyme. Digests were performed overnight at 37°C. Plugs were loaded onto a 1.0% agarose gel prepared using pulsed field certified agarose (Biorad) in 0.5×TBE. The running conditions  were determined by the auto algorithm function of the CHEF Mapper (Biorad). The following conditions were consistent in each case: 0.5× TBE, 14°C, 1.0% agarose. Separation parameters for the Southern blots in the current manuscript were as follows: HindIII to confirm presence of a common 3 kb fragment and insert size determined by PFGE. BAC clone 2272M5 (Acc. No. AQ745776) was selected for subcloning and sequencing. Cloning vector pBluescript II was digested with HindIII before phosphatase treatment using Calf intestinal phosphatase (NEB). BAC 2272M5 was digested with HindIII and fragments subcloned into pBluescript II, before blue-white screening using standard techniques BAC clones were identified that matched DXZ4 at both ends by BLAST using a single DXZ4 monomer sequence (Acc. No. S60754). BACs were obtained from Invitrogen and DNA isolated using the Qiagen plasmid Midi kit. BACs were digested with At the end of the PFGE run, the gel was rinsed with water before staining with ethidium bromide (1 ug/ml) at room temperature for 30 minutes. The gel was washed twice with water for 15 minutes each and an image captured. The gel was then treated with 0.25 M HCl for 15 minutes before denaturing for 30 minutes . DNA was transferred to Hybond-N+  overnight by standard Southern blotting EcoRI digested total genomic DNA.A DXZ4 probe was prepared by PCR amplification of regions of DXZ4 using oligonucleotides listed in the Supporting Information . The PCRHybridization was performed overnight at 60°C using Expresshyb (Clontech). Blots were washed the following day at 60°C using two 8-minute washes in 2×SSC, 0.1%SDS followed by one wash of 8 minutes in 0.2×SSC, 0.1%SDS. The probe was detected using anti-DIG-alkaline phosphatase, blocking, wash and detection buffers according to the manufacturers instructions (Roche). Signals were detected by exposure to photographic film (Kodak).Figure S1Comparison of DXZ4 hybridization patterns between PvuII and XbaI PFGE. Southern blots of PFGE separated DNA from 11 independent individuals cut with either XbaI or PvuII and hybridized with a DXZ4-DIG probe. Recognition sequences for either restriction endonuclease are not present in the DXZ4 array and therefore give near identical hybridizing patterns. Sizes in kb are given to the right of each blot. The first PvuII site is 24 kb closer to the array on the distal edge accounting for the smaller sized hybridizing fragments.(TIF)Click here for additional data file.Figure S2BAC clone insert size determination. Ethidium bromide stained 1.0% agarose gel showing restriction endonuclease digestion of DXZ4 BAC clone 2272M5 separated by PFGE. Separation performed at 14°C for 26 hours in 0.5× TBE, separating for 20–200 kb on a CHEF Mapper (Biorad). Markers and sizes are indicated, as are the restriction enzymes used that cut in the vector backbone, but not the DXZ4 array. NotI cuts twice in pBeloBAC11, excising the BAC insert, accounting for the smaller fragment size.(TIF)Click here for additional data file.Figure S3Southern blot of PvuII digested DNA from members of CEPH family 1345. Southern blot of PFGE separated DNA from CEPH family 1345 digested with PvuII and hybridized with a DXZ4-DIG probe. The top portion of the blot has been darkened in Photoshop in order to clearly see the 284 kb extra band (top arrow) also observed with XbaI. The middle arrow points to the additional 234 kb band and the lower arrow points to the additional 227 kb band.(TIF)Click here for additional data file.Table S1DXZ4 variation in genome build HG19. Summary of SNPs and microsatellite alleles in complete ∼3 kb DXZ4 monomers that define the DXZ4 array in human genome build hg19. Coordinates of SNPs are given relative to the reference sequence of subclone 35. Variants that do not appear in BAC 2272M5 are highlighted in red (only first appearance in the table is highlighted). The largest allele of the (CT) microsatellite is highlighted in green.(DOCX)Click here for additional data file.Table S2DXZ4 variation in monomer submitted by Giacalone et al. Summary of SNPs and microsatellite alleles in the single DXZ4 monomer sequence submitted by Giacalone and colleagues (DOCX)Click here for additional data file.PCR Primers S1DNA sequence of oligonucleotide primers used in current study.(DOCX)Click here for additional data file.


Our study demonstrates that diabetic patients have increased myocardial edema in the infarct segment at an early time point post acute myocardial infarction which persists till the subacute phase. This may lead to deleterious left ventricular remodeling and worse outcome in these patients.Diabetes is associated with worse left ventricular remodeling and poor prognosis in patients post acute myocardial infarction (AMI). The impact of diabetes on microvascular injury parameters including myocardial edema and hemorrhage post AMI is unknown. Our objective was to characterize the evolution of myocardial edema and hemorrhage post AMI in patients with and without diabetes.Sixty patients were enrolled post AMI and underwent cardiac magnetic resonance on a GE Signa Excite, 1.5T scanner with a 8-channel receive coil at 48 hours and 3 weeks. T2 maps were computed from a previously validated cardiac-gated spiral imaging sequence with T2 preparations yielding TEs=2.9,24.3,88.2,184.2ms to assess myocardial edema. The T2* sequence was a multiecho acquisition with 8 echoes (between 1.4 and 12.7ms) acquired at TR=14.6ms. Delayed hyperenhancement was also performed. We retrospectively reviewed and stratified patients into those that did and did not have diabetes (type 1 or type 2).We compared 15 diabetics versus 45 non-diabetics . At this time interval, the mean T2* was equivalent in the IS of both patient groups .Our study demonstrates that diabetic patients have increased myocardial edema in the IS at an early time point post AMI which persists till the subacute phase (3 weeks). The degree of myocardial hemorrhage does not appear to be influenced by diabetes status. Diabetes increases the area at risk during AMI which may lead to deleterious left ventricular remodeling and worse outcome in these patients.Dr. Connelly is supported by a Heart and Stroke Foundation of Canada Phase 1 Clinician Scientist Award. Dr. Wright receives research funding from GE Healthcare. Dr. Dick is supported by the Heart and Stroke Foundation of Canada. This work was supported in part by a Canadian Institutes of Health Research (CIHR) operating grant and the Ontario Research Fund.


Arterial partial pressure for oxygen and carbon dioxide, and oxygen saturations were measured in the jugular and arterial blood  . The subjects were exposed to hypoxia , inspiration of 100% oxygen (n = 8), atmospheric air (n = 37), hypercapnia  and asked to hyperventilate  with separate controls. By linear regression the contribution from arterial and jugular blood to ScO2 was estimated and R-squared (R2) and root mean square error (RMSE) between ScO2 and the arterial fraction in the reference saturation were calculated . Only data points where SjO2 ≤ ScO2 ≤ SaO2 were included. All reference saturations were calculated, e.g., ScapO2 = 0.50 · SaO2 + 0.50 · SjO2. The following equation, 0 = ScO2 – [a · SaO2 + (a − 1) · SjO2], was used to calculate the arterial fraction (a) for which the difference between ScO2 and the reference saturation was zero.Thirty seven subjects [age 27(9) years, height 181(10) cm, mass 79(13) kg, mean  with a RMSE of 4.233. In contrast, ScapO2  had a R2 of 0.606  and also for that ratio the RMSE was the lowest (2.70) . In addition, the mismatch between ScO2 and the reference ratio was more likely to be zero when a more arterial weighted reference was applied  Figure . The cald Figure . Thus, ocO2 values and the calibration ratio  is observed in five different NIRS devices used to determine cerebral oxygenation in healthy subjects exposed to isocapnic hypoxemia . Thus, the mismatch between the calibration ratio and ScO2 is aggravated when only jugular venous oxygen saturation is altered, which indicates that ScO2 accounts for more than 25% arterial blood and that evaluations of reference saturations for NIRS must also involve conditions known to affect arterial oxygen content.Similar to these findings, no eligible mismatch between ScO2 readings (Rasmussen et al., cO2 readings may be variation in skull thickness and amount of cerebrospinal fluid (Yoshitani et al., cO2 because of competitive absorption of light (Madsen et al., The arterial to venous balance within the brain may differ between individuals and explains the inter-individual variation in absolute ScO2 values are not comparable and exhibit large variations when compared to the calibration ratio, NIRS offers a unique non-invasive method for assessment of cerebral oxygen delivery vs. consumption and its clinical utility relies on changes from baseline rather than on absolute values (Murkin et al., cO2 is limited when skin blood flow is affected either by scalp ischemia or administration of sympathomimetic agents that affect skin blood flow (Davie and Grocott, Despite absolute SIn summary, this report suggests that the reference saturations applied when using Invos cerebral oximetry should be weighted more to arterial hemoglobin saturation than accepted by anatomical models.All authors contributed equally to the design, data analysis, and interpretation, drafting the manuscript and critical revision. All authors approved the final version before submission.


Mutations in the BRCA1 and BRCA2 genes predispose women to breast and ovarian cancer. The large majority of the alterations identified in these genes are point mutations and small insertion/deletion. However, an increasing number of large genomic rearrangements (LGRs) are being identified, especially in BRCA1. To date just a few large genomic rearrangements of BRCA1 gene have been reported in Poland. Technical limitations of conventional PCR-based methods are cause that gross rearrangements can be overlooked. It has been suggested that about 30% of mutations in the BRCA1 gene are missed by standard mutation detection methods. We screened for LGRs in BRCA1 and BRCA2 genes by Multiplex Ligation-dependent Probe Amplification (MLPA) in 200 unrelated patients with strong family history of breast and/or ovarian cancer negative for BRCA1 Polish founder mutation. We identified 3 different LGRs in BRCA1 gene. No large LGRs were detected in BRCA2 genes.


It was an observational cohort of pregnant HIV positive women ignorant of antiretroviral therapy with CD4 cell count of>350/mm3. Exclusion criteria: highly active antiretroviral therapy prior to pregnancy.Routine CD4 cell count assessment in HIV positive pregnant women completed by non exclusive measurement of the viral load by PCR /ARN in those with CD4 cell count>350/mm3, median: 528 (IQR: 421–625). 145 underwent measurement of viral load by PCR/RNA at a median gestational of 23 weeks of pregnancy (IQR: 19–28). Median viral load 4.4log10/ml, IQR (3.5–4.9).19/145(13%) had an undetectable viral load of=1.8log10/ml. 89/145(61%) had a viral load of=4 log10/ml and were eligible for maternal triple ARV prophylaxis.Between January and December 2010, CD4 cell count was systematically performed in all pregnant women diagnosed as HIV-infected (n=266) in a referral center of 25 antenatal clinics. 63% (N=170) had CD4 cell count>350/mm3 may require triple antiretroviral for prophylaxis of MTCT. Regardless of cost, such results are conclusive and may be considered in HIV high burden countries for universal access to triple antiretroviral prophylaxis in order to move towards virtual elimination of HIV MTCT.More than 6 in 10 pregnant HIV positive women with CD4 cell count of>350/mm Sub-Saharan Africa continues to record the highest burden of the HIV-1 epidemic, contributing more than 90% in the new cases of perinatally acquired HIV pediatric infections . Mother-3. Maternal baseline serum samples were quantified for HIV-1 RNA load using real Time reverse transcriptase PCR . All the samples were analyzed with the Laboratoire Centre Pasteur in Yaoundé acting as service provider at a unit cost of 30US$. ZDV was started for all the others non eligible for triple antiretroviral treatment at 14 weeks of pregnancy, while waiting for the result of the viral load.The study population was an observational cohort of pregnant HIV positive women. Pregnant women with<36 weeks of amenorrhea were enrolled in 25 antenatal clinics in the Djoungolo Health District. After opt-out counseling, serial HIV-1/2 algorithm antibody tests were done using Determine HIV-1/2 assay and SD bioline HIV 1/3 3.0  rapid kits on mothers' serum samples. For all HIV positive women, CD4 cell counts were measured in the referral laboratory. The method used a flow cytometer for measuring the percentage of CD4 T cells; an automated blood cell counter helped to measure the total number of lymphocytes. Results were produced in absolute numbers and in percentages. CD4 cell count screening was followed by non exclusive measurement of viral load when CD4 cell counts were>350/mm10 was considered as undetectable viral load. For this descriptive analysis we considered . A confidence interval of 95% was considered as the margin for percentage accuracy.The following qualitative variables were considered: maternal age, WHO clinical stages. Quantitative variables included: CD4 cell count and HIV Viral load. Viral load values were log10 transformed and stored in an Excel sheet form. A threshold of≤1.8 logAll interventions within this project were implemented under the supervision of the District Office of the Ministry of Public Health. Data collection and monitoring of the intervention was part of the routine activities of the Health District. Free measurement of HIV1 RNA viral load was offered to all the eligible mothers at the referral center where all activities were coordinated by the Center for HAART treatment and the local board in charge of fighting against HIV and AIDS.Mothers′ mean age (SD) was 27.0 years IQR 22.5–30 and 15/172(8.7%) were adolescents. All the mothers were classified in WHO clinical stage 1 or 2.3, IQR (442–646) and 68/170 had CD4 cell counts of between 350 and 500/mm3.Median CD4 cell count was 534 cell/mmMaternal viral load and transmission10. The median log10 viral load was 4.4(IQR 3.5–4.9). 13%, 95% CI (7.5–18) had an undetectable viral load. A viral load of>3log10 was recorded for 107/145(74%); 89/145(61%) had a viral load of≥4log10 and for 29/145  a very high level of viral load as usually observed during primary acute infection, was noted (≥5log10).3, making them eligible for maternal triple ARV prophylaxis as required elsewhere. Taking into account that an average of 4 in 10 HIV positive pregnant women are yet to be eligible for HAART because of CD4 cell count of<350/mm3, it may be deduced that among the remaining 6 in 10, at least 4 may require HAART for prophylaxis according to their HIV viral load. Altogether 8 in 10 women at least may be eligible for HAART during pregnancy based upon biological criteria. Such results vindicate the opinion of those who claim that universal access to maternal triple ARV prophylaxis during pregnancy regardless of CD4 cell count is a must and are consistent with the latest WHO recommendations for women not yet in need of treatment for themselves, namely option B [To our knowledge, this is the first study in Cameroon where viral load is systematically determined in pregnant women in order to assess the eligibility to more effective antiretroviral regimen especially at high levels of CD4 cell count. Our key finding is that the level of HIV viral load is≥4log 10/ml in more than 60% of HIV pregnant women, ignorant of HAART, with CD4 cell count>350/mmIn Sub-Saharan Africa, some countries have opted for triple antiretroviral therapy for all pregnant women according to the level of care . In the 10 in more than 60% newly diagnosed pregnant women in Cameroon with high levels of CD4 cell count. Such results suggest the promotion of the routine measurement of viral load during pregnancy in A option country or the switch to B option for all, in order to ensure larger access to maternal antiretroviral triple prophylaxis. This approach is likely to be more effective in lowering maternal viral load and reducing mother-to-child transmission of HIV.We concluded that antenatal serum HIV-1 RNA viral load, stands above 4log


Isolated aneurysm of subclavian or axillary artery is very rarely seen.Eight patients that were operated on between February 1998 and December 2007 due to true aneurysms of the subclavian and axillary arteries were examined. Six of the patients (75%) were male. Median age was 58 (38-73). Two of these patients had subclavian and 6 of them had axillary artery aneurysms. Median diameter of the aneurysm was 46 mm (30-60).Chief complaint was ischemic symptoms in 2 (25%) patients, non-disturbing mass in 3 (38%), feeling of compression in 2 (25%) and bleeding in 1 (12%) patient. Etiological factors include atherosclerosis in 5 patients (62%), a probable connective tissue disorder in 1 patient (13%) and idiopathic in 2 patients (25%). In these last 2 patients no atherosclerotic changes were prominent histopathologically and no other factors were identified; thus considered as idiopathic. One patient with a probable connective tissue disorder had no histopathological diagnosis. But he was phenotypically marfanoid and his past medical history was significant for surgery due to iliac artery aneurysm. Contrasted CT examination revealed no other aneurismatic segment preoperatively. Past medical history of another patient was also significant for surgery of contralateral axillary artery aneurysm. But histopathologic examination revealed atherosclerotic changes. Therefore this patient was not considered as having connective tissue disorder. One patient with ruptured axillary artery aneurysm was operated on emergently with a chief finding of bleeding.One of two cases with subclavian artery aneurysm that we operated on had acute ischemia findings of the upper extremity and the other had a non-disturbing mass. Consistent with the literature, 2 of our cases with axillary artery aneurysm had non-disturbing mass, 2 had feeling of compression, 1 had active bleeding and 1 had findings of acute ischemia.


Ccl2-Ccr2 and Cx3cl1-Cx3cr1 signalling for retinal pathology is of particular interest because of its potential role in age-related macular degeneration (AMD). Ccl2, Ccr2, and Cx3cr1 signalling defects impair macrophage trafficking, but have, in several conflicting studies, been reported to show different degrees of age-related retinal degeneration. Ccl2/Cx3cr1 double knockout (CCDKO) mice show an early onset retinal degeneration and have been suggested as a model for AMD. In order to understand phenotypic discrepancies in different chemokine knockout lines and to study how defects in Ccl2 and/or Cx3cr1 signalling contribute to the described early onset retinal degeneration, we defined primary and secondary pathological events in CCDKO mice. To control for genetic background variability, we compared the original phenotype with that of single Ccl2, Cx3cr1 and Ccl2/Cx3cr1 double knockout mice obtained from backcrosses of CCDKO with C57Bl/6 mice. We found that the primary pathological event in CCDKO mice develops in the inferior outer nuclear layer independently of light around postnatal day P14. RPE and vascular lesions develop secondarily with increasing penetrance with age and are clinically similar to retinal telangiectasia not to choroidal neovascularisation. Furthermore, we provide evidence that a third autosomal recessive gene causes the degeneration in CCDKO mice and in all affected re-derived lines and subsequently demonstrated co-segregation of the naturally occurring RD8 mutation in the Crb1 gene. By comparing CCDKO mice with re-derived −/−/Crb1Rd8/RD8, Cx3cr1−/−/Crb1Rd8/RD8CCl2 and −/−/Cx3cr1−/−/Crb1Rd8/RD8CCl2 mice, we observed a differential modulation of the retinal phenotype by genetic background and both chemokine signalling pathways. These findings indicate that CCDKO mice are not a model of AMD, but a model for an inherited retinal degeneration that is differentially modulated by Ccl2-Ccr2 and Cx3cl1-Cx3cr1 chemokine signalling.Microglia and macrophages are recruited to sites of retinal degeneration where local cytokines and chemokines determine protective or neurotoxic microglia responses. Defining the role of  Mononuclear phagocytes  are part of the myeloid cell lineage and are effector cells of the innate immune system. These cells all express CX3CR1 and are thought to be derived from different circulating monocyte populations CX3CR1 as a genetic risk factor for AMD An important role of these two chemokine pathways in AMD has been supported by the identification of variants of the chemokine receptor gene Ccl2, Ccr2 or Cx3c1 have reported that the abnormal trafficking and function of macrophages and microglia in these models is associated with a variable degree of age-related retinal and RPE degeneration Several conflicting studies on single chemokine knockout mice for Ccl2, Ccr2 and Cx3cr1 knockout mice shows that chemokine signalling defects lead to dysfunctional macrophages in the retina. However, the contributory role of these dysfunctional macrophages to photoreceptor or RPE degeneration is not clear. Some groups have not observed significant age-related retinal and RPE degeneration or any spontaneous CNV in Ccl2 knockout mice Ccl2 and Ccr2 single knockout mice Cx3cr1 knockout mice, accumulation of subretinal macrophages/microglia is associated with a marked, progressive age-related retinal degeneration. This suggests that Cx3cr1 signalling may play a more pronounced role for survival of photoreceptors and the RPE, while the role of Ccl2 in this process remains unresolved Ccl2/Cx3cr1 double knockout mice have been described as developing a more accelerated and severe, early onset retinal phenotype with high penetrance of rapid photoreceptor loss and RPE defects, which suggested that the combined knockout of both chemokine signalling pathways might act synergistically and may thus lead to an early onset retinal degeneration The age-related accumulation of subretinal macrophages in Ccl2 and Cx3cr1 signalling pathways may contribute to the development of the early onset retinal degeneration, we aimed to define primary and secondary pathological events during the age-related progression of the degeneration in CCDKO mice between 2 weeks and 22 months of age Ccl2, single Cx3cr1 and double Ccl2/Cx3cr1 knockout mice from the original CCDKO mouse line by backcrossing with C57Bl/6 mice. We housed all these lines under same lighting conditions in the same room to control for environmental factors including pathogen burden and in addition raised some CCDKO mice in darkness from birth. Through these experiments, we identified the primary pathological event in CCDKO mice in the outer nuclear layer of the retina and established the RD8/RD8Crb1 mutation as a third independent autosomal recessive locus as the cause for the early onset retinal degeneration which was not dependent on light. Furthermore, we observed differential modulatory effects of the genetic background, as well as of both chemokine signalling pathways on the manifestation of the early onset retinal degeneration and therefore show that CCDKO mice are not a model for AMD pathology. These finding also highlight a differential modulatory role of Ccl2-Ccr2 and Cx3cl1-Cx3cr1 chemokine signalling for retinal degeneration.In order to understand the reported phenotypic discrepancies in the different chemokine knockout mouse models and to study how defects in the two Ccl2/Cx3cr1 double knockout mice (CCDKO mice) used in this study were derived from two breeding pairs  that we obtained from the original line as described by Tuo et al. and were thankfully provided by Chi-Chao Chan and Jingsheng Tuo C57Bl/6 background C57Bl/6J OLA Hsd mice  that were imported as young adult mice at 6–8 weeks of age and housed in the same animal rooms next to CCDKO mice. For backcrossing experiments, CCDKO mice from our original homozygous line and C57Bl/6J Ola Hsd mice were used as founder animals (F0). Obtained offspring (F1) that were heterozygous for both, the Ccl2 and the Cx3cr1 alleles were interbred to obtain the F2 generation. F2- animals were genotyped for both cytokine loci and interbred with each other to establish new lines for all combination of cytokine genotypes that include homozygous wildtype (+/+/Cx3cr1+/+Ccl2) mice, new single knockout mice for Ccl2 (−/−/Cx3cr1+/+Ccl2) or Cx3cr1 (+/+/Cx3cr1−/−Ccl2) as well as a new double knockout line for both Ccl2 and Cx3cr1 (−/−/Cx3cr1−/−Ccl2).As control animals we used age-matched in vivo procedures, mice were anesthetized by a single intraperitoneal (IP) injection of a mixture of medetomidine hydrochloride , and ketamine (60 mg/kg body weight) in water. Whenever necessary, the pupils were dilated with 1 drop of 1% tropicamide. The animal experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and under the UK Home Office project licence (PPL 70/1279).For Ccl2 and Cx3cr1 respectively  using primer combinations distinguishing the wildtype and knockout alleles for ectively  [44]. Gequencing .Autofluorescence imaging was performed using a HRA2 scanning laser ophthalmoscope with a 55° angle lense as described previously To phenotype offspring during backcrosses for retinal degeneration, we used fundus images focused on the inner retinal layer and evaluated the phenotype of the animals qualitatively using the red free (barrier filter at 500 nm) and the autofluorescent channels of the HRA2. To more quantitatively evaluate the severity of the phenotype, we subsequently counted the number of autofluorescent lesions per fundus image in the autofluorescent images for each animal after these had been processed in Adobe Photoshop CS 2  using the auto level function. These counts include not only the typical disciform autofluorescent lesions from the inferior retina, but also include any autofluorescent signal from the whole fundus image including those from the superior retina.OCT imaging and fundus fluorescein angiography were performed during the same session using either the Spectralis™ HRA+OCT  for OCT imaging or the HRA2 for fundus fluorescein angiography. First OCT images were obtained from the animals using OCT+IR or OCT+AF channels to correlate the retinal position with the obtained two-dimensional optical section obtained by the OCT. Subsequently, the same animals were injected intraperitoneal with 200 µl of 2% fluorescein in phosphate buffered saline (PBS) and fundus fluorescein angiography images were taken using the autofluorescent channel of the HRA2 equipped with an Argon laser at 488 nm wavelength. Obtained 2-dimensional OCT images and autofluorescent fundus images were exported and processed in Adobe Photoshop CS 2 .Semithin histological morphometric analyses were performed as described previously CCDKO and C57Bl/6 wildtype mice between 2 weeks and 22 months of age were assessed under bright field microscopy using a 100× objective and a 10× ocular lense. Singular pathological events in the retina (e.g. misplacement of nuclei from the outer nuclear layer toward the inner or outer retina or disorganized photoreceptor columns) and in the RPE  were counted form the superior to the inferior end of the retina in each section. From these individual sums of damages per section we calculated the mean sum of outer nuclear and RPE damage from three sections per animal to obtain a representative measure of outer retinal or RPE damage score per animal similar to a scheme described by Hollyfield et al. and us To evaluate the pathological changes in the retina and the RPE over time sections from age-matched For 3-D analyses retinae fixed in 3% glutaraldehyde and 1% paraformaldehyde buffered to pH 7.4 with sodium cacodylate were rinsed in buffer, and then osmicated, en bloc stained with uranyl acetate and Waltons lead citrate using the method of West et al. Regions of interest (ROI) were cut from resin blocks following normal surveying by LM and TEM of toluidine blue stained semithin sections and unstained ultrathin sections. The isolated ROI's were then superglued onto a Leica cryopin, re-trimmed to place the region of interest within a mesa of height ∼1 mm and side 0.5 mm and sputter coated with 5 nm gold palladium. Next, the specimen was locked into the specimen holder of the Gatan 3 View ultramicrotome mounted to the opened chamber door of a Zeiss Sigma variable pressure field emission scanning electron microscope and the diamond knife advanced until the full face of the block was being sectioned at 200 nm increments. At this point the microscope was evacuated and the block face imaged using Gatan's low voltage backscatter detector at 2–4 kV and chamber pressure of 10–30 Pa to suppress charging artifacts. Finally the pixel dwell time, magnification and chamber pressure were optimised to yield a focused ROI and the microtome programmed to automatically cut a maximum of 999×100 nm thick sections with intervening image acquisitions. In this way a stack of 999 images spanning an axial distance of 99.9 µms were digitally acquired at 4096×4096 pixel resolution in Digital Micrograph format. Three-dimensional reconstruction and labeling of the vascular lumen and Bruch's membrane was obtained using the Amira 5.3.3 software .Eyes for retinal and RPE/choroidal flat mounts were briefly fixed in 4% paraformaldehyde (PFA)/PBS before dissection and post-fixed again in 4% PFA/PBS for a total of 1 hour. After blocking with PBS/1% BSA /5% nonspecific goat serum  including 0.3% Triton X-100 for permeabilisation for 1 hour the flat mounts were incubated over night at 4°C with a 1∶500 dilution of anti Iba1 antibody  in blocking solution to label microglia and with primary TRITC-conjugated lectin at a 1∶10 dilution  to label endothelial cells. After washing with 3–4 times with PBS, 1∶500 dilution of goat anti-rabbit AlexaFluor 488 nm-conjugated secondary antibody  was used to visualize the Iba1 antibody. After washing three times with PBS, retinal and RPE/choroidal flat mounts were mounted with fluorescence mounting medium containing Hoechst 33342  and images were obtained using a confocal laser scanning microscope . The obtained Z-stack images were processed for 3D data visualization using Imaris software .Statistical analyses were performed using GraphPad Prism 5 for Windows .CCDKO line C57Bl/6 mice between 1 month and 22 months of age. Autofluorescent fundus imaging by scanning laser ophthalmoscopy (AF-SLO) demonstrated faint abnormal autofluorescent fundus lesion at 1 months of age  and the outer plexiform layer (OPL) which corresponded spatially to the disciform autofluorescent fundus lesions in the inferior retina  or in complete darkness (luminescence <0.5 lx). As an additional control group we included wildtype C57Bl/6 mice raised under 12 h/12 h light/dark cycle conditions. Both groups of CCDKO mice showed a very similar inferior localisation of autofluorescent lesions at 8 weeks of age  do not modulate the retinal phenotype in CCDKO mice significantly during the first 8 weeks of life.The inferior localization of the retinal degeneration observed in s of age , while ce groups . We did CCDKO mice we compared the localisation of AF-SLO and OCT lesions (C57Bl6 and supplementary CCDKO).To further characterise the nature of the autofluorescent lesions in  lesions  with thaCCDKO mice demonstrated relatively normal ramified microglia which showed a slightly more swollen cell body than microglia in C57Bl/6 mice at the same retinal layer (white arrows in CCDKO) versus 3A (C57Bl/6)). In the outer retina, however, CCDKO mice revealed microglia cells that have migrated into the outer nuclear layer (CCDKO) and are positioned around circular columns of photoreceptor nuclei dropping out of the ONL  or the inner segment (IS) area (C57Bl6). The observed degenerating columns of photoreceptor cells in CCDKO mice together with the surrounding recruited microglia correspond in location, size and configuration well with the disciform shape of the autofluorescent fundus lesions observed in AF-SLO images  area C57Bl6. CCDKO mice, we examined superior-inferiorly-oriented sagittal semithin sections for all age groups of CCDKO mice and control C57Bl/6 mice. CCDKO mice demonstrate an early mislocalisation of photoreceptor nuclei in the inferior outer nuclear layer at two weeks of age indicating a very early onset of retinal changes in the inferior outer retina . The age-dependent increase in RPE damage in CCDKO mice was significantly higher compared to C57Bl/6 mice indicating a disease related increase of RPE damage in CCDKO mice  bright field microscopy revealed grey spots in the inferior retina of 1 months old CCDKO animals , Cx3cr1 single knockout mice +/+/Cx3cr1−/−Ccl2), and the re-derived Ccl2/Cx3cr1 double knockout mice (−/−/Cx3cr1−/−Ccl2) we established affected and an unaffected mouse lines that either exhibit the typical autofluorescent fundus lesions at the age of 8 weeks or not  allowed us to establish 6 independent chemokine knockout mouse lines. For each chemokine genotype including s or not . To ensus or not . This stCrb1-RD8 mutation in all affected lines, allowed us to subsequently use these lines to assess the differential modulatory effect of the genetic background and the two chemokine loci, Ccl2 and Cx3cr1, on the phenotype of the RD8 retinal degeneration in offspring from the F2 generation. As a quantitative measure for the retinal degeneration phenotype, we decided to use the number of autofluorescent fundus lesions in AF-SLO fundus images at the age of 8 weeks  that these fluorescent fundus lesion are an early in vivo indicator for typical primary pathological events in the outer retina which led to the recruitment of microglia. Thus, assessing the number of these lesions might be a good measure for a potential modulatory effect of the chemokine knockouts on the retinal degeneration phenotype in vivo.The genetic proof of a third autosomal recessive locus by breeding, genotyping and phenotyping of the lines as well as the presence of  8 weeks . We haveCCDKO line showed significantly higher number of autofluorescent fundus lesions than normal C57Bl/6 mice and all other 6 newly established chemokine knockout mouse lines (Ccl2 or Cx3cr1 as well as the newly established “affected” chemokine double knockout mouse (Ccl2−/−/Cx3cr1−/−/RD8/RD8Crb1), which has the same genotype as the original CCDKO line. This clearly indicates that the retinal degeneration caused by a homozygous RD8/RD8Crb1 mutation in these mouse lines is attenuated by an increasing C57Bl/6 genetic background.At 8 weeks of age, the original RD8/RD8Crb1 mutation indeed causes the early onset retinal phenotype since all mouse lines that carry the RD8/RD8Crb1 mutation  that do not carry the RD8/RD8Crb1 mutation  and −/−/Crb1RD8/RD8Cx3cr1 mice (Cx3cr1−/− (affected) F3) both showed similar numbers of autofluorescent fundus lesions, which was for both lines significantly higher that the number of autofluorescent fundus lesions in −/−/Crb1RD8/RD8Ccl2 mice (Ccl2−/− (affected) F3.By comparing the affected mouse lines with each other we observed that the re-derived −/−/Crb1RD8/RD8Cx3cr1) or the double chemokine knockout (−/−/Cx3cr1−/−/Crb1RD8/RD8Ccl2) leads to a more pronounced degenerative phenotype of the RD8 mutation than the RD8 mutation in combination with the knockout of Ccl2 (−/−/Crb1RD8/RD8Ccl2) alone. Furthermore this also shows, that there is no significant additive affect of knocking Ccl2 out in addition to Cx3cr1, since the double chemokine knockout of Ccl2 and Cx3cr1 (−/−/Cx3cr1−/−/Crb1RD8/RD8Ccl2) is not significantly increased compared to the single knockout of Cx3cr1 (−/−/Crb1RD8/RD8Cx3cr1) (This suggests that absence of Cx3cr1 in either the single (RD8/RD8) .Ccl2 signalling alone might also have a modulatory effect on the RD8 retinal degeneration, we evaluated littermates from the backcrosses that all carried the RD8/RD8Crb1 mutation but were either homozygous wildtype (+/+Ccl2/RD8/RD8Crb1), heterozygous (+/−Ccl2/RD8/RD8Crb1) or homozygous knockout (−/−Ccl2/RD8/RD8Crb1) for the Ccl2 locus . All offspring with any of the Ccl2 genotype combinations showed a significantly smaller number of autofluorescent lesions in the fundus compared to the original CCDKO mice , littermates from the backcross that carried the homozygous RD8 mutation, but were wildtype (+/+/Crb1RD8/RD8Ccl2) or heterozygous (+/−/Crb1RD8/RD8Ccl2) for Ccl2 showed a significantly higher number of lesions in the fundus, while Ccl2 deficient littermates (−/−/Crb1RD8/RD8Ccl2) show no significant difference compared with all three groups at 8 weeks of age. Therefore this data suggests that Ccl2 does not have a significant effect on the manifestation of the early onset retinal degeneration in mice that carry a homozygous RD8 mutation, although we cannot exclude a mildly protective effect of Ccl2 deficiency.To clarify, whether absence of l2 locus . These aDKO mice . This re alleles . CompareCcl2/Cx3cr1 double knockout (CCDKO) mouse line and to understand whether and how these two chemokine pathways contribute to the retinal degeneration CCDKO mouse line CCDKO line and suggested an involvement of Müller cells in disease progression CCDKO knockout mouse lines as well as in all re-derived “affected” Ccl2 and Cx3cr1 and Ccl2/Cx3cr1 double knockout mice, is caused by a third independent autosomal recessive locus. Re-derived Ccl2 or Cx3cr1 single knockout lines and re-established Ccl2/Cx3cr1 double knockout mice which do not carry the third locus exhibited no signs of an early onset retinal phenotype. This indicates that neither Ccl2 nor Cx3cr1 deficiency alone nor the combined double knockout of both chemokines leads to a pronounced early onset retinal degeneration. In addition we showed by genomic sequencing that the naturally occurring homozygous RD8 mutation co-segregates with the early onset retinal phenotype in all affected chemokine lines. This naturally occurring RD8 (RD8/RD8Crb1) mutation is a single base pair deletion of a cytosine in exon 9 of the Crb1 gene that causes a frame shift and premature stop codon in the Crb1 protein which lead to a truncated transmembrane and cytoplasmic domain of Crb1 RD8/RD8Crb1 mice leads to very similar phenotypic features as we have observed in CCDKO mice. These include the early onset inferior fundus lesions, a discontinuous outer limiting membrane, an early dropout of nuclei from the outer nuclear layer as well as the subsequent focal age-related retinal dysplasia which is secondarily associated with vascular lesions in the outer retina CCDKO mice is independent from light is consistent with previous data for −/−Crb1 knockout mice. These mice, when raised in complete darkness for 6 months also show a similar degree of retinal degeneration as −/−Crb1 mice raised under normal lighting conditions (100 lux) −/−Crb1 mice CCDKO mice are about 100fold lower than light levels necessary to modulate the phenotype of this type of retinal degeneration. These data suggest, that the inferior location of the retinal degeneration in Crb1 mutant mice  is not due to the influence of light, but due to an unknown factor that restricts the photoreceptor degeneration to the inferior retina. These pronounced similarities in the phenotype as well as the co-segregation of the RD8/RD8Crb1 mutation with the early onset retinal degeneration in the different re-derived chemokine knockout mice indicate that the retinal degeneration observed in original CCDKO mice is the consequence of the RD8/RD8Crb1 mutation and not due to the combined double knockout of Ccl2 and Cx3cr1 as previously reported In this study, we aimed to identify primary and secondary pathological events during the previously described early onset retinal degeneration in RD8/RD8Crb1 mutation as the underlying cause for the early onset retinal degeneration in CCDKO mice enables us to better interpret the observed secondary degenerative events in original CCDKO mice. The two major secondary events observed in the −/−/Cx3cr1−/−/Crb1RD8/RD8Ccl2 mice with age include RPE changes and closely associated vascular lesions in the area of retinal degeneration. The vascular lesions contained tortuous and dilated vessels in the area of retinal degeneration and showed an increasing penetrance of late phase hyperfluorescence in fluorescein angiography which indicates a low grade vascular inflammation and a low grade late vascular leakiness of retinal vessels within the lesions. Using serial block-face scanning electron microscopy (SB-SEM) and 3D reconstruction, we demonstrated that the retinal vessels within the degenerate area break through the RPE layer and come in close contact with Bruch's membrane, but do not penetrate it. This abnormal location of retinal vessels seems to induce a secondary response of RPE cells which migrate along those vessels into the retina and ensheath them within the RPE/vascular lesion. However, the fact that not all aged CCDKO mice showed the occurrence of the late phase hyperfluorescence in the fluorescein angiography suggests that the manifestation of this vascular phenotype is variable and thus likely not a primary consequence of the RD8 mutation, but rather a secondary process dependent on the individual severity of the progression of the retinal degeneration. Based on the clinical observations  as well as on the basis of our ultrastructural observations, that vessels of retinal origin grow underneath the RPE but do not penetrate Bruch's membrane, we propose that the vessels within the RPE/vascular lesions in the −/−/Cx3cr1−/−/Crb1RD8/RD8Ccl2 mice are similar to retinal telangiectasia rather than choroidal neovascularisation, which was reported to occur in about 10–15% of the original CCDKO mouse line above the age of 3 months CCDKO mice The identification of the CCDKO mice are also very similar to those in aged RD8/RD8Crb1 mice RD8/RD8Crb1 mice might show similar vascular lesions as described here and do not develop CNV. Our data supports the view that vascular lesions in patients with Crb1 mutations, that show Coat's-like exudates in the inferior region of the retina, are likely to be more similar to retinal telangiectasia with similar vascular features as observed in this study, than choroidal neovascularisation RD8/RD8Crb1 mice as well as in patients with Crb1 mutations. A similar process in which retinal vessels are located adjacent to Bruch's membrane and are covered by RPE cells has been described in late stage retinal degeneration in rhodopsin knockout mice Crb1, but might be more common during late stages of other severe retinal degenerations.Since the late stage secondary changes in CCDKO mice to a C57Bl/6 genetic background attenuates the observed retinal degeneration in CCDKO mice. The data suggest that the genetic background of C57Bl/6 is a strong modulator of the manifestation of retinal degeneration in all affected chemokine knockout mice carrying the RD8/RD8Crb1 mutation and indicates that other genetic factors influence the observed phenotype. The presence of other genetic modifiers is also supported by the high degree of phenotypic variability observed within each affected mouse lines. Our findings are consistent with the original characterisation of RD8/RD8Crb1 mice, which demonstrated that the retinal degeneration is strongly modulated by backcrosses into C57Bl/6, CAST/EiJ or C3HfB6/Ga mice. Interestingly, only the discontinuous outer limiting membrane phenotype in RD8/RD8Crb1 mice remained preserved after backcrossing RD8/RD8Crb1 mutation in −/−/Cx3cr1−/−/Crb1RD8/RD8Ccl2 mice in this study as well.Our data demonstrates that genetic backcrossing of the Ccl2 and/or Cx3cr1 chemokine signalling differentially modulates the severity of the retinal degeneration caused by the RD8/RD8Crb1 mutation at 8 weeks of age. This is supported by the observation that all Cx3cr1 deficient lines that carry the RD8/RD8Crb1 mutation, including the −/−/Cx3cr1−/−/Crb1RD8/RD8Ccl2 and −/−/Crb1RD8/RD8Cx3cr1 mice, exhibit a more severe retinal degeneration compared to the −/−/Crb1RD8/RD8Ccl2 line of the same backcross generation and age. In contrast, Ccl2 deficiency (−/−/Crb1RD8/RD8Ccl2) alone does not significantly influence the retinal degeneration caused by the RD8/RD8Crb1 mutation, although we can not exclude a slight protective effect of it based on the trend for a reduced number of autofluorescent lesions in Ccl2 deficient mice (−/−/Crb1RD8/RD8Ccl2) compared to heterozygous (+/−/Crb1RD8/RD8Ccl2) or wildtype (+/+/Crb1RD8/RD8Ccl2) littermates. Ccl2 deficiency also does not significantly add to the exacerbated retinal degeneration seen in −/−/Crb1RD8/RD8Cx3cr1 mice suggesting that the effects of Ccl2 and Cx3cr1 signalling defects do not act synergistically on the manifestation of the retinal degeneration in RD8/RD8Crb1 mice. A modulatory influence of either of the two chemokine pathways and in particular of Cx3cr1 signalling on retinal degenerations, is further supported by the observation that Iba1+ microglia cells, which express Cx3cr1 in the retina Our data demonstrates that the genetic inactivation of either CCDKO line and in other affected chemokine lines in this study, the defective formation of the outer limiting membrane due to mutations in Crb1 acts as an endogenous trigger for the local upregulation of inflammatory mediators. This hypothesis is consistent with the increased expression of Cx3cl1 in retinal and choroidal vascular endothelial cells, Müller cells, RPE as well as photoreceptors after inflammatory processes Ccl2 in Müller cells within affected retinal areas after focal light injury and after retinal detachment Ccl2 as well as Cx3cl1 provide chemotactic cues for the local recruitment of microglia and systemic monocytes to the primary focal lesion site Cx3cr1 deficient mice in combination with the RD8/RD8Crb1 mutation, or may act mildly protective as eventually indicated by the trend towards a slight reduction in lesion size in Ccl2 deficient mice which also carry the RD8/RD8Crb1 mutation.Our findings support the hypothesis that in original Cx3cr1 knockout mice in the brain, where an increased neurotoxicity of brain microglia on surrounding neurons has been observed after systemic inflammation induced by LPS Cx3cr1 deficiency leads to a reduced dynamic behavior of retinal microglia during immune surveillance as well as during injury response Cx3cr1 deficient microglia can be neurotoxic in the retina as well and have the capacity to act as modulators of retinal degeneration as suggested by our observation. Therefore, we hypothesise that the additional neurotoxic effect of Cx3cr1 deficiency on the retinal degeneration caused by the RD8/RD8Crb1 mutation might be due to an impaired migratory capability and a subsequent reduced removal of dying photoreceptors by Cx3cr1-deficient microglia which might result in the production of more neurotoxic inflammatory mediators including Ccl5, TNFa or IL-6, which may negatively affect photoreceptor cell survival, and have all been previously shown to be upregulated in CCDKO mice Cx3cr1 deficiency, Ccl2 deficiency does not significantly modulate the manifestation of the RD8/RD8Crb1 phenotype. This minor role of Ccl2 on the RD8 retinal degeneration seems consistent with our previous observation that Ccl2 deficiency does not lead to a significant associated age-related retinal degeneration despite an accumulation of subretinal, dysfunctional macrophages Ccl2 in this process or by a compensatory effect of other chemokine ligands that can bind to and activate CCR2 −/−/Crb1RD8/RD8Ccl2 mice may suggest a mild protective effect of Ccl2 deficiency, which would be in line with Ccl2's role for the transendothelial migration of pro-inflammatory monocytes across the blood brain barrier Ccl2 and Ccr2 deficiencies for the formation of atherosclerosis that lead to a reduction of the formation of foam cells due to a reduced recruitment of systemic myeloid cells to the site of the lesion in the periphery Further support for this idea comes from convincing evidence for a microglia-mediated neurotoxicity in Ccl2 and Cx3cr1 chemokine signalling for the inherited retinal degeneration caused by the RD8/RD8Crb1 mutation and suggests that these two chemokine pathways not only modulate age-related degenerative processes or acute inflammation in the retina, but also contribute to the pathology of inherited retinal degenerations. Therefore it seems likely that these two chemokine signalling pathways and maybe the innate immune status of myeloid cells might act as a modifier for CRB1 mutations in humans and thus contribute to the high phenotypic variability observed in patients with different CRB1 mutations that either lead to Lebers congenital amaurosis (LCA), early onset childhood retinal dystrophy or juvenile onset retinitis pigmentosa CCL2 levels and reduced expression of Cx3cr1 on non-classical monocytes in aged probands as well as further increased levels of CCL2 in aqueous humor samples from patients with exudative AMD CX3CL1-CX3CR1 signalling to increased CCL2-CCR2 signalling systemically with age which might contribute to an increased susceptibility to retinal degeneration with age and during disease progression of multi-factorial age-related retinal diseases including AMD and diabetes. Understanding further the role and interaction of chemokine signalling pathways for the control of monocytes and microglia cell in degenerative processes of the retina might help to develop more targeted therapeutic approaches to modulate the progression of inherited and other age-related, multi-factorial retinal degenerations.Taken together, our study provides evidence for a differential modulatory role of Movie S1C57Bl/6. Animation of 3D-reconstructions (IMARIS) of confocaol Z-stack images taken from the inferior area of a retinal flat mount of a C57Bl/6 mouse at 2 months of age. The volume of the z-stack  comprises the deep retinal vascular plexus, the outer nuclear layer and part of the inner segments. The deep retinal vascular plexus was labeled with Tritc-lectinB4 and is represented in red, while retinal microglia labeled with Iba1 are shown in green. At the top of the z-stack microglia located at the level of the deep retinal vascular plexus show a normal ramified morphology. These cells are not present in the outer retina. The red and green Imaris surfaces in the area of the inner segments at the bottom of the z-stack represent autofluorescent signals from photoreceptors.(AVI)Click here for additional data file.Movie S2CCDKO. Animation of 3D-reconstructions (IMARIS) of confocaol Z-stack images taken from the inferior area of a retinal flat mount of a CCDKO mouse at 2 months of age. The volume of the z-stack  comprises the deep retinal vascular plexus, the outer nuclear layer and part of the inner segments. The deep retinal vascular plexus was labeled with Tritc-lectinB4 and is represented in red, while retinal microglia labeled with Iba1 are shown in green. At the top of the z-stack microglia located at the level of the deep retinal vascular plexus show thicker cell bodies compared to wildtype. In addition microglia are also observed in the outer retina in distinct columns. These microglia columns are observed in areas were the inner segment autofluorescence (at the bottom of the z-stack) is absent indicating altered photoreceptor positions or drop out.(AVI)Click here for additional data file.


We have previously proposed that uterine caspase-3 may modulate uterine contractility in a gestationally regulated fashion. The objective of this study was to determine the mechanism by which uterine caspase-3 is activated and consequently controlled in the pregnant uterus across gestation. Utilizing the mouse uterus as our gestational model we examined the intrinsic and extrinsic apoptotic signaling pathways and the endoplasmic reticulum stress response as potential activators of uterine caspase-3 at the transcriptional and translational level. Our study revealed robust activation of the uterine myocyte endoplasmic reticulum stress response and its adaptive unfolded protein response during pregnancy coinciding respectively with increased uterine caspase-3 activity and its withdrawal to term. In contrast the intrinsic and extrinsic apoptotic signaling pathways remained inactive across gestation. We speculate that physiological stimuli experienced by the pregnant uterus likely potentiates the uterine myocyte endoplasmic reticulum stress response resulting in elevated caspase-3 activation, which is isolated to the pregnant mouse myometrium. However as term approaches, activation of an elevated adaptive unfolded protein response acts to limit the endoplasmic reticulum stress response inhibiting caspase-3 resulting in its decline towards term. We speculate that these events have the capacity to regulate gestational length in a caspase-3 dependent manner. Premature labor is the leading cause of neonatal mortality, however to date, few effective and broadly applicable interventions are available for the prevention of preterm birth Due to the potential importance of uterine CASP3 activity in regulating gestational length, this study has focused on determining the factors that may regulate uterine CASP3 during pregnancy with the hope that we can ultimately understand the mechanism by which uterine quiescence across is maintained across gestation, allowing pregnancies to be carried to tern. We speculate that by determining the factors that allow the pregnant uterus to transition from a quiescent to a contractile phenotype in normal term labor, that we may gain insight into the mechanisms that are perturbed allowing for the onset of pre-term birth. In this study we have defined that the uterine myocyte endoplasmic reticulum stress response (ERSR) and its related unfolded protein response (UPR) are the likely regulators of uterine myocyte CASP3 activity during pregnancy. Physiological stimuli such as fluxes in protein synthesis, cellular differentiation, hypoxia and glucose deprivation have been described to result in the accumulation of misfolded proteins and perturbation of ER homeostasis resulting in the activation of the ERSR and its adaptive UPR We propose that physiological and potential uterotonic stimuli that the pregnant uterus experiences across gestation may potentiate CASP3 activation through the ERSR thereby maintaining the pregnant uterus in a quiescent state. However, towards the end of pregnancy we suggest that increased activation of the adaptive UPR limits the uterine ERSR, thereby reducing CASP3 apoptotic potential resulting in its gestational decline towards term. Ultimately these events initially limit the uterine myocyte contractile potential from early to mid-gestation through CASP3 activation, however towards term they permit reconstitution of the uterine myocyte contractile architecture.In summary this study examined three potential signaling pathways in the pregnant mouse uterus that may have the capacity to regulate CASP3 activity. Utilizing a gestational series of mouse uteri from E6–E19 we examined the presence of the ERSR in the pregnant uterus at the transcriptional and translational level. The traditional canonical intrinsic and extrinsic pathways, which initiate through transmembrane receptors at the cell surface or through the cytoplasmic release of the mitochondrial protein cytochrome C (CYCS), respectively All chemicals were obtained from Sigma Aldrich, St Louis MO unless otherwise indicated. Recombinant mouse Bid (truncated and full length) (Cat# Pro-644) was purchased from ProSpec-Tany Technogene Ltd.  and recombinant active caspase 8 (CASP8) (Cat# CC123) was obtained from Millipore Inc, .All animal studies were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. Timed-pregnant female CD-1 mice (6–8 weeks old) were obtained from Charles River and housed according to IACUC guidelines. Uterine tissues  were harvested between 0800–1000 h on E6, E8, E10, E11, E12, E13, E15, E16, E17, E18 and E19. The uterine horn was cleared of all embryonic material and maternal decidua. The isolation of mitochondrial and cytosolic fractions, were performed on fresh uterine tissues, processed immediately after harvest as described below. For immunofluorescence studies, the uteri were fixed in 4% PFA overnight and subsequently embedded in paraffin blocks for sectioning. The remaining uterine tissue was washed in 1×PBS and flash frozen for subsequent protein and mRNA analysis.2, 5 mM KCL, and 0.1% Triton ×-100) and 1×protease/phosphatase inhibitor cocktail (Complete Mini EDTA free/PhosStoP)  using an IKA homogenizer . The homogenate was centrifuged at 5000 RPM for 10 min at 4°C, and the supernatant was retained as the cytoplasmic fraction. The pellet was washed in NE1 and then resuspended in ice-cold NE2 buffer , 500 mM NaCl, 1.5 mM MgCl2, and 0.2 mM EDTA (pH 8.0)) with 1×protease/phosphatase inhibitor cocktail and was incubated on ice for 30 min, vortexed every 5 min, and centrifuged at 10,000 RPM for 10 min at 4°C. The supernatant was retained as the nuclear fraction. Our previous published data in the human myometrial cell Cytoplasmic and nuclear protein extracts were prepared from frozen uterine tissue as described previously In order to isolate pure preparations of intact mitochondria and overcome problems associated with mitochondrial rupture in frozen tissues, we used freshly harvested uterine tissue for subcellular fractionation. The fresh mitochondrial and cytosolic fractions  were isolated from uterine tissues using a protocol modified from previously described methods Equal amounts of protein were loaded on NuPAGE pre-cast gradient gels  and transferred on to Hybond-P PVDF membranes . The membranes were blocked in 5% non-fat milk prepared in 1× Tris Buffered Saline with Tween-20 for 1 hr at room temperature and then incubated with the primary antibodies overnight. This was followed by incubation with horseradish peroxidase-conjugated secondary antibodies diluted in 5% milk-1×TBS-T buffer. Immunoreactive bands were visualized using an ECL detection system (ThermoScientific Rockford IL). The concentrations of primary antibodies and their sources are as follows, Cell Signaling Technologies -HSPA5 (#3177 1:1000), DDIT3 (#5554 1: 200), full length and cleaved CASP12 (#2202 1:500), PDI (#3501 1:1000), full length and cleaved CASP3 (#9665 1:1000), COX4I1 (#4850 1:1000), GAPDH (#2118 1:2000), full length and truncated BID (#2003 1:500); Santa Cruz Biotechnology – CYCS (sc-7159 1:1000), XBP1 (sc-7160 1:500). Thermo Scientific – NCOA3 (#PA1-845 1:2000). The immunoreactive cytoplasmic, nuclear, cytosolic and mitochondrial bands obtained by immunoblotting were quantified using ImageJ (NIH Bethesda MD) and normalized to PDI, NCOA3, COX4I1 and CYCS, respectively as their protein concentrations were found to remain relatively unchanged in the pregnant mouse uterus across gestation.Paraffin embedded uterine tissues were sectioned at 5 µm thickness and collected on Superfrost Plus slides . Paraffin sections were deparaffinized in xylene and rehydrated through an alcohol series. For DDIT3 and HSPA5, antigen retrieval was performed by boiling the sections for 15 minutes in 10 mM citrate buffer pH 6.0. For cleaved CASP3 immunohistochemical analysis, uterine tissue sections were incubated with proteinase k (10 µg/ml) for 10 minutes at room temperature. Tissue sections were then blocked in 1 X PBS containing 5% normal donkey serum and incubated in the primary antibody overnight at 4°C at the following concentrations DDIT3 1:100, cleaved CASP3 , HSPA5 1:100. Sections were then washed in 1×PBS and incubated in the fluorescent secondary antibody for 1 hr at room temperature. Donkey anti-rabbit secondary antibodies conjugated to either Alexa flour 488 or Cy3  were used at a dilution of 1∶500 in 1×PBS. The sections were then washed and incubated with 4′,6-diamidino-2-phenylindole (DAPI) to stain nuclei and mounted in gelvatol. Images were collected at 63× on a Leica DMRBE  using a Q-Imaging Micro Publisher 5.0 RTV . Serial sections were used for immunoflourescence as differences in antigen retrieval methods for the CASP3 and DDIT3 antibodies precluded dual immunofluorescence analysis. Companion-blocking peptides were utilized to pre-absorb each primary antibody, at each gestational stage utilized in this study, thereby acting as negative controls for auto-fluorescence and false positives.Rplp0 was used as a housekeeping gene as we have found Rplp0 levels to remain stable across gestation in the pregnant mouse uterus Rplp0. Expression data were analyzed using the ΔΔCt method RNA was isolated from pulverized uterine tissues using the RNeasy Mini Kit  according to the manufacturer’s instructions. Total RNA was converted to cDNA using the High Capacity cDNA Reverse Transcription Kit . Primers were designed using NCBI Primer Blast to obtain amplicons from 50–170 bp. . Statistical analysis of immunoblots and Q-PCR were performed with StatPlus:mac software 2009 version . Data are represented as mean ± standard error (SE) of the mean. The results were subjected to a one-way ANOVA followed by pairwise comparison (Student-Neuman-Keuls method) to determine differences between groups. Significance was set at P<0.05.Cytoplasmic proteins were isolated from uterine tissues of pregnant mice from gestation E6 to E19  as described in the Materials and Methods. To determine the levels of full length and active CASP3 immunoblotting was performed . As seenThe hallmark of an active extrinsic caspase signaling pathway is the appearance of cleaved CASP8 and tBID Downstream from CASP8 in the extrinsic pathway of caspase activation is the cleavage of the Bcl-2 family protein, BID To examine whether uterine CASP3 activation occurred through the intrinsic caspase activation pathway, we determined whether the pregnant uterine myocytes released the 15 kDa CYCS from the mitochondria to the cytosol indicating a loss of mitochondrial integrity Cytoplasmic and nuclear protein extracts isolated from pregnant mouse uterine tissues from E6–E19 were examined for markers of the ERSR by immunoblotting. The presence and gestational regulation of the transcription factor DDIT3, a marker for activation of ERSR The expression of the chaperone HSPA5, a marker of the adaptive UPR To determine the localization of DDIT3, active CASP3 and HSPA5 in the pregnant mouse uterus across gestation , we performed immunofluorescence analysis on formaldehyde fixed paraffin embedded sections of mouse uterine tissues as described in the Materials and Methods. Immunohistochemical analysis of DDIT3 confirmed its nuclear localization in uterine myocytes of the pregnant mouse uterus  and thouAtf4, Ddit3, Casp12, Atf6 and Hspa5 across gestation in the pregnant mouse uterus was performed utilizing Q-PCR analysis. ATF4 is a transcription factor that is involved in the induction of Ddit3 expression occurring as a result of ER stress Atf4 and Ddit3 transcript did not significantly change through the duration of pregnancy. Levels of full length Casp12 demonstrated approximately a 1.4-fold induction at E6 and E8 in comparison with the rest of gestation but this increase was not statistically significant . Due to poor sensitivity of commercially available antibodies we were unable to perform immunohistochemical or immunoblotting analysis for ATF4 or ATF6.Examination of the mRNA levels of nificant . We alsonificant . Hspa5 eWe hypothesize that activation of the ERSR and the resulting UPR in the uterus are central in the non-apoptotic triggering and management of uterine CASP3 activity during pregnancy. We have previously determined that uterine CASP3 activation during pregnancy may have the capacity to effectively regulate the timing of parturition, through modulation of the uterine myocyte contractile architecture The first indication that ER stress may act as a potential mechanism controlling uterine CASP3 came from the observation that although we had robust CASP3 activity we were unable to detect its mechanism of activation through either the classic intrinsic or extrinsic apoptotic signaling pathways , 3 and 4Atf4, DditT3 and Casp12 in the pregnant mouse did not reveal any significant changes across gestation (Hspa5 mRNA levels across gestation (Atf6, which has been shown to induce Hspa5 expression. Elevated levels of HSPA5 assist in clearing the backlog of accumulated proteins thereby in the context of the pregnant uterus suggest an active resolution of the uterine ERSR occurs towards term. Immunohistochemical analysis seen in Recently an alternate, mitochondrial independent mechanism of CASP3 activation has been described, where CASP3 is cleaved and activated as a direct result of ER resident CASP12 activation. estation , confirmestation . Chaperoestation . We alsoestation  mirroredestation . We alsoin vivo. We speculate that in the instances of pre-term birth an inappropriate ERSR or an inability to host an appropriate UPR may trigger a precocious withdrawal of uterine CASP3 allowing for a premature increase in uterine contractile responsiveness. With the growing recognition of an association of ER stress with human disease and with increased understanding of the fundamental mechanisms regulating ER stress, novel targets for drug discovery and new strategies for therapeutic preventative intervention are beginning to emerge and may ultimately be utilized to reveal indicators for and the resolution of pre-term birth.In summary, we propose that throughout pregnancy the uterine myocyte must monitor, tolerate and adapt to intrinsic and extrinsic uterotonic stimuli


Candida isolates from HIV patients with oral candidiasis and to correlate it with the CD4 count of the patients.Oral candidiasis is a clinical predictor for progression to AIDS. Antifungal drug resistance is becoming a major problem with this immunedepleted population. Considering the above facts, the study was conducted to speciate and to determine the susceptibility pattern of the Samples collected from (n=150) the lesion using sterile cotton swabs in HIV patients with oral candidiasis. Isolation and speciation were done by standard mycological procedures. Antifungal susceptibility was determined by Microbroth dilution method, as per the CLSI guidelines. Estimation of CD4+ T lymphocyte of the patients was done by FACS count system.Candida albicans 118 (78%) was the most common species followed by Candida tropicalis 17 (11%), Candida krusei 8 (5%), Candida parapsilosis 6 (4%), Candida glabrata 2 (1%) and Candida guilliermondi 1 (1%) . By microbroth dilution 18 (11.8%) isolates were fluconazole resistant, 23 (15.1%) were itraconazole resistant and all were amphotericin susceptible. Of the 150 patients, 106 (70.6%) had CD4 count <200 cells/ µl. Azole resistant was more common in patients with CD4 count <200 cells/ µl.Of the 150 samples, two revealed a mixture accounting for the 152 isolates. Candida albicans is the most frequently isolated species. Non- albicans Candida species are emerging as important pathogens with increasing rates of azole resistance and with increased immunosuppression. This emphasizes the need for speciation and determination of susceptibility pattern of the Candida isolates from HIV patients with oropharyngeal candidiasis.


Reduction aortoplasty is an alternative method of treatment for ascending aorta aneurysm without involvement of the aortic root especially in high risk patients to decrease the cross clamp time, our study is to evaluate the early results and outcome of longitudinal reduction aortoplasty supported by pericardial patch at our institute.From 2006 till end of 2011, 37 patients with aneurysm of the ascending aorta without involvement of the aortic root underwent longitudinal reduction aortoplasty supported by pericardial patch which was sewed to the lateral wall of the ascending aorta by 5/0 prolene. The average age was 70.9 year ±4.2, females were 28 (75.7%), the average ejection fraction was 42.1%. The size of the ascending aorta was less than 55 mm in all patients; all patients underwent other cardiac procedure at the same time. 33 patients (89%) aortic valve replacement, 22 patients (59.5%) aortic valve replacement with CABG, 4 patients (11%) CABG alone, 7 patients (19%) were redo aortic valve replacement. Mean follow-up was 2 year.Preoperative mortality was in 2 cases 5.4%, both because of heart failure because of low ejection fraction preoperatively. One year survival was 91.9%. Reoperation done in 3 cases one because of hematoma collection beneath the pericardial patch after 3 weeks of the primary operation, the other 2 was because of valve complications. No redilataion seen in any patient till the time we did this study.Reduction aortoplasty is safe alternative to the standard replacement of the ascending aorta for patients without involvement of the aortic root with mild to moderate dilatation, can be done either supported with pericardial patch or unsupported, we think that supported aortoplasty may decrease the risk of redilataion and the risk for reoperation, our early results are encouraging.


An accurate diagnosis of the morphology of the root canal system is a pre-requisite for successful root canal treatment. Frequently, root canals are left untreated because the clinicians fail to identify their presence, particularly in teeth that have anatomical variations or additional root canals. In this report a maxillary lateral incisor with two roots has been described. Many anatomical studies have declared that maxillary incisors always have a single root, while variations in the number of lateral canals and/or position of apical foramen are reported 2]3]4][3][4]2][[4][3][4][3][4]2]. As indiA brief literature review revealed 11 cases reporting maxillary lateral incisors with two roots 9]10][1[10]19][[1[10]1913]14][[14][13][[[14][13][[1[10]19Neville et al. used the term supernumerary roots when describing the presence of additional roots on a tooth compared with the classical description in dental anatomy. The most frequently affected teeth are the permanent molars  from either arch and mandibular cuspids and premolars .Periapical radiography is an essential tool in diagnosing internal anatomy of a tooth. The use of shift cone angle radiographic technique and parallel angle radiograph to identify superimposed roots and overlapping and unidentified canals has been advocated. In this case report, a rare case of maxillary lateral incisor with two roots is described.A 20 year-old male was referred to the clinic for root canal treatment of the maxillary right lateral incisor (#7). The medical history was found to be non-contributory. Patient’s chief complaint was pain in relation to the upper right anterior teeth region. Clinical examination showed a pit over the cingulum region. Tooth also had grade I mobility and was tender both on palpation and percussion. No sinus tract or fistula was found. Radiographic examination using bisecting angle technique revealed widening of periodontal ligament space and well-defined periapical radiolucency around the lateral incisor as well as the central incisor .Based on the clinical and radiographic evidences, the tooth was diagnosed as having chronic apical periodontitis with an acute episode.The tooth was isolated with rubber dam  and anaesthetized with 1:200000 solution of Xylocaine with adrenaline  using an aspirating syringe. The access was created initially with a no. 2 round bur and then the cavity was enlarged to a triangular outline with round end tapered bur TR 13 . A small standardized #06 K-file  was used to negotiate each of the two separate root canals. Conventional working length radiograph was taken . The mesSodium hypochlorite 3%  alternated with 17% EDTA gel  was used for irrigation to facilitate shaping. The canals were then dried with sterile paper points , filled with mixed calcium hydroxide  and 0.2% chlorhexidine  slurry; the coronal cavity was sealed with Cavit G . The access opening for the adjoining central incisor was also performed on the same day with calcium hydroxide and chlorhexidine dressing. A week later, tooth was non-tender to percussion and the canals were dry. The canals were obturated with laterally condensed gutta-percha . Resin based AH Plus  sealer was used for obturation. The central incisor showed exudate, so it was treated again with calcium hydroxide/chlorhexidine dressing.A final radiograph for the lateral incisor  was thenAfter 1 year on follow-up visit, the patient reported complete alleviation of symptoms along with reduction in mobility. The recall radiograph showed resolution of the periapical pathology .When a maxillary incisor presents with two roots or two root canals, conditions such as fusion, gemination, dens in dente, palatogingival or distolingual groove and some variation in the normal development of Hertwig's epithelial root sheath must be considered 27]30].[30].27][.[30].27]Gemination is an anomaly in which the tooth germ divides during the development of the tooth, resulting in the formation of a double crown with single root, and in the case of fusion, the crown of two separate tooth buds fuse during development resulting in a bifid crown with two root canals in one root. In this case clinical examination as well as the pre-treatment radiographs revealed a crown of normal size and shape when compared with the contra lateral side. Therefore a diagnoses of fusion (single larger crown) or germination (fused or joined crown) can be disregarded 30]..30].There are few reports of maxillary lateral incisor with dens in dente and dens invaginatus showing two roots 6]8]9][8][9]6][[9][8][9][8][9]6]11]. In . In 11].Another developmental anomaly, which may appear similar to this case radiographically, is palatogingival or distolingual groove, but clinical examination ruled it out 27]..27].According to Bhasker  normal r


Life expectancies (LEs) of patients in UK Collaborative HIV Cohort (UK CHIC) stratified by CD4 count at start of antiretroviral therapy (ART) have been estimated [1] but not gains in years of life in response to ART. We estimated LE associated with attained CD4 count and viral suppression at different durations of ART. Patients in UK CHIC aged > 20 years who started ART in 2000 to 2008 (excluding person who injects drugs) were followed to end of 2010. All-cause mortality was ascertained from clinic notes and by linkage to national records. We used the nearest CD4 count before ART and the last in each of years 1 to 5 of ART and determined whether patients were virally suppressed (HIV-1 RNA < 400 copies/mL) in the past year for those remaining under follow-up. Poisson models were used to estimate mortality rates by sex, age, latest CD4 count  and viral suppression for each duration of ART. Abridged life tables were constructed from age-specific mortality rates to estimate LE for ages 20 to 85 years. Results are presented as the average number of years that will be lived after exact age 35 years. A total of 17,021 patients started ART from 2000 to 2008 of whom 708 (4.2%) died; 3956 (23%) were lost to study follow-up. There was no difference in mortality between those with attained CD4 350 to 499 and ≥ 500. On starting ART, male LE at exact age 35 was 36, 44 and 42  years for attained CD4 < 200, 200 to 349,≥350, respectively; after 5 years on ART, it was 22, 42 and 46  years, respectively. Only 17% of patients had CD4 ≥ 350 at ART start, compared with 78% of patients on ART for > 5 years. The difference in LE between suppressed versus unsuppressed patients was around 11 years. The figure shows that both CD4 count and viral suppression contribute to changes in LE. Male patients that increased their CD4 in the 1st year of ART from < 200 to 200–349 or ≥ 350 gained 6 and 11 years of LE to 42 and 48 years, respectively, with similar rises for women. Overall, LE was 4 years greater for those on ART for > 5 years compared with those starting ART. Individuals that attain viral suppression and a CD4 count > 350 within 1 year of ART start have a normal LE with 35-year olds estimated to live to over 80 years on average. LE in patients with CD4 count < 200 beyond 5 years on ART drops by 15 years. Estimated LE may be biased by under-ascertainment of deaths, missing CD4 measurements and extrapolation beyond available data.