Individuals with end-stage renal disease (ESRD) have excess risk of various cancer types. However, the total burden of human papillomavirus-related cancers remains unknown.We performed a nationwide observational cohort study during 1994–2010.For each person with ESRD, we sampled 19 population controls (without ESRD) matched on age, gender and municipality. Participants were followed until first diagnosis of human papillomavirus-related cancer, death, emigration, or 31 December 2010, whichever came first.Human papillomavirus-related cancers were extracted from Danish medical administrative databases. We considered cancers of the cervix, vulva, vagina, penis, anus, and subsets of head and neck cancers as human papillomavirus-related. We calculated incidence rates of human papillomavirus-related cancer and used Poisson regression to identify risk factors for human papillomavirus-related cancer.Among 12,293 persons with ESRD and 229,524 population controls we identified 62 and 798 human papillomavirus-related cancers, respectively. Incidence rates of human papillomavirus-related- cancer were 102 per 100,000 person-years  among persons with ESRD and 40.8 per 100,000 person-years  among population controls. ESRD patients had 4.54  fold increased risk of anal cancer and 5.81 fold  increased risk of vulvovaginal cancer. Adjusted for age, comorbidity, and sex, ESRD patients had 2.41  fold increased risk of any human papillomavirus-related cancer compared with population controls. Compared with dialysis patients renal transplant recipients had an age-adjusted non-significant 1.53  fold higher risk of human papillomavirus-related cancer.Persons with ESRD have excess risk of potentially vaccine-preventable human papillomavirus-related cancers. Oncogenic types of human papillomavirus (HPV) can cause cancer of the cervix, vagina, vulva, penis, anus, and a subset of cancers of the head and neck -3. AmongWhile most studies have focused on heterogeneous cancer types among patients with ESRD, few studies have assessed the total burden of HPV-related cancers. In light of the recent development in prophylactic HPV vaccination, the interest in HPV-related cancers has been renewed. Vaccines against the two most important oncogenic types of HPV 16 and 18) are now licensed. Among younger healthy individuals HPV vaccination lowers the risk of pre-cancer lesions of the cervix and the risk of anal, vulva, vaginal, and penile infections, with the HPV-types included in the vaccines  and 18 a. FurtherA recently published large American cohort study found no increased risk of cervical cancer among solid organ transplant recipients, which contrasts the findings reported by Vajdic and colleagues among Australian kidney transplant recipients ,10. ThesThe benefit and cost-effectiveness of HPV immunization will depend on the total burden of HPV-related cancer, the proportion of ESRD patients with active HPV infection at the time of vaccination, and the immunogenicity of the vaccines among these patients. Thus, improved knowledge of the epidemiology of HPV-related cancers among ESRD patients are of great importance for the decisions of “if” “who”, and “when”, to vaccinate.The main objectives of this Danish nationwide population-based cohort study were to compare the risk of HPV-related cancers in the general population with that of patients with ESRD, and to identify risk factors for HPV-related cancers in patients with ESRD.We performed a population-based, observational cohort study among ESRD patients in Denmark during 1 January 1994–31 December 2010. In Denmark, the prevalence of chronic renal failure requiring renal replacement therapy, i.e. dialysis or transplantation is 0.08% and in 2010 approximately 4,700 persons were living with ESRD . TreatmeWe defined ESRD patients as patients treated with renal replacement therapy  for at least 90 days. Prevalent ESRD patients as of 1 January 1994 or incident thereafter, who were resident in Denmark and at least 14 years of age at the time of their first treatment for ESRD were identified in the DNR and included in the study. For each ESRD patient we sampled 19 population controls matched on age (within 1 year of date of birth), municipality, and gender from the Civil Registration System (CRS) at the day of inclusion in the ESRD registry. Since 1968, the CRS has provided all Danish citizens with a unique 10 digit civil registration number, which allows for accurate linkage between different databases .We obtained information on vital status, sex, date of birth, date of death, and residence of ESRD patients and their population controls from the CRS, which is updated within a week of person’s birth, death, or emigration. Population controls were used for comparative analysis. ESRD patients and population controls diagnosed with HPV-related cancer prior to study participation were excluded.th edition of the International Classification of Diseases (ICD-10) as outlined in Table We classified cancers of the anus, vulva, vagina, cervix, penis, and a subset of head and neck cancers as HPV-related . Cancer ases ICD-0 as outlth version (ICD-8) was used. The Danish Cancer Registry (DCR) contains data on incident cancers in Denmark since 1943.The DCR is nationwide and contains ICD-10 diagnoses (as part of the ICD-0 classification) since 1978 [The Danish National Registry of Patients (DNRP) contains information of all hospital admissions in Denmark since 1977 and all outpatient contacts since 1995 ,28. Varince 1978 . Originaage . Information on cause of renal failure was obtained from the DNR database. Causes of renal failure were categorized as follows: Glomerulonephritis, Diabetes Mellitus types I and II, Chronic Interstitial Nephritis, Hypertension and Chronic renal failure without specification, Polycystic Kidney Disease, Vasculitis, and Other. The Charlson’s Comorbidity index (CCI) was used to quantify comorbidity. The CCI is a weighted index based on 19 major disease categories each assigned with a weight . CCI is a validated measure of co-morbidity [Based on date of birth in the CRS we generated a time varying covariate orbidity ,31. We dFor each ESRD patient and his/her population controls time at risk started at 1 January 1994 or the date of first treatment for ESRD, whichever came last. Time at risk ended at the date of first diagnosis of a HPV-related cancer, death, emigration, or 31 December 2010, whichever came first.. In these analyses, renal replacement therapy i.e. dialysis and transplant recipients with functioning grafts was treated as a time varying covariate. Thus, if the mode of renal replacement therapy was changed during follow-up, a person would contribute to risk time in both subgroups. IR’s were compared between the two subgroups with and without stratification for age. For the latter analyses Poisson regression was used: Adjustment for sex, comorbidity and cause of renal failure only caused minor effect on the risk estimates and therefore neither of these variables were included in the final analyses.We calculated incidence rates (IRs) and 95% confidence intervals (95% CIs) of HPV-related cancers with and without stratification for the five anatomic regions outlined in Table We used Poisson regression to identify potential risk factors for HPV-related cancer among ESRD patients. The following variables were included: Sex, age , and the CCI index score. Among the study population 24 patients with ERSD and 227 population controls were HIV positive. Among HIV-positive persons in our study population, only two (population controls) developed a HPV-related cancer during the study period and we therefore chose not to adjust for HIV status in our analyses. While age and sex are well-known risk factors for cancer, the CCI is not an established risk factor for HPV-related cancer. We therefore calculated adjusted risk estimates with and without the CCI. In the risk factor analysis, we did not stratify for anatomical location of HPV-related cancers.We used the Stata® statistical software  version 11.0 for statistical analysis. The study was approved by the Danish Data Protection Agency.We excluded 92 ESRD patients (and their matched controls) diagnosed with a HPV-related cancer prior to study participation. A total of 442 population controls diagnosed with HPV-related cancer prior to study participation were also excluded. Persons who died or emigrated before the study period did not contribute to risk time. Consequently 12,293 ESRD patients and 229,524 population controls were included in the study.Table Median time at risk was 3.35 (maximum 17.0) years and 7.85 (maximum 17.0) years among ESRD patients and population controls, respectively. Median age of ESRD patients at the date of their first treatment with renal replacement therapy was 61.6 years.During the study period we identified a total of 62 cases of HPV-related cancers among ESRD patients and 798 cases among population controls Table .Table Among ESRD patients, transplant recipients with functioning grafts had an unadjusted 1.83  fold higher risk of HPV-related cancer compared to dialysis patients. When adjusted for age IRR fell to 1.53  comparing transplant recipients to dialysis patients Table .Table In this nationwide, population-based study, we found that the risk of HPV-related cancer overall was 2.5 fold increased among ERSD patients compared to matched population controls. Interestingly, ESRD patients had a more than fourfold increased risk of anal cancer and female ESRD patients had a nearly six fold increased risk of cancers of vulva and vagina. Major risk factors for HPV-related cancer were age and gender. When adjusted for age, transplant recipients (with a functioning graft) had a modest 1.53 fold increased risk of HPV-related cancer compared to dialysis patients. Although not significant we cannot rule out an increased risk of HPV-related cancer among transplant recipients due to the low number of cancers in the two groups.Our study is one of few population-based studies to investigate the burden of HPV-related cancer among unselected individuals with ESRD. The major strengths of our study are the use of population-based nationwide cohorts with high degrees of completeness and minimal loss to follow-up. Because we considered only the first diagnosis of HPV-related cancer our estimates were not biased by multiple episodes of HPV-related cancer occurring in highly susceptible individuals. Further, HPV-related cancer may increase the risk of development of ESRD and therefore lead to increased risk of HPV-related cancer among ESRD patients.Our study had some limitations. The relatively low number of HPV-related cancer limited our opportunities to adjust for potential risk factors and the width of confidence interval complicates the comparison between transplant recipients and patients on dialysis. However, large follow-up time and careful matching yielded estimates of high statistical precision and validity.We did not have access to information on use of immunosuppressive drugs among ESRD patients and therefore we could not assess the effect of individual drug regimens on the risk of HPV-related cancer. Likewise, we were unable to identify dialysis patients receiving immunosuppressive therapy for their renal disease. Whereas HPV is considered mandatory for development of cervical cancer the etiological fraction of HPV differ among other cancer types considered as HPV-related. If the etiological fractions of HPV are higher among ESRD patients than among population controls this may have led to underestimation of the importance of HPV among ESRD patients in our study. We restricted our analysis to the cancers most strongly associated with HPV in order to minimize the risk of misclassification of non-HPV-cancers as HPV-related. Whereas diagnoses in hospital discharge registries may be revised and therefore not reflect true cancers, cancer registries have a degree of underreporting. We chose to include cancers identified solely in the DNRP in this study. In the DCR, ICD-7 diagnoses were used until 2004. Subsequently diagnoses have been electronically recoded from ICD-7 to ICD-10. In terms of HPV-related head and neck sites, this may cause some misclassification during the recoding process due to the very specific cancer sites (ICD-10) used to identify these cancers. In these cases DNRP may be more accurate than the DCR. In the remaining cases the validity of cancers identified in the DNPR may be lower than cancers identified in the DCR. Previous studies have indicated high completeness of the DCR and DNP for breast cancer , urologiIn a previous study, Birkeland and colleagues reported a 8.6 fold increased risk of cervical cancer among female kidney transplant recipients . In a moFor non-cervical HPV-related cancer among kidney transplant recipients standardized incidence rate-ratios reported by Engels were in concordance with our findings in ESRD patients and the findings by Vajdic and colleagues ,10.The cancer risk among dialysis patients is less well studied than among kidney transplant recipients. A previous study suggested that dialysis patients had a modest 1.18 fold increased cancer risk overall, whereas the risk of cervical cancer, penile cancer, and cancer of the oral cavity, was between 1.7 and 4.0 fold increased . HoweverOverall we estimate the external validity of our results to be high due to the population based design, choice of study period, the high quality of the databases used for the study, and the overall concordance with existing literature. Nevertheless differences in renal populations exist and should always be taken into account when applying our results to other renal populations.Our results suggest that efforts to prevent HPV-related cancer should focus on dialysis patients as well as transplant recipients. Despite nationwide free of charge screening programs for cervical cancer, ESRD patients are still at increased risk of these cancers. Whether attendance to cervical screening differs among ESRD patients and the control population in our study population is unknown. Given the high IRs of vulvovaginal cancer it is important that the clinicians performing cervical smears are also observant to early signs of these cancers in ESRD patients For non-cervical HPV-related cancers no validated screening programs are currently available. Anal cancer screening have been considered among HIV infected men, but low sensitivity of cytology, high prevalence of anal intraepithelial neoplasia, and uncertainty of the natural history of these lesions complicates development of a screening program. For these cancers, early detection therefore relies on clinician’s and patients’ awareness on early signs and symptoms of cancer.Two HPV vaccines are commercially available ,41. BothOur study demonstrates that persons with ESRD have a markedly increased risk of HPV-related cancer compared to matched population controls. Patients with end-stage renal disease had a 4.5 fold increased risk of anal cancer and an almost 6 fold increased risk of vulvovaginal cancer. Transplant recipients only had a modest non-significant 1.53 fold increased risk of HPV-related cancer compared to dialysis patients. Age and female sex were most important risk factors for HPV-related cancer among persons with end-stage renal disease. Future studies of safety, immunogenicity, and efficacy of prophylactic HPV-vaccines among ESRD patients are needed to clarify the benefit of HPV-vaccination in this population.The authors declare that they have no competing interests.SJF, BJ, and OSS conceived the study idea. SJF, OSS, LSD, BJ, LJØ and UF designed the study. OSS and BJ collected the data. LSD, OSS, and UFF analysed the data. All authors interpreted the data. LSD wrote the first draft, and all authors critically reviewed and edited the manuscript and approved the final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2369/14/137/prepub


Recently, it has been shown that human ejaculate enhances human immunodeficiency virus 1 (HIV-1) infectivity. Enhancement of infectivity is conceived to be mediated by amyloid filaments from peptides that are proteolytically released from prostatic acid phosphatase (PAP), termed Semen-derived Enhancer of Virus Infection (SEVI). The aim of this study was to test the range of HIV-1 infectivity enhancing properties of a large number of individual semen samples (n = 47) in a TZM-bl reporter cell HIV infection system. We find that semen overall increased infectivity to 156% of the control experiment without semen, albeit with great inter- and intraindividual variability (range -53%-363%). Using transmission electron microscopy, we provide evidence for SEVI fibrils in fresh human semen for the first time. Moreover, we confirm that the infectivity enhancing property can be inhibited by the major green tea ingredient epigallocatechin-3-gallate (EGCG) at non-toxic concentrations. The median inhibition of infection by treatment with 0.4 mM EGCG was 70.6% (p < 0.0001) in our cohort. Yet, there were substantial variations of inhibition and in a minority of samples, infectivity enhancement was not inhibited by EGCG treatment at all. Thus, topical application of EGCG may be a feasible additional measure to prevent the sexual transmission of HIV. However, the reasons for the variability in the efficacy of the abrogation of semen-mediated enhancement of HIV-1 infectivity and EGCG efficacy have to be elucidated before therapeutic trials can be conducted. HIV-infection is an imminent health issue, with an estimated 33 million individuals infected worldwide according to UNAIDS . GloballRecently, it has been reported that human ejaculate acts as a potent enhancer of HIV infectivity . This enInterestingly, it has been previously demonstrated that epigallocatechin-3-gallate (EGCG), the major active constituent of green tea, can inhibit the infectivity enhancing effect of SEVI, possibly by interference with de novo SEVI formation or by degradation of present preformed PAP-derived amyloid fibrils . This obin vitro experiments carried out either with pooled human semen or with fibrils formed from synthetic PAP-fragment peptides (PAP248-286) -thymidin incorporation assay and in a flow cytometry-based apoptosis/necrosis assay utilizing Annexin-V and 7-AAD  was ruled out in a [D Figure .We next wanted to verify the efficacy of EGCG treatment for synthetic SEVI. Synthetic SEVI enhanced HIV-infectivity drastically, leading to a 40-fold (4000%) increase in the infection rate in our assay when applied in a concentration of 250 μg/ml. Treatment with 0.4 mM EGCG completely abolished this effect Figure .in vivo situation, we incubated TZM-Bl cells directly with semen from an HIV-positive, highly viremic donor in presence or absence of 0.4 mM EGCG  , most liIn the above mentioned study, Kim et al. have demonstrated that semen-mediated HIV-1 infectivity enhancement correlates with SEVI/PAP248-286 levels measured by ELISA utilizing antisera from guinea pigs and rabbits . Centrif2+ exhibits inhibitory effects on semen proteases [2+, and thus tend to have a higher capacity to enhance HIV infectivity.Possible causes for the observed heterogeneity of the HIV-infectivity enhancing potency of semen, besides the amount of SEVI fibrils present, could be the variable abundance of cationic polypeptides that inhibit HIV-1 infection  and/or nroteases , it is iOur results confirm and extend the original findings by Hauber et al., who first described the inhibition of semen-mediated enhancement of HIV-infectivity by EGCG for a limited number of individual semen samples . While iClinically, the systemic administration of EGCG has been proven to be safe and well tolerated in several studies -26. TopiIn agreement with Münch et al., it has to be pointed out that our results may underestimate the potency of SEVI for in vivo infectivity enhancement . The quaIn summary, we demonstrate for the first time that fresh human semen contains fibrils with clear resemblance to fibrils formed from synthetic SEVI. Moreover, we demonstrate that the semen-meditated enhancement of HIV-1 infectivity is highly variable and that EGCG can indeed effectively abrogate this activity at non-toxic concentrations in the majority of semen samples. Our study highlights the high degree of variation of HIV-1 infectivity enhancement by individual semen samples (as well as its inhibition by EGCG), even of longitudinal samples originating from identical donors. While EGCG holds potential as a possible microbicide, these variations have to be taken into account and further elucidated before therapeutic trials may be conducted.EMBL accession no. AAB60640), in PBS was agitated at 37°C and 1, 200 rpm for 2-3 days (i.e. until the solution became turbid) to initiate fibril formation [All semen samples were liquefied for 30 min and kept frozen at -20°C until they were used in the experiments. PBS supplemented with 100 units/ml penicillin, 100 mg/ml streptomycin was used to dilute semen as indicated. A solution of chemically synthesized SEVI was prepared as described earlier . Briefly4 cells were seeded in microtiter wells in a volume of 100 μl. After 24 h, cells were infected with HIV-1  according to the manufacturers protocol. Data were collected on a FACS Canto flow cytometer .Cellular viability in presence of EGCG was tested in a [For transmission electron microscopy analysis, semen and a solution of synthetic SEVI (5 mg/ml) were encapsulated in capillary microtubes and processed for ultrathin sectioning as described earlier ,32.t-test analysis as indicated in the figure legends. Correlations were determined using the two-tailed Pearson correlation coefficient. Graphpad Prism version 5 was used for all calculations. For all analyses, p-values of less than 0.05 were considered significant.We determined the statistical significance of differences using Student's The authors declare that they have no competing interests.The work presented here was carried out in collaboration between all authors. JvL and JSzW defined the research theme. SF, PH, JSzW, JvL conceived and designed the study. JSzW provided most of the funding. SF, PH, IH, VM and AT carried out most of the laboratory experiments. SF, PH, JSzW and JvL analyzed the data and interpreted the results. PH and JSzW wrote the first draft. JVL, WS and HH gave important input to the manuscript. HH and BH carried out the transmission electron microscopy experiments. All authors read and approved the final manuscript.


Uptake of prevention of mother-to-child HIV transmission (PMTCT) programs remains challenging in sub-Saharan Africa because of multiple barriers operating at the individual or health facility levels. Less is known regarding the influence of program-level and contextual determinants. In this study, we explored the multilevel factors associated with coverage in single-dose nevirapine PMTCT programs.® Donation Programme (VDP) from 269 sites in 20 PMTCT programs and 15 sub-Saharan countries from 2002 to 2005. Site performance was measured using a nevirapine coverage ratio (NCR), defined as the reported number of women receiving nevirapine divided by the number of women who should have received nevirapine . Data on program-level determinants were drawn from the initial application forms, and country-level determinants from the Demographic and Health Surveys (DHS) and the World Bank (World Development Indicators). Multilevel linear mixed models were used to identify independent factors associated with NCR at the site-, program- and country-level.We analyzed aggregate routine data collected within the framework of the ViramuneOf 283,410 pregnant women attending ANC in the included sites, 174,312 women (61.5%) underwent HIV testing after receiving pre-test counselling, of whom 26,700 tested HIV positive (15.3%), and 22,591 were dispensed NVP (84.6%). Site performance was highly heterogeneous between and within programs. Mean NCR by site was 43.8% (interquartile range: 19.1-63.9). Multilevel analysis identified higher HIV prevalence , higher proportion of persons with knowledge of PMTCT , higher health expenditure as a proportion of Gross Domestic Product  and lower percentage of rural population  as significant country-level predictors of higher NCR at the p<0.05 level. A medium ANC monthly activity (30-100/month) was the only site-level predictor found .Heterogeneity of nevirapine coverage between sites and programs was high. Multilevel analysis identified several significant contextual determinants, which may warrant additional research to further define important multi-level and potentially modifiable determinants of performance of PMTCT programs. Since the HIV epidemic began, children have borne a significant part of the burden of morbidity and mortality. Mother-to-child transmission (MTCT) of HIV, occurring principally during labour, was responsible for approximately 390,000 new infections in 2010 . In thisDespite considerable progress made thus far, improving prevention program effectiveness in the field still remains a complex challenge, as suggested by discrepancies often observed between anticipated and actual uptake of services ,11. In lA better knowledge of these multidimensional constraints could help improve final coverage and guarantee program success. In addition, there is still great interest in documenting lessons learned from the implementation of single-dose NVP programs, given probable similarities between such challenges and those that programs based on more complex ARV interventions might encounter. Consequently, the objective of this study was to assess the site-, program- and country-level characteristics associated with NVP coverage levels achieved by PMTCT programs conducted in sub-Saharan African countries.® Donation Programme (VDP). Viramune® (nevirapine) has been offered to developing countries since 2000 by Boehringer Ingelheim. Axios International has been responsible for the management of NVP distribution and monitoring of participating PMTCT programs. All sub-Saharan African countries were eligible for participation in VDP. The nevirapine donation was available to governments directly, as well as Non Governmental Organisations (NGOs), international organisations and public and private health care providers with government approval.We used aggregate monitoring and evaluation data collected within the framework of the ViramuneAmong VDP participating programs , we selected those with data available by site or single-site programs. During the study period (January 2002 to December 2005), a total of 269 sites were included, participating in 20 PMTCT programs in 15 sub-Saharan countries . No statistically significant differences between selected and not-selected VDP programs were identified with respect to their main characteristics, including institution status, mean number of sites, mean ANC per program per month and mean HIV prevalence .To assess the overall performance of PMTCT sites, a coverage indicator was calculated. The NVP coverage ratio (NCR) was defined as the [observed number of women receiving NVP] divided by [the expected number of women who should have received NVP]. This denominator was calculated as the number of women attending antenatal care (ANC) visits multiplied by the observed HIV seroprevalence at each site (number of HIV-positive women/ number of HIV-tested women).Following acceptance to VDP, participating institutions were required to submit a progress report every six to 12 months with the following data for each site: number of women registered in antenatal care, number of HIV-tested and HIV-positive women and number of women receiving NVP tablets. These data did not permit assessment of the number of newborn NVP doses provided. The mean NCR over the period of follow-up was calculated for each site from all available progress reports. Predictors assessed at the site level included the site-observed HIV prevalence, the site activity (number of women receiving ANC), and the type of health facility .Inclusion in VDP required programs to submit a descriptive application form and receive approval by a body of independent experts. We collected the following data in application forms: type of responsible institution or organisation (public/private) and number of sites included.Variables collected at the national level were drawn from the Demographic and Health Surveys (DHS)  and the s) were used to assess univariate correlation of NCR with intermediate process indicators .Descriptive statistics were computed for the characteristics of the PMTCT programs and are provided as medians (Interquartile Range [IQR]) for quantitative variables and as percentages for qualitative variables. NCR dispersion across sites was assessed by using univariate boxplots. Non-parametric Spearman correlation rank tests (rnull) model without any predictor. The proportional change in the variance (PCV) was then computed in the final model for each level: PCV was defined as the proportion of variance “explained” from the variance initially estimated in the null-model. A two-tailed P-value of less than 0.05 was considered to be significant. All statistical analyses were performed using Stata, version 11.0 .Multilevel linear mixed models were used in univariate and multivariate analyses to identify factors associated with NCR, accounting for the hierarchical clustering of sites (level 1) nested in programs (level 2) within countries (level 3). The following multivariate modeling strategy was applied: predictors associated with NCR at the p<0.2 level in univariate analysis were entered sequentially, starting from level 1 to level 2 and 3, using a backward stepwise method at each level. Beta coefficients are given for each predictor variable, indicating the adjusted difference in NCR (0–100) between categories for qualitative variables or per 1-point difference in predictor value for continuous variables. Rural population proportion and total healthcare expenditure group-level predictors were modeled continuously after checking for linearity of the regression coefficients. The initial variance partition between levels was explored in an empty  and women treated rate .Of the 283,410 pregnant women attending antenatal care, 174,312 women (61.5%) were offered, accepted and underwent HIV testing after receiving pre-test counselling, of whom 26,700 (15.3%) tested HIV positive. After post-counselling, 22,591 women (84.6%) were eventually dispensed NVP. Using these figures, the global NCR was 52.0%. Considering the performance rates per site, the mean testing coverage was 61.5% (IQR 42.2 - 83.8), the mean treatment rate was 70.2% (IQR 50.0 - 98.6) while the mean NCR per site was 43.8% (IQR 19.1 – 63.9). NCR differed greatly across sites ranging from 0.0 to 1.0, and across programs (0.07 to 0.94) as shown in Figure Results from univariate multilevel analysis of PMTCT performance by site-, program- and country-level predictors are shown in Table Multivariate analysis using linear mixed models Table  identifinull-model including no site- or program/country-level predictors, 58.5% was found at the site-level and 41.5% at the program/country level (2.7% [program-level] + 38.8% [country-level]). To determine the proportion of variance explained by the predictors retained in the final model, we computed the PCV for each level: PCV was 99.9% for the program/country level whereas only 0.22% of the site-level variance was explained by the final model.Of the initial total variance of NCR measured in the In this study, we analysed aggregate data from 283,410 pregnant women attending 269 PMTCT sites. We identified several factors operating at the country-level that significantly influenced nevirapine coverage in women, including PMTCT knowledge, HIV prevalence, rural population proportion and proportion of GDP dedicated to healthcare expenditure. These factors were independent of site-level factors, such as the site activity or the type of health facility, and program-level factors, such as the status of the institution or the program burden measured as the number of sites included. To our knowledge, this study is the first to explore the contextual determinants of PMTCT program performance within the formalized framework of multilevel modeling. The contextual predictors identified explained almost 100% of the initial variance at the program/country level. Our data also showed a huge heterogeneity of performance between sites within the same programs.Among 269 PMTCT sites, we found a nevirapine coverage ratio of only 44%. Our result is only somewhat more optimistic than a study by Temmerman et al. reporting a NVP administration rate of 20%, using a similar whole-ANC-population-based calculation process . The takSpecific PMTCT and general comprehensive knowledge about AIDS were significantly associated with higher nevirapine coverage, whereas general awareness about AIDS – as measured by the proportion of persons who had heard of AIDS – and accepting attitudes or stigma were not significantly linked to NCR. This is consistent with previous reports showing knowledge as an important factor of success in antiretroviral adherence  or breasThe national prevalence of HIV remained significant after accounting for knowledge about AIDS and healthcare expenditure. In this context, the interpretation of this indicator might be of interest because it likely encapsulates several other contextual features  or other factors not assessed in our analysis. Thus, we may interpret national HIV prevalence as an awareness and readiness marker indicative of both population and health services.Rural population as a percent of total population was negatively associated with NCR. This result is consistent with the loss to follow-up of pregnant women , as mateNational health expenditure as a proportion of GDP was an independent and positive predictor of nevirapine coverage, probably suggesting an overall positive impact of increased investment in the organization and infrastructure of health services. This finding may confirm the importance of overall health system strength to ensure effective PMTCT services in the field, where performance is notably linked to sites’ infrastructure and testing capacities ,14 as ilAt the site-level, the highest and lowest nevirapine coverage results could be observed within the same program, emphasizing challenges to the on-the-ground feasibility of PMTCT programs. Beyond infrastructure and testing capacities, lack of sufficient and well trained staff have been suggested to explain some of the discrepancies observed between sites. Integration of voluntary counselling and testing (VCT) services in pre-existing ANC settings represents an undeniable additional workload for in-place health workers. At the same time and for economic reasons, human resources have not always risen adequately ,20,25. EWe consider the generalizability of our findings in the context of global PMTCT programs. We included VDP programs with data available by site or one-site programs, which raises the question of the representativeness of the sites included, both in regard to the whole VDP program and to other existing PMTCT programs. We did not identify any significant difference between included and not-included VDP programs regarding their main characteristics, which suggests limited selection bias within the VDP program itself. As for the other PMTCT programs, our program panel cannot be considered as fully representative of all programs, implemented either within the same countries or elsewhere in sub-Saharan Africa and/or based on different antiretroviral drugs and regimen designs. However, our analysis was conducted on a range of sites and programs, including primary or secondary/tertiary health facilities with various ANC activities within programs from several different sub-Saharan African regions, thereby supporting the external validity of our results. Secondly, a possible selection bias cannot be excluded in regards to the pregnant women included in this study, since only women actually attending ANC services were considered. Finally, our findings stem from an early era in large scale implementation of PMTCT services, when antiretroviral interventions were based on much simpler regimens than the multi-drug regimens currently recommended ,10. HoweOur study has several limitations that should be noted. In particular, we were not able to control whether a pregnant woman actually took the NVP dose she was dispensed in PMTCT services. Specifically, we lacked the ability to directly measure the drug compounds contained in cord blood samples, which is an accurate and useful means to counter the limited quality of reported medical data when assessing program coverage . NCR tenIn conclusion, we observed a wide range of coverage outcomes among PMTCT sites and programs. To explain these discrepancies, we identified several country-level factors, including knowledge about PMTCT and AIDS or the proportion of GDP allocated to healthcare, adjusting for a variety of site- and program-level characteristics. Our findings illustrate the potential role of surrounding contextual and global health system features, in addition to local characteristics of sites. While qualitative and quantitative research is still needed at the site level, the overall understanding of factors associated with program performance would benefit from further research that attempts to identify more distant but important and potentially modifiable determinants of success.ANC: Antenatal care; DHS: Demographic and Health Surveys; GDP: Gross Domestic Product; HIV: Human Immunodeficiency Virus; IQR: Interquartile Range; NCR: Nevirapine Coverage Ratio; NGO: Non Governmental Organization; NVP: Nevirapine; PCV: Proportional Change in Variance; PMTCT: Prevention of Mother-To-Child Transmission of HIV; VDP: Viramune Donation Programme.The authors declare that they have no competing interests.All authors participated in the study conception and design. MHB, JS and JL participated in the data acquisition and extraction. EA and JL performed the statistical analysis and interpretation of data. EA made the drafting. All authors participated in the critical revision of the manuscript and have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/13/286/prepub


Evidence from anatomical and functional imaging studies have highlighted major modifications of cortical circuits during adolescence. These include reductions of gray matter (GM), increases in the myelination of cortico-cortical connections and changes in the architecture of large-scale cortical networks. It is currently unclear, however, how the ongoing developmental processes impact upon the folding of the cerebral cortex and how changes in gyrification relate to maturation of GM/WM-volume, thickness and surface area. In the current study, we acquired high-resolution (3 Tesla) magnetic resonance imaging (MRI) data from 79 healthy subjects  between the ages of 12 and 23 years and performed whole brain analysis of cortical folding patterns with the gyrification index (GI). In addition to GI-values, we obtained estimates of cortical thickness, surface area, GM and white matter (WM) volume which permitted correlations with changes in gyrification. Our data show pronounced and widespread reductions in GI-values during adolescence in several cortical regions which include precentral, temporal and frontal areas. Decreases in gyrification overlap only partially with changes in the thickness, volume and surface of GM and were characterized overall by a linear developmental trajectory. Our data suggest that the observed reductions in GI-values represent an additional, important modification of the cerebral cortex during late brain maturation which may be related to cognitive development. A large body of work during last two decades has highlighted the importance of adolescence for the continued maturation of cortical circuits While modifications in the volume of GM/WM have been extensively characterized, relatively little evidence exists on maturational changes in the folding of the cortical surface. The cerebral cortex in humans has as one of its distinguishing characteristics a highly convoluted folding pattern that leads to a significantly increased cortical surface. For example, the surface area of the human cortex is per average ten times larger than that of the macaques monkey but only twice as thick There is evidence that the cortical folding pattern is subject to developmental changes. After 5 months in utero, cortical folds appear and continue to develop at least into the first postpartum year This finding is supported by recent MRI-studies which have investigated GI-values during brain maturation. Raznahan et al. In the present study, we sought to comprehensively characterize the development of gyrification during adolescence through investigating whole-brain GI-values in MRI-data. In addition, we obtained GM-parameters  as well as WM-volume estimates to determine the relationship between age-dependent changes in gyrification and GM/WM parameters. Our results show widespread reduction in GI-values which occur in overlapping but also distinct areas of GM-change, such as in precentral, temporal and frontal regions, which highlight the ongoing anatomical modification of the cerebral cortex during adolescence.85 right-handed participants  between the ages of 12 and 23 years were recruited from local high-schools and the Goethe University Frankfurt and were screened for the presence of psychiatric disorders, neurological illness and substance abuse. Written informed consent was obtained from all participants. For participants younger than 18 years, written consent was given by their parents. The Hamburger-Wechsler intelligence testing battery (HAWI-E/K) 3, slices: 176 and a voxel size of 1×1×1.1 mm3.Structural magnetic resonance images were obtained with a 3-Tesla Siemens Trio scanner , using a CP head coil for RF transmission and signal reception. We used a T1-weighted three-dimensional (3D) Magnetization Prepared Rapid Acquisition Gradient Echo (MPRAGE) sequence with the following parameters: time repetition (TR): 2250 ms., time echo (TE): 2.6 ms., field of view (FOV): 256×256 mmhttp://surfer.nmr.mgh.harvard.edu) MRI-data were processed with the surface and volume pipeline of the FreeSurfer-software version 5.1.0  on the tesselated surface A 3-D lGI was computed The regional WM-volume below parcellated cortical GM-regions were estimated. Each white matter voxel was labeled to the nearest cortical GM-voxel with a distance limit of 5 mm resulting in 33 WM-volumes of the corresponding 33 gyral labeled GM areas The estimated intracranial Volume (eTIV) in the FreeSurfer pipeline was derived from an atlas normalization procedure. Through the Atlas Scaling Factor (ASF), which represents a volume-scaling factor to match an individual to an atlas target, calculations of each eTIV were performed The analyses steps are summarized in 2 and age3 effects for all anatomical parameters which were controlled for the influence of age, gender and eTIV.In the first step, we investigated whole-brain lGI-values, cortical thickness, cortical surface area and volume of GM in a vertex-by-vertex analysis. A General Linear Model (GLM) was employed to analyze the effect of age on the different anatomical parameters . All analyses were performed while controlling for the effects of gender and eTIV. We employed a false discovery rate approach (FDR) http://surfer.nmr.mgh.harvard.edu/fswiki/mri_surfcluster). In addition, Cohen's d To obtain estimates of area size, we selected vertices with the largest lGI-values and their corresponding Talairach coordinates and applied the automatic mri_surfcluster function in FreeSurfer (−4) were assigned to FreeSurfers gyral based areas In a second step, we examined Pearson correlation coefficients between age-dependent lGI-effects and changes in cortical thickness, cortical surface area and GM/WM-volume. To include WM-volume data, parcellation based regional analyses were performed. Four vertices from the vertex-by-vertex analyses per hemisphere with pronounced age-lGI effects , left superior-frontal , left inferior-temporal , left lateral-orbitofrontal  and right precentral cortex , right pars triangularis , right rostral-middlefrontal  and superior parietal . No significant effects of gender were found for changes in lGI-values at a FDR at 0.005 and age-related reductions in gyrification followed nonlinear (cubic) trajectories  , Table 1ectories .2, p = 10−7.13, BA 6, 8 and 9) and rostral-middle-frontal  cortices in the left hemisphere and in the precentral cluster in the right hemisphere  . Moreover, we found age-dependent, bilateral decreases in GM-volume which were localized to the superior-frontal  lobe in the left hemisphere and to the pars orbitalis  and to the inferior-parietal  lobe of the right hemisphere .Cortical thickness decreased most prominently in the superior-frontal (area size = 2608.63 mm and 45) . The cormisphere . GM-volu2, p = 10−9.64, BA 4), caudal middle frontal  and supramarginal  clusters in the left hemisphere. Surface area decreased in the right hemisphere most prominently in precentral , inferior parietal  and superior parietal  cortices . No significant effects of gender were found for changes in cortical thickness, GM-volume and surface area at a FDR at 0.005For surface area, we found a significant reduction in precentral , left superior-parietal  and left pericalcarine  clusters. For the right hemisphere, effects were observed in a precentral region , postcentral  and in superiorfrontal cortices .We found 16 (left hemisphere) and 7 Clusters (hemisphere) where agerrelated . The str2, p = 10−6.54, BA 8, 9, 10, 11, 45, 46 and 47), superior-parietal  and pericalcarine  cluster for the left hemisphere. In the right hemisphere, strongest cubic age and lGI relations were found in a precentral , caudal-middlefrontal  and middle-temporal cluster .Cubic effects of age on lGI were found in 18 (left hemisphere) and 7 Clusters (right hemisphere). Regions with the strongest cubic effects were localized in a large superior-frontal , paracentral  and inferior-parietal  cortices.Agemisphere . Stronge2, p = 10−4.55, BA 31), a part of the gyrus inferior frontalis-pars opercularis-  and the banks of superior temporal sulcus  were characterized by a significant age3 and lGI relationship  and temporal (BA 20 and 21) cortices, which is consistent with data from previous longitudinal studies Although the precentral cluster, which extended to pre-/post-central gyrus, supramarginal gyrus as well as to the superior parietal cortex, has been less consistently involved in adolescent brain maturation, there is evidence to suggest that these brain areas may be related to ongoing changes in cognition and behavior. A recent study by Ramsden et al. A second region of pronounced changes in IGl-values was the frontal cortex which has been consistently linked to changes in anatomy and behavior during adolescence. In the present study, decreased lGI-values were found in the frontal pole (BA 10), orbitofrontal cortex (BA 11) and the inferior frontal gyrus (BA 47). A large body of work has indicated that these regions are centrally involved in the behavioral modifications during adolescence, such as the improvements in cognitive inhibition Finally, substantial reductions in gyrification were found in a cluster corresponding to BA 19, 20 and 37 which comprises early visual areas and cortical regions dedicated to object recognition. In addition to modifications in higher cognitive functions, adolescence is also associated with improvements in neural oscillations elicited by simple and complex visual stimuli Strong quadratic effects of age on lGI were found in left superior-frontal  and righthemispheric frontal (BA 8) clusters, which is in line with a previous study by  and Autism Spectrum Disorders (ASDs), are associated with abnormal cortical folding patterns. Specifically, participants with WS are characterized by reductions in the depth of sulci in parieto-occipital regions which are prominently involved in the visuo-constructive deficits Schizophrenia is a severe psychiatric disorder with a typical onset during the transition from adolescence to adulthood which also involves aberrant gyrification. Post-mortem Because our data strongly suggest that cortical folding undergoes major modifications during adolescence, one possibility is that in addition to early neurodevelopmental influences, abnormal brain development during adolescence contributes to the aberrant anatomy of the neocortex and the manifestation of cognitive dysfunctions and clinical symptoms.The findings support the view that the adolescence involves fundamental changes in the architecture of the cerebral cortex. Specifically, we can show that cortical folding patterns undergo pronounced change which involves a reduction in gyrification across large areas of the cerebral cortex, in particular in precentral, frontal and temporal regions. Future studies need to establish the functional relevance of these modification for concurrent changes in behavior, cognition and physiology through correlations with neuropsychological data and functional brain imaging methods, such as fMRI and MEG.Figure S1Nonlinear age effects on the local gyrification index (lGI) in a whole brain, vertex-by-vertex analyses projected onto an average template brain. Top Row: Age2 effects are illustrated for the left hemisphere (left) and right hemisphere (right) from lateral and medial views. Bottom Row: Correlations between age3 and lGI are shown for the left (left) and right hemisphere (right) from lateral and medial views. Blue colors indicate a significant decrease of lGI-values with increasing age, whereas warmer colors are coded for an increase in lGI. All analyses were performed by controlling for the effects of gender, eTIV and age (linear). Note: No significant correlations between age3 and lGI were found by controlling for the effects of gender, eTIV, age (linear) and age2.(TIFF)Click here for additional data file.Figure S2Nonlinear age effects on GMV in a whole brain, vertex-by-vertex analyses projected onto an average template brain. Left: Age2 effects on GMV for the left hemisphere from lateral and medial view. Right: Effects of age3 are illustrated for the left hemisphere from lateral and medial view. Blue colors indicate a significant decrease of GMV with increasing age, whereas warmer colors are coded for an increase in GMV. All analyses were performed by controlling for the effects of gender, eTIV and age (linear). Note: No significant correlations between age3 and GMV were found by controlling for the effects of gender, eTIV, age (linear) and age2.(TIFF)Click here for additional data file.


Long-term breast-cancer survivors have a highly elevated risk (1 in 6 at 20 years) of contralateral second breast cancer. This high risk is associated with the presence of multiple pre-malignant cell clones in the contralateral breast at the time of primary breast cancer diagnosis. Mechanistic analyses suggest that a moderate dose of X-rays to the contralateral breast can kill these pre-malignant clones such that, at an appropriate Prophylactic Mammary Irradiation (PMI) dose, the long-term contralateral breast cancer risk in breast cancer survivors would be considerably decreased.To test the predicted relationship between PMI dose and cancer risk in mammary glands that have a high risk of developing malignancies. We tested the PMI concept using MMTV-PyVT mammary-tumor-prone mice. Mammary glands on one side of each mouse were irradiated with X-rays, while those on the other side were shielded from radiation. The unshielded mammary glands received doses of 0, 4, 8, 12 and 16Gy in 4-Gy fractions. In high-risk mammary glands exposed to radiation doses designed for PMI (12 and 16 Gy), tumor incidence rates were respectively decreased by a factor of 2.2  at 12 Gy, and a factor of 3.1  at 16 Gy, compared to those in the shielded glands that were exposed to very low radiation doses. The same pattern was seen for PMI-exposed mammary glands relative to zero-dose controls.The pattern of cancer risk reduction by PMI was consistent with mechanistic predictions. Contralateral breast PMI may thus have promise as a spatially targeted breast-conserving option for reducing the current high risk of contralateral second breast cancers. For estrogen-receptor positive primary tumors, PMI might optimally be used concomitantly with systemically delivered chemopreventive drugs such as tamoxifen or aromatase inhibitors, while for estrogen-receptor negative tumors, PMI might be used alone. Breast cancer is one of the most common types of cancer in the US, and as many as one in eight women may develop breast cancer during their lifetime . Long teFor estrogen-receptor positive tumors, adjuvant tamoxifen reduces contralateral breast cancer risks by as much as 40%  - which The general concept behind chemopreventive approaches is that carcinogenesis is a multi-stage process involving the gradual accumulation of pre-malignant clones, with chemopreventive drugs intervening at one or more stages in this process . We haveOf course ionizing radiation is also carcinogenic, and can induce new breast tumors ,17.The nIn fact, a mechanistically-based analysis  of the tcontralateral breast of breast cancer patients with such a moderate radiation dose might be an effective option for safely reducing contralateral breast cancer risks in breast cancer survivors [The ipsilateral breast requires a high radiation dose in order to eradicate large numbers of remaining primary tumor cells. However the reasoning here suggests  that a murvivors . We haveurvivors .Although the number of pre-malignant target cells in the contralateral breast is not well defined, because the dependence of the required radiation dose on this number is logarithmic rather than linear , any reaThus, PMI of the whole contralateral breast, performed at the same time as standard post-lumpectomy radiotherapy to the ipsilateral breast, and with an optimally chosen intermediate radiation dose, might be a potentially breast-conserving option to significantly decrease the high second cancer risks in the contralateral breast of long-term breast cancer survivors . BecauseHere we have tested the PMI hypothesis in an animal model for high-risk breast cancer – the FVB/N-Tg(MMTV-PyVT)634Mul/J (abbreviated as MMTV-PyVT) female transgenic mouse . Our goaThis study was approved by Columbia University IACUC, under Animal Care Protocol AC-AAAD3951, and all mouse work was performed in accordance with IACUC guidelines. Mammary tumor incidence rates were measured as a function of radiation dose in: 1) irradiated mammary glands, 2) shielded mammary glands exposed only to low doses of scattered radiation, and 3) unexposed control animals.in vivo, minimizing mouse numbers and follow-up time. To conduct proof of principle experiments regarding the PMI hypothesis, we selected the FVB/N-Tg(MMTV-PyVT)634Mul/J female mouse (MMTV-PyVT) , from thThe near hemi-body irradiation geometry used here is illustrated in One hundred female MMTV-PyVT mice, divided into 5 groups of 20 mice each, were used. The mice were between 41 and 43 days old at the start of irradiation. Each group respectively received total x-ray doses to the unshielded mammary glands of 0, 4, 8, 12 and 16 Gy, delivered in 4-Gy fractions. The maximum number of fractions per day was two, 6 hours apart. In summary, the unexposed control mice received a single sham-irradiation, the 4 Gy exposed mice received a single irradiation with 4 Gy, and the 8 Gy exposed mice received two 4 Gy irradiations 6 hours apart on the same day. The 12 Gy exposed mice received two 4 Gy irradiations 6 hours apart on the first day, and a third 4 Gy irradiation on the second day. The 16 Gy exposed mice received two 4 Gy irradiations 6 hours apart on the first day, and two more 4 Gy irradiations 6 hours apart on the second day. The two highest doses (12 and 16 Gy in 4-Gy fractions) were designed to be in the range that would sterilize most or all of the pre-existing pre-malignant and/or early malignant cells in each mouse mammary gland , whiThe x-ray irradiations were carried out using an XRAD-320 irradiator  operated at 320 kVp and 12.5 mA, with a filter composed of 1.5 mm Al, 0.25 mm Cu and 0.75 mm Sn. The dose rate was 0.86 Gy/min. Immediately prior to irradiation, the mice were anesthetized with a 0.2 ml intraperitoneal injection of a solution of 10 mg/ml ketamine and 1 mg/ml xylazine. Control mice, receiving 0 Gy, were anesthetized and sham-irradiated.vs. unshielded mammary glands is similar to the ratio of doses in the contralateral vs. the irradiated breast in human breast radiotherapy [Doses were measured using microMOSFET  solid-stotherapy .The mice were maintained with 50 mg/kg amoxicillin in the drinking water, and their health and body weights were monitored regularly. After irradiation, the mouse mammary glands were palpated at 2-3 day intervals. Appearance times and the sizes of mammary tumors were recorded. Mice were sacrificed according to Columbia University animal care guidelines when they met either of the following criteria: tumor diameter >20 mm, tumor size exceeding 10% of body weight, tumor ulceration, debilitating diarrhea, progressive dermatitis, rough hair coat, hunched posture, lethargy or persistent recumbence, coughing, labored breathing, neurologic signs , bleeding from any orifice, self-induced trauma, and any condition interfering with eating or drinking . All mice eventually developed mammary tumors, and most had multiple tumors. The first tumor to present was invariably the one which caused the mouse to be sacrificed when it grew to a large size, and tumors which appeared later were still small at the time of sacrifice. Consequently, the incidence rate of the first detected tumor was selected as the most relevant endpoint in this study, and these data are presented here. The goal of this study was to assess how radiation dose affected the cumulative incidence rate (per mouse-day at risk) of the first detected palpable mammary tumors. This was done by comparing the cumulative tumor incidence rates in each radiation dosage group, on each mouse side (unshielded or shielded), with the rate in the unirradiated control group, using Poisson regression, as implemented by the WinPepi software package . The datCumulative tumor incidence rates on different mouse sides were also compared within each radiation dosage group, using the 2-tailed mid-P approach  with BonThe potential presence of a monotonic trend in tumor incidence rate as function of radiation dose was evaluated by the Poisson trend statistic (Eq. 3.12 in Ref. ). TemporAll the data pertaining to the mice used in this study, including body weights, dates of irradiation, tumor detection and sacrifice, are available upon request.Each irradiated mouse received a fractionated radiation dose (varying from 4 to 16 Gy in different mice) to the unshielded mammary glands, and a very much lower dose (<1 Gy) to the shielded glands (0.2-0.9 Gy) caused by x-ray scatter. As seen in earlier studies of radiation-induced mammary cancer , the temBy contrast, the cumulative tumor-incidence rates (per mouse-day at risk) showed a clear dose response. Both relative to the zero dose controls, and relative to the corresponding mammary glands that received only scattered doses, tumor incidence rates initially increased with increasing dose to the irradiated mammary gland, but then decreased at higher doses to below the background levels. These results are summarized in We focus here on comparisons between tumor incidence rates in the PMI-irradiated mammary glands and in the corresponding shielded mammary glands; as described above, this is designed to model a comparison of cancer rates in a human contralateral breast exposed to PMI vs. cancer rates in the same breast receiving a typical scatter dose after standard radiotherapy to the ipsilateral breast. At the doses which were designed to be relevant for PMI  the tumor incidence rates in the unshielded PMI-exposed glands were significantly lower than those in the shielded glands: the rate ratios between the PMI-exposed glands and the shielded glands were 0.45  at 12 Gy and 0.32  at 16 Gy . These same patterns were apparent for the tumor rates in the PMI-exposed glands relative to zero-dose controls . AlthougThe measured dose response patterns  are consWhile there are similarities, there are of course differences between the high-risk animal breast cancer model used here and the contralateral breast in a breast cancer patient. Firstly, the mice were irradiated at an earlier time of life (early puberty) compared to most women (who develop breast cancer at a median age ~60): In fact both for rodents  and humaSecondly, it is unclear whether the relevant cells in the mouse model should be considered as pre-malignant or fully malignant, or perhaps a combination of these; this should not, however, affect the dose-effect patterns seen here, as killing of either would be expected to reduce cancer risk – and indeed the target cells for contralateral breast cancer in humans are also likely to be heterogeneous in their development. With these caveats, the results shown here are consistent with the predicted pattern of cancer risks in the PMI dose range. Specifically, we have provided proof-of-principle experimental confirmation that there is likely to be a “window of opportunity” in PMI dose (schematized in Contralateral whole breast PMI may thus have promise as a spatially-targeted breast-conserving option for reducing the current high risk of contralateral second breast cancers in long-term breast cancer survivors. The potential efficacy of PMI is likely to be independent of estrogen receptor status: thus for estrogen-receptor positive primary tumors, PMI might optimally be used concomitantly with systemically delivered chemopreventive drugs such as tamoxifen or aromatase inhibitors, while for estrogen-receptor negative tumors, PMI might be used alone.Should PMI prove successful, the potential impacts could be major. For example, there are currently more than 2.6 million breast cancer survivors alive in the US : applyin


Betaine (glycine betaine or trimethylglycine) plays important roles as an osmolyte and a methyl donor in animals. While betaine is reported to suppress expression of proinflammatory molecules and reduce oxidative stress in aged rat kidney, the effects of betaine on the central nervous system are not well known. In this study, we investigated the effects of betaine on lipopolysaccharide (LPS)-induced memory impairment and on mRNA expression levels of proinflammatory molecules, glial markers, and GABA transporter 2 (GAT2), a betaine/GABA transporter.Mice were continuously treated with betaine for 13 days starting 1 day before they were injected with LPS, or received subacute or acute administration of betaine shortly before or after LPS injection. Then, their memory function was evaluated using Y-maze and novel object recognition tests 7 and 10-12 days after LPS injection , respectively. In addition, mRNA expression levels in hippocampus were measured by real-time RT-PCR at different time points.Repeated administration of betaine  prevented LPS-induced memory impairment. GAT2 mRNA levels were significantly increased in hippocampus 24 hr after LPS injection, and administration of betaine blocked this increase. However, betaine did not affect LPS-induced increases in levels of mRNA related to inflammatory responses. Both subacute administration  and acute administration (1 hr after LPS injection) of betaine also prevented LPS-induced memory impairment in the Y-maze test.These data suggest that betaine has protective effects against LPS-induced memory impairment and that prevention of LPS-induced changes in GAT2 mRNA expression is crucial to this ameliorating effect. Betaine (glycine betaine or trimethylglycine) is widely distributed in plants and microorganisms as well as in various dietary sources ,2. Some In humans, betaine is obtained from the diet  or from Betaine/GABA transporter-1 (BGT-1), the mouse transporter homologue of which is known as GABA transporter 2 (GAT2), is an integral membrane transporter capable of utilizing both betaine and GABA as substrates ,9. The dLipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, is used to experimentally induce memory impairment, neuroinflammatory responses, and oxidative stress such as increases in mRNA levels of interleukin (IL)-1ß and IL-6 , heme oxIn this study, we investigated the effects of betaine on LPS-induced memory impairment using the Y-maze and novel object recognition tests. We also examined the effect of betaine on LPS-induced changes in mRNA expression levels of proinflammatory molecules, glial markers, and GAT2 using real-time RT-PCR.Male ddY strain mice  were used. The mice were kept in a regulated environment  under a 12-h light/dark cycle (lights on 7:45 a.m.) and given food and tap water ad libitum. The experimental protocols concerning the use of laboratory animals were approved by the animal ethics board of Meijo University and followed the guidelines of the Japanese Pharmacological Society ; the Interministerial Decree of May 25th, 1987 ; and the National Institutes of Health Guide for the Care and Use of Laboratory Animals . All efforts were made to minimize animal suffering and to reduce the number of animals used.Escherichia coli 0111:B4  was dissolved in 0.9% saline and administered intracerebroventricularly (i.c.v.) into the lateral ventricle of the mouse brain according to the method of Haley & McCormick [Betaine hydrochloride  was dissolved in 0.9% saline and injected subcutaneously (s.c.). Lipopolysaccharide from cCormick  at a doscCormick  and we cFirst, we investigated whether betaine alleviated LPS-induced memory impairment using the Y-maze and novel object recognition tests, which were carried out 7 and 10-12 days after the LPS injection , respectively. Time schedules of behavioral experiments were referred to a previous report , which sImmediate working memory was assessed by recording spontaneous alternation behavior during a single session in a Y-maze  made of The novel object recognition test, which was described previously , was useFor real-time RT-PCR, mice were sacrificed after the administration of LPS and/or betaine. Immediately after their decapitation, their hippocampi were rapidly dissected according to the method of Glowinski & Iversen  and immeStatistical analysis was performed, and the figures were produced using Prism 5 for Mac OS X . It could not be assumed that the behavioral data were sampled from a Gaussian distribution; therefore, the data are expressed as median and interquartile range values. Significance was evaluated using the Mann-Whitney U-test for comparisons between two groups, and Kruskal-Wallis non-parametric one-way ANOVA followed by Bonferroni's test were used for multiple comparisons. The expression levels of each mRNA are shown as mean ± S.E.M. An unpaired t-test  was used to compare two groups, and one-way ANOVA followed by Dunnett's test was used for multiple comparisons. The criterion for significance was p < 0.05.In the Y-maze test, LPS treatment  significantly decreased the percentage of alternations 7 days after LPS injection , a marker of astrocytes, were investigated. LPS treatment (30 μg/mouse) significantly increased mRNA expression levels of CD11b, CD45, and GFAP 24 hr after injection  significantly increased mRNA expression for GAT2 24 hr after injection  significantly decreased the percentage of alternations in the Y-maze test  also increased mRNA expression levels of the microglial markers CD11b and CD45, and the astrocytic marker GFAP; however, betaine also did not prevent the LPS-induced increases in mRNA levels for these glial markers. Our results indicate that betaine does not suppress mRNA expression of proinflammatory molecules or glial markers, and the mechanism behind the ameliorating effects of betaine on memory impairment is not mediated by the expression of these genes, which is the mechanism by which betaine suppresses the expression of proinflammatory molecules and increased oxidative stress in aged rat kidney ,7. This Four different subtypes of GAT have been cloned and are termed GAT1, GAT2, GAT3, and GAT4 in mice  . GAT2/BGIt is known that the changes that occur during the early phase after LPS treatment are crucial to delayed neuronal impairment such as the memory impairment shown in this study. To elucidate the mechanisms underlying the effects of betaine, we considered that administration of betaine during the early phase after LPS injection might be necessary for preventing LPS-induced memory because mRNA expression levels for GAT2 transiently increased after LPS injection and recovered by 48 hr after LPS injection. Interestingly, either subacute  or single (1 hr after the LPS injection) administration of betaine prevented LPS-induced memory impairment, but this effect was not seen when betaine was given 1 hr before LPS injection. Consistent with betaine's effect in alleviating LPS-induced delayed memory impairment, betaine also significantly reduced LPS-induced increases in GAT2 mRNA levels in hippocampus. These data suggest that during the early period after LPS injection, betaine plays a crucial role in preventing LPS-induced neuronal dysfunction. On the other hand, a single administration of betaine, 1 hr before LPS injection, did not prevent LPS-induced memory impairment. This finding that betaine has a neuroprotective effect on delayed memory impairment even when administered after LPS injection has important therapeutic implications. Excitotoxicity has been implicated in the etiology of ischemic stroke and chronic neurodegenerative disorders. Hence, the development of novel neuroprotective molecules that ameliorate excitotoxic brain damage is being vigorously pursued. Indeed, betaine attenuates glutamate-induced neurotoxicity in primary cultured brain cells . MontoliBetaine improves LPS-induced memory impairment and blocks LPS-induced increases in mRNA expression for GAT2; however, betaine does not prevent LPS-induced increases in mRNA expression of proinflammatory molecules or glial markers. These results suggest that betaine has protective effects against LPS-induced memory impairment that are mediated through unique mechanisms involving betaine actions on GAT2, which is involved in the development of memory impairment, without affecting proinflammatory molecules or glial markers.The authors declare that they have no competing interests.MT carried out the behavioral experiments. YN and AE carried out the real-time RT-PCR. MM participated in the design of the study, performed the statistical analysis, drafted the manuscript, and helped to carry out the behavioral experiments and real-time RT-PCR. MH conceived the study, participated in its design and coordination, and helped to draft the manuscript. All of the authors have read and approved the final manuscript.


CTLA4 blocking monoclonal antibodies provide a low frequency but durable tumor responses in patients with metastatic melanoma, which led to the regulatory approval of ipilimumab based on two randomized clinical trials with overall survival advantage. The similarly fully human anti-CTLA4 antibody tremelimumab had been developed in the clinic at a fixed rate infusion, resulting in very prolonged infusion times. A new formulation of tremelimumab allowed testing a shorter infusion time.A phase 1 multi-center study to establish the safety and tolerability of administering tremelimumab as a 1-hour infusion to patients with metastatic melanoma. Secondary endpoints included pharmacokinetic and clinical effects of tremelimumab.No grade 3 or greater infusion-related adverse events or other adverse events preventing the administration of the full tremelimumab dose were noted in 44 treated patients. The overall side effect profile was consistent with prior experiences with anti-CTLA4 antibodies. Objective tumor responses were noted in 11% of evaluable patients with metastatic melanoma, which is also consistent with the prior experience with CTLA4 antagonistic antibodies.This study did not identify any safety concerns when tremelimumab was administered as a 1-hour infusion. These data support further clinical testing of the 1-hour infusion of tremelimumab. . The mean (SD) Cmax was 322 (SD 62) μg/mL and mean t1/2 was 470 hours (19.6 days). No subject had measurable HAHA suggesting minimal immunogenicity of tremelimumab after multiple 1-hour intravenous infusions. Taken together, these data indicate that the shorter infusion time did not alter the PK of tremelimumab, since the PK parameters are similar to those after a 5-hour infusion of tremelimumab [Figure limumab .This study was conducted to establish the safety and tolerability of administering tremelimumab as a one-hour infusion. The use of a shorter infusion is of importance to patient convenience. The main finding of this study is that no grade 3 or greater infusion-related AEs, or other AEs preventing the full tremelimumab dose from being administered, were reported during the infusion period in any of the study subjects. The one-hour infusion safety and tolerability profile of tremelimumab was consistent with that observed to date in other studies with longer infusion times of this antibody. Therefore, we conclude that the one-hour infusion of tremelimumab is suitable for further clinical testing.Bo Huang, Erjian Wang, Poe-Hirr Hsyu, Jesus Gomez-Navarro, Diana Gerhardt and Margaret A. Marshall were employees of Pfizer at the time of this study.AR, JAC, MSG, APA, TFL, DHL, BC, JAG, KL and RG treated patients and provided data. BH was the study statistician. EW and PHH performed pharmacokinetic studies, JG-N, DG and MAM were study clinicians from the sponsor. AR and MAM wrote the manuscript. All authors read and approved the final manuscript.


The yield of mycobacterial blood cultures for multidrug-resistant (MDR) and extensively drug-resistant tuberculosis (XDR-TB) among drug-resistant TB suspects has not been described.Mycobacterium tuberculosis bacteremia and the additive yield of mycobacterial blood cultures compared to sputum culture.We performed a retrospective, cross-sectional analysis to determine the yield of mycobacterial blood cultures for MDR-TB and XDR-TB among patients suspected of drug-resistant TB from rural South Africa. Secondary outcomes included risk factors of M. tuberculosis, of which 20 (49%) were XDR-TB and 8 (20%) were MDR-TB. One hundred fourteen (88%) patients were known to be HIV-infected. Patients on antiretroviral therapy were significantly less likely to have a positive blood culture for M. tuberculosis (p = 0.002). The diagnosis of MDR or XDR-TB was made by blood culture alone in 12 patients.From 9/1/2006 to 12/31/2008, 130 patients suspected of drug-resistant TB were evaluated with mycobacterial blood culture. Each patient had a single mycobacterial blood culture with 41 (32%) positive for Mycobacterial blood cultures provided an additive yield for diagnosis of drug-resistant TB in patients with HIV from rural South Africa. The use of mycobacterial blood cultures should be considered in all patients suspected of drug-resistant TB in similar settings. Tuberculosis (TB) is the leading cause of mortality among people living with HIV worldwide ,2. Drug-Mycobacterium tuberculosis are needed in settings with a high prevalence of drug resistance.Prompt diagnosis and treatment are essential to improve drug-resistant TB outcomes, but TB diagnosis in patients with HIV co-infection is challenging, particularly in resource-limited settings . HIV-infM. tuberculosis can vary between 2%-64% depending on the population of study and suspicion for extrapulmonary TB  27-38) and 8 (6%) patients were less than 12 years of age  had no prior history of TB, although 89 (69%) patients were failing drug-susceptible TB treatment at the time of blood culture collection. The median duration of TB treatment for these patients was 8.0 weeks (IQR 4.0-20.0). Forty-five (35%) patients were suspected to have extrapulmonary TB.M. tuberculosis was isolated in 41 (32%)  were drug-resistant TB. Specifically, 20 (49%) specimens were XDR-TB and 8 (20%) were MDR-TB  specimens. In the six study patients with Cryptoccocus species or NTM detected in the blood, five were currently receiving first-line TB therapy at the time of blood culture collection yet none of the patients had culture documentation of M. tuberculosis.Of the remaining positive blood cultures, NTM were found in 3 (6%) specimens and M. tuberculosis bacteremia who were extrapulmonary TB suspects, OR 2.1 ; adjusted OR 2.3 . Among HIV-infected patients, those on antiretroviral therapy for any duration at the time of blood culture collection were significantly less likely to have M. tuberculosis bacteremia, OR 0.29 ; adjusted OR 0.22  (Table M. tuberculosis was cultured from the blood in patients as young as 8 years (MDR-TB) and as old as 62 years (XDR-TB).There were significantly more patients with M. tuberculosis bacteremia, there were 23 patients that also had a sputum sample collected for comparison (Table M. tuberculosis bacteremia, in patients with both a blood and a sputum culture positive for M. tuberculosis, only 24% were suspected of extrapulmonary TB.Of the 41 patients with M. tuberculosis blood cultures. Importantly, among patients with MDR or XDR-TB bacteremia, in over half of those in whom a sputum culture was unavailable, the blood culture was the only means of drug-resistant TB diagnosis. Bacteremia with XDR-TB was more common than MDR-TB, but reflective of community trends from sputum diagnosis in Tugela Ferry [We found that among a predominantly HIV-infected population of patients suspected of drug-resistant TB, MDR-TB and XDR-TB were isolated in nearly 70% of all positive la Ferry .M. tuberculosis bacteremia is likely to be present in drug-resistant TB suspects at higher rates than clinically suspected. Thus, we feel that these results are generalizable to other populations in sub-Saharan Africa where TB/HIV co-infection rates are high and the incidence of drug-resistant TB may be increasing.Current guidelines suggest the use of mycobacterial blood cultures may be beneficial in suspected extrapulmonary TB, but do not address the use in all HIV-infected persons or those suspected of drug-resistant TB . A recenThe bulk of the additive yield for MDR and XDR-TB in blood compared to sputum cultures was found in patients that did not have a sputum sample collected. The most common reason to not have a sputum sample collected in this hospital is the patient's inability to expectorate due to an absence of cough or marked physical disability; however, due to the retrospective nature of this study, we cannot confirm the reasoning for an individual patient. Standard of practice in other settings is to collect two or more sputum samples for microscopy and/or TB culture as a means of increasing yield. Further prospective study is warranted to determine how multiple sputum samples would affect the comparative yield to blood culture in similar populations with advanced HIV. Blood is an easily accessible fluid and carries the additional advantage of not requiring cold storage for transport. Additionally, the cost of analyzing a mycobacterial blood culture with the National Health Services Laboratory in South Africa is no more expensive than MGIT analysis of a sputum specimen.M. tuberculosis bacteremia were not suspected to have extrapulmonary TB. Mycobacterial culture of lymph node aspirates and pleural fluid were available to clinicians during this study period [M. tuberculosis were suspected of extrapulmonary TB. Thus, our findings suggest that many patients with pulmonary TB in this setting may also harbor unrecognized M. tuberculosis bacteremia. Detection of otherwise occult M. tuberculosis bacteremia regardless of DST, in a patient without suspected extrapulmonary TB may prompt a more exhaustive search for an extrapulmonary focus which could alter treatment and carry important implications for monitoring and clinical outcome.In this study, the majority of patients with y period , yet in M. tuberculosis bacteremia. Earlier studies of M. tuberculosis bacteremia in similar populations in Africa were carried out prior to widespread availability of antiretrovirals and therefore this association could not have been documented until now [M. tuberculosis bacteremia. One explanation is, in accord with national guidelines, the CD4 count is only checked twice annually; thus the CD4 count recorded may be falsely low for those patients that initiated antiretroviral therapy within the prior six months. Alternatively, in some patients early restoration of lymphocyte function may precede restoration of total lymphocyte count.Patients on antiretroviral medication at the time of blood culture collection were significantly less likely to have ntil now ,21. Our ntil now . NotablyM. tuberculosis bacteremia may not have been captured. It is also a possibility that blood cultures may have been preferentially pursued in patients in whom a diagnosis was not as readily made by sputum analysis. Therefore, only prospective study of simultaneous blood and rigorous collection of multiple sputum samples in all drug-resistant TB suspects would allow determination of a true incremental yield in this setting.One of the primary limitations of the study, given its retrospective design, was that the decision of blood culture collection was dependent upon the attending clinician and therefore additional patients suspected of drug-resistant TB may not have had blood cultures sent and were not included in the study. Additional factors that influenced the decision to pursue the investigation for M. tuberculosis bacteremia. The adjunctive use of mycobacterial blood cultures should be considered in all patients suspected of drug-resistant TB, particularly in those unable to expectorate. In many regions of Africa and the developing world, culture and DST are not routinely performed for the diagnosis of TB despite the inferior sensitivity of routine sputum microscopy and the inability of microscopy to detect drug-resistant TB. Expanded access to culture and DST of sputum in South Africa has been projected to save 47,955 lives and avert 7,721 new MDR-TB cases over the next 10 years [In summary, mycobacterial blood cultures diagnosed MDR and XDR-TB in a substantial number of patients predominately HIV-infected and suspected of drug-resistant TB from rural South Africa. Bacteremia with drug-susceptible and drug-resistant TB was not restricted to patients suspected of extrapulmonary TB, as many patients with sputum culture confirmed pulmonary TB also had 10 years . Our finThe authors declare that they have no competing interests.SKH conceived of the study design, performed data analysis and drafted the manuscript. TAT, NRG and SS participated in study design, data analysis and critical revision of the manuscript. FJE, APM and PB participated in study design, acquired patient related data, and provided critical revision of the manuscript. YC and LR performed specimen processing, culture and drug-susceptibility testing. UL and GF participated in study design and critical revision of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/344/prepub