HIV-1 Genetic Diversity and Its Impact on Baseline CD4+T Cells and Viral Loads among Recently Infected Men Who Have Sex with Men in Shanghai,China

The HIV-1 epidemic among men who have sex with men (MSM) has been spreading throughout China. Shanghai, a central gathering place for MSM, is facing a continuously increasing incidence of HIV-1 infection. In order to better understand the dynamics of HIV-1 diversity and its influence on patient’s immune status at baseline on diagnosis, 1265 newly HIV-1-infected MSM collected from January 2009 to December 2013 in Shanghai were retrospectively analyzed for genetic subtyping, CD4+T cell counts, and viral loads. HIV-1 phylogenetic analysis revealed a broad viral diversity including CRF01_AE (62.13%), CRF07_BC (24.51%), subtype B (8.06%), CRF55_01B (3.24%), CER67_01B (0.95%), CRF68_01B (0.4%), CRF08_BC (0.08%) and CRF59_01B (0.08%). Twenty-four unique recombination forms (URFs) (1.98%) were identified as well. Bayesian inference analysis indicated that the introduction of CRF01_AE strain (1997) was earlier than CRF07_BC strain (2001) into MSM population in Shanghai based on the time of the most recent common ancestor (tMRCA). Three epidemic clusters and five sub-clusters were found in CRF01_AE. Significantly lower CD4+T cell count was found in individuals infected with CRF01_AE than in those infected with CRF07_BC infection (P<0.01), whereas viral load was significantly higher those infected with CRF01_AE than with CRF07_BC (P<0.01). In addition, the patients with >45 years of age were found to have lower CD4+T cell counts and higher viral loads than the patients with <25 years of age (P<0.05). This study reveals the presence of HIV-1 subtype diversity in Shanghai and its remarkable influence on clinical outcome. A real-time surveillance of HIV-1 viral diversity and phylodynamics of epidemic cluster, patient’s baseline CD4+T cell count and viral load would be of great value to monitoring of disease progression, intervention for transmission, improvement of antiretroviral therapy strategy and design of vaccines.     

TLR2-Modulating Lipoproteins of the Mycobacterium tuberculosis Complex Enhance the HIV Infectivity of CD4+ T Cells

Co-infection with Mycobacterium tuberculosis accelerates progression from HIV to AIDS. Our previous studies showed that M. tuberculosis complex, unlike M. smegmatis, enhances TLR2-dependent susceptibility of CD4+ T cells to HIV. The M. tuberculosis complex produces multiple TLR2-stimulating lipoproteins, which are absent in M. smegmatis. M. tuberculosis production of mature lipoproteins and TLR2 stimulation is dependent on cleavage by lipoprotein signal peptidase A (LspA). In order to determine the role of potential TLR2-stimulating lipoproteins on mycobacterial-mediated HIV infectivity of CD4+ T cells, we generated M. smegmatis recombinant strains overexpressing genes encoding various M. bovis BCG lipoproteins, as well as a Mycobacterium bovis BCG strain deficient in LspA (ΔlspA). Exposure of human peripheral blood mononuclear cells (PBMC) to M. smegmatis strains overexpressing the BCG lipoproteins, LprF (p<0.01), LprH (p<0.05), LprI (p<0.05), LprP (p<0.001), LprQ (p<0.005), MPT83 (p<0.005), or PhoS1 (p<0.05), resulted in increased HIV infectivity of CD4+ T cells isolated from these PBMC. Conversely, infection of PBMC with ΔlspA reduced HIV infectivity of CD4+ T cells by 40% relative to BCG-infected cells (p<0.05). These results may have important implications for TB vaccination programs in areas with high mother-to-child HIV transmission.     

Hierarchy Low CD4+/CD8+ T-Cell Counts and IFN-γ Responses in HIV-1+ Individuals Correlate with Active TB and/or M.tb Co-Infection

Objective

Detailed studies of correlation between HIV-M.tb co-infection and hierarchy declines of CD8+/CD4+ T-cell counts and IFN-γ responses have not been done. We conducted case-control studies to address this issue.

Methods

164 HIV-1-infected individuals comprised of HIV-1⁺ATB, HIV-1⁺LTB and HIV-1⁺TB^- groups were evaluated. Immune phenotyping and complete blood count (CBC) were employed to measure CD4+ and CD8+ T-cell counts; T.SPOT.TB and intracellular cytokine staining (ICS) were utilized to detect ESAT6, CFP10 or PPD-specific IFN-γ responses.

Results

There were significant differences in median CD4+ T-cell counts between HIV-1⁺ATB (164/μL), HIV-1⁺LTB (447/μL) and HIV-1⁺TB^- (329/μL) groups. Hierarchy low CD4+ T-cell counts (<200/μL, 200-500/μL, >500/μL) were correlated significantly with active TB but not M.tb co-infection. Interestingly, hierarchy low CD8+ T-cell counts were not only associated significantly with active TB but also with M.tb co-infection (P<0.001). Immunologically, HIV-1⁺ATB group showed significantly lower numbers of ESAT-6-/CFP-10-specific IFN-γ+ T cells than HIV-1⁺LTB group. Consistently, PPD-specific IFN-γ+CD4+/CD8+ T effector cells in HIV-1⁺ATB group were significantly lower than those in HIV-1⁺LTB group (P<0.001).

Conclusions

Hierarchy low CD8+ T-cell counts and effector function in HIV-1-infected individuals are correlated with both M.tb co-infection and active TB. Hierarchy low CD4+ T-cell counts and Th1 effector function in HIV-1+ individuals are associated with increased frequencies of active TB, but not M.tb co-infection.     

Renal Transplant Recipients Treated with Calcineurin-Inhibitors Lack Circulating Immature Transitional CD19+CD24hiCD38hi Regulatory B-Lymphocytes

BackgroundCD19⁺CD24^hiCD38^hi transitional immature B-lymphocytes have been demonstrated to play an important role in regulating the alloimmune response in transplant recipients. Here, we analyzed the effect of calcineurin inhibition on these peripherally circulating regulatory B-cells (Breg) in renal transplant recipients receiving cyclosporine A (CsA) or tacrolimus.MethodsPBMCs from healthy subjects (HS) (n = 16) and renal transplant recipients (n = 46) were isolated. Flow cytometry was performed for CD19, CD24, CD38 and IL-10 either after isolation or after 72 hours of co-culture in presence of PMA/Ionomycin and TLR9-ligand in presence or absence of increasing concentrations of tacrolimus or CsA.ResultsThe amount of CD19⁺ B-cells among lymphocytes was ∼9.1% in HS, ∼3.6% in CsA (n = 11, p<0.05) and ∼6.4% in TAC (n = 35, p<0.05) treated patients. Among B-cells, a distinct subset of Breg was found to be 4.7% in HS, 1.4% in tacrolimus treated patients and almost blunted in patients receiving CsA. Similarily, ∼4% of B-cells in HS and even fewer in CsA or tacrolimus treated patients produced IL-10 (0.5% and 1.5%, p<0.05) and this was confirmed both in non-transplanted CsA-treated healthy subjects and in in vitro co-culture experiments. Among 29 patients with <1% of Breg, 9 cases (31%) displayed an allograft rejection in contrast to only one case of rejection (6%) among 17 patients with >1%.ConclusionCalcineurin inhibitors reduce number and IL-10 production of Bregs in the peripheral circulation of both renal transplant recipients and non-transplanted healthy subjects. CNI induced Breg reduction is not restricted to a solid organ transplant setting and is not mediated by co-medication with steroids or MPA. A low proportion of Breg cells is associated with an elevated frequency of allograft rejection events.     

A Suitable Streptomycin-Resistant Mutant for Constructing Unmarked In-Frame Gene Deletions Using rpsL as a Counter-Selection Marker

The streptomycin counter-selection system is a useful tool for constructing unmarked in-frame gene deletions, which is a fundamental approach to study bacteria and their pathogenicity at the molecular level. A prerequisite for this system is acquiring a streptomycin-resistant strain due to rpsL mutations, which encodes the ribosomal protein S12. However, in this study no streptomycin resistance was found to be caused by rpsL mutations in all 127 clinical strains of Klebsiella pneumoniae isolated from liver abscess patients. By screening 107 spontaneous mutants of streptomycin resistance from a clinical strain of K. pneumoniae, nucleotide substitution or insertion located within the rpsL was detected in each of these strains. Thirteen different mutants with varied S12 proteins were obtained, including nine streptomycin-dependent mutants. The virulence of all four streptomycin-resistant mutants was further evaluated. Compared with the parental strain, the K42N, K42T and K87R mutants showed a reduction in growth rate, and the K42N and K42T mutants became susceptible to normal human serum. In the mice LD₅₀ (the bacterial dose that caused 50% death) assay, the K42N and K42T mutants were ∼1,000-fold less lethal (∼2×10⁵ CFU) and the K87R mutant was ∼50-fold less lethal (∼1×10⁴ CFU) than the parental strain (∼2×10² CFU). A K42R mutant showed non-observable effects on the above assays, while this mutant exhibited a small cost (P<0.01) in an in vitro growth competition experiment. In summary, most of the K. pneumoniae strains with streptomycin resistance caused by rpsL mutations are less virulent than their parental strain in the absence of streptomycin. The K42R mutant showed similar pathogenicity to its parental strain and should be one of the best choices when using rpsL as a counter-selection marker.     

Prevalence of Anal Human Papillomavirus Infection and Risk Factors among HIV-positive Patients in Tokyo,Japan

Background

Oncogenic human papillomavirus (HPV) infection, particularly multiple HPV types, is recognized as a necessary cause of anal cancer. However, a limited number of studies have reported the prevalence of anal HPV infection in Asia. We determined the prevalence, genotypes, and risk factors for anal HPV infection in Japanese HIV-positive men who have sex with men (MSM), heterosexual men, and women.

Methods

This cross-sectional study included 421 HIV-positive patients. At enrollment, we collected data on smoking, alcohol, co-morbidities, drugs, CD4 cell counts, HIV RNA levels, highly active anti-retroviral therapy (HAART) duration, sexually transmitted infections (STIs), and serological screening (syphilis, hepatitis B virus, Chlamydia trachomatis, Entamoeba histolytica). Anal swabs were collected for oncogenic HPV genotyping.

Results

Oncogenic HPV rate was 75.9% in MSM, 20.6% in heterosexual men, and 19.2% in women. HPV 16/18 types were detected in 34.9% of MSM, 17.7% of heterosexual men, and 11.5% of women. Multiple oncogenic HPV (≥2 oncogenic types) rate was 54.6% in MSM, 8.8% in heterosexual men, and 0% in women. In univariate analysis, younger age, male sex, MSM, CD4 <100, HIV viral load >50,000, no administration of HAART, and having ≥2 sexually transmitted infections (STIs) were significantly associated with oncogenic HPV infection, whereas higher smoking index and corticosteroid use were marginally associated with oncogenic HPV infection. In multivariate analysis, younger age (OR, 0.98 [0.96–0.99]), MSM (OR, 5.85 [2.33–14.71]), CD4 <100 (OR, 2.24 [1.00–5.01]), and having ≥2 STIs (OR, 2.81 [1.72–4.61]) were independently associated with oncogenic HPV infection. These 4 variables were also significant risk factors for multiple oncogenic HPV infection.

Conclusions

Among Japanese HIV-infected patients, approximately two-thirds of MSM, one-fifth of heterosexual men, and one-fifth of women have anal oncogenic HPV infection. Younger age, MSM, ≥2 STIs, and immunosuppression confer a higher risk of infection with oncogenic HPV and multiple oncogenic types.     

Quantitative Assessment of Intra-Patient Variation in CD4+ T Cell Counts in Stable,Virologically-Suppressed,HIV-Infected Subjects

Objectives

Counts of absolute CD4+ T lymphocytes (CD4+ T cells) are known to be highly variable in untreated HIV-infected individuals, but there are no data in virologically-suppressed individuals. We investigated CD4+ T cell variability in stable, virologically-suppressed, HIV-1 infected adults on combination antiretroviral therapy (cART).

Methods

From a large hospital database we selected patients with stable virological suppression on cART for >3 years with >10 CD4+ T cell measurements performed over a further >2 years; and a control group of 95 patients not on cART.

Results

We identified 161 HIV-infected patients on cART without active HCV or HBV infection, with stable virological suppression for a median of 6.4 years. Over the study period 88 patients had reached a plateau in their absolute CD4+ T cell counts, while 65 patients had increasing and 8 patients had decreasing absolute CD4+ T cell counts. In patients with plateaued CD4+ T cell counts, variability in absolute CD4+ T cell counts was greater than in percent CD4+ T cells (median coefficient of variation (CV) 16.6% [IQR 13.8-20.1%] and CV 9.6% [IQR 7.4-13.0%], respectively). Patients with increasing CD4+ T cell counts had greater variability in absolute CD4+ T cell counts than those with plateaued CD4 T cell counts (CV 19.5% [IQR 16.1-23.8%], p<0.001) while there was no difference in percent CD4+ T cell variability between the two groups. As previously reported, untreated patients had CVs significantly higher than patients on cART (CVs of 21.1% [IQR 17.2-32.0%], p<0.001 and 15.2% (IQR 10.7-20.0%), p<0.001, respectively). Age or sex did not affect the degree of CD4+ variation.

Conclusions

Adults with stable, virologically-suppressed HIV infection continue to have significant variations in individual absolute CD4+ T cell and percent CD4+ T cell counts; this variation can be of clinical relevance especially around CD4+ thresholds. However, the variation seen in individuals on cART is substantially less than in untreated subjects.     

Urine from Treated Cattle Drives Selection for Cephalosporin Resistant Escherichia coli in Soil

The U.S. Food and Drug Administration recently issued new rules for using ceftiofur in food animals in part because of an increasing prevalence of enteric bacteria that are resistant to 3^rd-generation cephalosporins. Parenteral ceftiofur treatment, however, has limited effects on enteric bacteria so we tested the hypothesis that excreted ceftiofur metabolites exert significant selection pressure for ceftiofur-resistant Escherichia coli in soil. Test matrices were prepared by mixing soil with bovine feces and adding urine containing ceftiofur metabolites (CFM) (0 ppm, ∼50 ppm and ∼100 ppm). Matrices were incubated at 23°C or 4°C for variable periods of time after which residual CFM was quantified using a bioassay. Bla _CMY-2 plasmid-bearing ceftiofur resistant (cef^R) E. coli and one-month old calves were used to study the selection effects of CFM and transmission of cef^R bacteria from the environment back to animals. Our studies showed that urinary CFM (∼13 ppm final concentration) is biologically degraded in soil within 2.7 days at 23°C, but persists up to 23.3 days at 4°C. Even short-term persistence in soil provides a >1 log₁₀ advantage to resistant E. coli populations, resulting in significantly prolonged persistence of these bacteria in the soil (∼two months). We further show that resistant strains readily colonize calves by contact with contaminated bedding and without antibiotic selection pressure. Ceftiofur metabolites in urine amplify resistant E. coli populations and, if applicable to field conditions, this effect is far more compelling than reported selection in vivo after parenteral administration of ceftiofur. Because ceftiofur degradation is temperature dependent, these compounds may accumulate during colder months and this could further enhance selection as seasonal temperatures increase. If cost-effective engineered solutions can be developed to limit ex vivo selection, this may limit proliferation for ceftiofur resistant enteric bacteria while preserving the ability to use this important antibiotic in food animal production.     

Estimating the Size of HIV Key Affected Populations in Chongqing,China, Using the Network Scale-Up Method

Objectives

To estimate the average social network size in the general population and the size of HIV key affected populations (KAPs) in Chongqing municipality using the network scale-up method (NSUM).

Methods

A general population survey was conducted in 2011 through a multistage random sampling method. Participants aged between 18 and 60 years were recruited. The average social network size (c) was estimated and adjusted by known population method. The size of HIV KAP in Chongqing municipality was estimated using the adjusted c value with adjustment for the transmission effect using the scaled respect factor.

Results

3,026 inhabitants of Chongqing agreed to the survey, and 2,957 (97.7%) completed the questionnaire. The adjusted c value was 310. The estimated size of KAP was 28,418(95% Confidence Interval (CI):26,636∼30,201) for female sex workers (FSW), 163,199(95%CI:156,490∼169,908) for clients of FSW, 37,959(95%CI: 34,888∼41,030) for drug users (DU), 14,975(95%CI:13,047∼16,904) for injecting drug users (IDU) and 16,767(95%CI:14,602∼18,932) for men who have sex with men (MSM). The ratio of clients to FSW was 5.74∶1, and IDU accounted for 39.5% of the DU population. The estimates suggest that FSW account for 0.37% of the female population aged 15–49 years in Chongqing, and clients of FSW and MSM represent 2.09% and 0.21% of the male population aged 15–49 years in the city, respectively.

Conclusion

NSUM provides reasonable population size estimates for FSW, their clients, DU and IDU in Chongqing. However, it is likely to underestimate the population size of MSM even after adjusting for the transmission effect.     

Role of Toll-Like Receptor 4 in Colorectal Carcinogenesis: A Meta-Analysis

Objective

This meta-analysis was performed to evaluate the role of toll-like receptor 4 (TLR-4) in colorectal carcinogenesis.

Methods

The PubMed, CISCOM, CINAHL, Web of Science, Google Scholar, EBSCO, Cochrane Library, and CBM databases were searched from inception through November 1st, 2013 without language restrictions. Odds ratios (ORs) or standardized mean differences (SMD) with their 95% confidence intervals (CI) were calculated.

Results

Fourteen case-control studies met the inclusion criteria for this meta-analysis. A total of 1,209 colorectal cancer (CRC) cases and 1,218 healthy controls were involved in this meta-analysis. Two common polymorphisms (299 A>G and 399 C>T) in the TLR-4 gene, TLR-4 mRNA and protein expression were assessed. Our meta-analysis results revealed that the TLR-4 399 C>T polymorphism might increase the risk of CRC (allele model: OR = 1.77, 95%CI = 1.32∼2.36, P<0.001; dominant model: OR = 1.83, 95%CI = 1.32∼2.52, P<0.001; respectively). However, we found no correlation between the TLR-4 299 A>G polymorphism and CRC risk (all P>0.05). A subgroup analysis by ethnicity suggested that TLR-4 genetic polymorphisms were associated with an increased risk of CRC among Asians (allele model: OR = 1.50, 95%CI = 1.19∼1.88, P = 0.001; dominant model: OR = 1.49, 95%CI = 1.16∼1.92, P = 0.002; respectively), but not among Caucasians and Africans (all P>0.05). Furthermore, our results showed that TLR-4 mRNA and protein levels in CRC patients were higher than those in healthy controls (TLR-4 mRNA: SMD  = 2.51, 95%CI  = 0.98∼4.05, P = 0.001; TLR-4 protein: OR  = 4.75, 95%CI  = 1.16∼19.36, P = 0.030; respectively).

Conclusion

Our findings provide empirical evidence that TLR-4 may play an important role in colorectal carcinogenesis. Thus, TLR-4 is a promising potential biomarker for the early diagnosis of CRC.     

Intestinal Parasitosis in Relation to CD4+T Cells Levels and Anemia among HAART Initiated and HAART Naive Pediatric HIV Patients in a Model ART Center in Addis Ababa,Ethiopia

BackgroundIntestinal parasites (IPs) are major concerns in most developing countries where HIV/AIDS cases are concentrated and almost 80% of AIDS patients die of AIDS-related infections. In the absence of highly active antiretroviral therapy (HAART), HIV/AIDS patients in developing countries unfortunately continue to suffer from the consequences of opportunistic and other intestinal parasites. The aim of the study was to determine the prevalence of intestinal parasites in relation to CD4+ T cells levels and anemia among HAART initiated and HAART naïve pediatric HIV patients in a Model ART center in Addis Ababa, Ethiopia.MethodsA prospective comparative cross-sectional study was conducted among HAART initiated and HAART naive pediatric HIV/AIDS patients attending a model ART center at Zewditu Memorial Hospital between August 05, 2013 and November 25, 2013. A total of 180 (79 HAART initiated and 101 HAART naïve) children were included by using consecutive sampling. Stool specimen was collected and processed using direct wet mount, formol-ether concentration and modified Ziehl-Neelsen staining techniques. A structured questionnaire was used to collect data on socio-demographic and associated risk factors. CD4+ T cells and complete blood counts were performed using BD FACScalibur and Cell-Dyn 1800, respectively. The data was analyzed by SPSS version 16 software. Logistic regressions were applied to assess any association between explanatory factors and outcome variables. P values < 0.05 were taken as statistically significant.ResultsThe overall prevalence of IPs was 37.8% where 27.8% of HAART initiated and 45.5% of HAART naive pediatric HIV/AIDS patients were infected (p < 0.05). Cryptosporidium species, E. histolytica/dispar, Hook worm and Taenia species were IPs associated with CD4+ T cell counts <350 cells/μμL in HAART naive patients. The overall prevalence of anemia was 10% in HAART and 31.7% in non-HAART groups. Hook worm, S. stercoralis and H. nana were helminthes significantly associated with anemia in non-HAART patients [AOR, 95% CI: 4.5(1.3, 15.2), P< 0.05]. The prevalence of IPs in non-HAART patients was significantly associated with eating unwashed/raw fruit [AOR, 95%CI: 6.3(1.2, 25.6), P<0.05], open field defecation [AOR, 95%CI: 9.3(1.6, 53.6), P<0.05] and diarrhea [AOR, 95%CI: 5.2(1.3, 21.3), P<0.05]. IPs significantly increased in rural residents [AOR, 95%CI: 0.4(0.1, 0.9, P<0.05)].ConclusionThe overall prevalence of intestinal parasites significantly differed by HAART status and cryptosporidium species were found only in HAART naïve patients with low CD4+ T cell counts. Anemia was also more prevalent and significantly associated with IPs in non-HAART patients. This study identified some environmental and associated risk factors for intestinal parasitic infections. Therefore, Public health measures should continue to emphasize the importance of environmental and personal hygiene to protect HIV/AIDS patients from infections with intestinal parasites and maximize the benefits of HAART.     

DR*W201/P65 Tetramer Visualization of Epitope-Specific CD4 T-Cell during M. tuberculosis Infection and Its Resting Memory Pool after BCG Vaccination

Background

In vivo kinetics and frequencies of epitope-specific CD4 T cells in lymphoid compartments during M. tuberculosis infection and their resting memory pool after BCG vaccination remain unknown.

Methodology/Findings

Macaque DR*W201 tetramer loaded with Ag85B peptide 65 was developed to directly measure epitope-specific CD4 T cells in blood and tissues form macaques after M. tuberculosis infection or BCG vaccination via direct staining and tetramer-enriched approach. The tetramer-based enrichment approach showed that P65 epitope-specific CD4 T cells emerged at mean frequencies of ∼500 and ∼4500 per 10⁷ PBL at days 28 and 42, respectively, and at day 63 increased further to ∼22,000/10⁷ PBL after M. tuberculosis infection. Direct tetramer staining showed that the tetramer-bound P65-specific T cells constituted about 0.2–0.3% of CD4 T cells in PBL, lymph nodes, spleens, and lungs at day 63 post-infection. 10-fold expansion of these tetramer-bound epitope-specific CD4 T cells was seen after the P65 peptide stimulation of PBL and tissue lymphocytes. The tetramer-based enrichment approach detected BCG-elicited resting memory P65-specific CD4 T cells at a mean frequency of 2,700 per 10⁷ PBL.

Significance

Our work represents the first elucidation of in vivo kinetics and frequencies for tetramer-bound epitope-specific CD4 T cells in the blood, lymphoid tissues and lungs over times after M. tuberculosis infection, and BCG immunization.     

Correlations of MTHFR 677C>T Polymorphism with Cardiovascular Disease in Patients with End-Stage Renal Disease: A Meta-Analysis

ObjectiveThis meta-analysis was conducted to evaluate the correlations of a common polymorphism (677C>T) in the methylenetetrahydrofolate reductase (MTHFR) gene with risk of cardiovascular disease (CVD) in patients with end-stage renal disease (ESRD).MethodThe following electronic databases were searched without language restrictions: Web of Science (1945∼2013), the Cochrane Library Database (Issue 12, 2013), MEDLINE (1966∼2013), EMBASE (1980∼2013), CINAHL (1982∼2013) and the Chinese Biomedical Database (CBM) (1982∼2013). Meta-analysis was performed using STATA statistical software. Odds ratios (ORs) with their 95% confidence intervals (95%CIs) were calculated.ResultsEight cohort studies met all inclusion criteria and were included in this meta-analysis. A total of 2,292 ESRD patients with CVD were involved in this meta-analysis. Our meta-analysis results revealed that the MTHFR 677C>T polymorphism might increase the risk of CVD in ESRD patients (TT vs. CC: OR = 2.75, 95%CI = 1.35∼5.59, P = 0.005; CT+TT vs. CC: OR = 1.39, 95%CI = 1.09∼1.78, P = 0.008; TT vs. CC+CT: OR = 2.52, 95%CI = 1.25∼5.09, P = 0.010; respectively). Further subgroup analysis by ethnicity suggested that the MTHFR 677C>T polymorphism was associated with an elevated risk for CVD in ESRD patients among Asians (TT vs. CC: OR = 3.38, 95%CI = 1.11∼10.28, P = 0.032; CT+TT vs. CC: OR = 1.44, 95%CI = 1.05∼1.97, P = 0.022; TT vs. CC+CT: OR = 3.15, 95%CI = 1.02∼9.72, P = 0.046; respectively), but not among Africans or Caucasians (all P>0.05).ConclusionOur findings indicate that the MTHFR 677C>T polymorphism may be associated with an elevated risk for CVD in ESRD patients, especially among Asians.     

Prime-Boost Vaccination with SA-4-1BBL Costimulatory Molecule and Survivin Eradicates Lung Carcinoma in CD8+ T and NK Cell Dependent Manner

Subunit vaccines containing universal tumor associated antigens (TAAs) present an attractive treatment modality for cancer primarily due to their safety and potential to generate long-term immunological responses that can safeguard against recurrences. However, TAA-based subunit vaccines require potent adjuvants for therapeutic efficacy. Using a novel form of the 4-1BBL costimulatory molecule, SA-4-1BBL, as the adjuvant of choice, we previously demonstrated that a single vaccination with survivin (SVN) as a bona fide self TAA was effective in eradicating weakly immunogenic 3LL tumors in >70% of C57BL/6 mice. The present study was designed to i) assess the therapeutic efficacy of a prime-boost vaccination and ii) investigate the mechanistic basis of vaccine efficacy. Our data shows that a prime-boost vaccination strategy was effective in eradicating 3LL lung carcinoma in 100% of mice. The vaccine efficacy was correlated with increased percentages of CD8⁺ T cells expressing IFN-γ as well as potent killing responses of both CD8⁺ T and NK cells in the absence of detectable antibodies to ssDNA as a sign of autoimmunity. Antibody depletion of CD8⁺ T cells one day before vaccination completely abrogated therapeutic efficacy, whereas depletion of CD4⁺ T cells had no effect. Importantly, NK cell depletion had a moderate (∼50% reduction), but significant (p<0.05) effect on vaccine efficacy. Taken together, these results shed light on the mechanistic basis of the SA-4-1BBL/SVN subunit vaccine formulation in a lung carcinoma model and demonstrate the robust therapeutic efficacy of the prime-boost immunization strategy with important clinical implications.     

Natural Polymorphisms in Tap2 Influence Negative Selection and CD4∶CD8 Lineage Commitment in the Rat

Genetic variation in the major histocompatibility complex (MHC) affects CD4∶CD8 lineage commitment and MHC expression. However, the contribution of specific genes in this gene-dense region has not yet been resolved. Nor has it been established whether the same genes regulate MHC expression and T cell selection. Here, we assessed the impact of natural genetic variation on MHC expression and CD4∶CD8 lineage commitment using two genetic models in the rat. First, we mapped Quantitative Trait Loci (QTLs) associated with variation in MHC class I and II protein expression and the CD4∶CD8 T cell ratio in outbred Heterogeneous Stock rats. We identified 10 QTLs across the genome and found that QTLs for the individual traits colocalized within a region spanning the MHC. To identify the genes underlying these overlapping QTLs, we generated a large panel of MHC-recombinant congenic strains, and refined the QTLs to two adjacent intervals of ∼0.25 Mb in the MHC-I and II regions, respectively. An interaction between these intervals affected MHC class I expression as well as negative selection and lineage commitment of CD8 single-positive (SP) thymocytes. We mapped this effect to the transporter associated with antigen processing 2 (Tap2) in the MHC-II region and the classical MHC class I gene(s) (RT1-A) in the MHC-I region. This interaction was revealed by a recombination between RT1-A and Tap2, which occurred in 0.2% of the rats. Variants of Tap2 have previously been shown to influence the antigenicity of MHC class I molecules by altering the MHC class I ligandome. Our results show that a restricted peptide repertoire on MHC class I molecules leads to reduced negative selection of CD8SP cells. To our knowledge, this is the first study showing how a recombination between natural alleles of genes in the MHC influences lineage commitment of T cells.     

Thermal Inactivation of Desiccation-Adapted Salmonella spp. in Aged Chicken Litter

Thermal inactivation of desiccation-adapted Salmonella spp. in aged chicken litter was investigated in comparison with that in a nonadapted control to examine potential cross-tolerance of desiccation-adapted cells to heat treatment. A mixture of four Salmonella serovars was inoculated into the finished compost with 20, 30, 40, and 50% moisture contents for a 24-h desiccation adaptation. Afterwards, the compost with desiccation-adapted cells was inoculated into the aged chicken litter with the same moisture content for heat treatments at 70, 75, 80, 85, and 150°C. Recovery media were used to allow heat-injured cells to resuscitate. A 5-log reduction in the number of the desiccation-adapted cells in aged chicken litter with a 20% moisture content required >6, >6, ∼4 to 5, and ∼3 to 4 h of exposure at 70, 75, 80, and 85°C, respectively. As a comparison, a 5-log reduction in the number of nonadapted control cells in the same chicken litter was achieved within ∼1.5 to 2, ∼1 to 1.5, ∼0.5 to 1, and <0.5 h at 70, 75, 80, and 85°C, respectively. The exposure time required to obtain a 5-log reduction in the number of desiccation-adapted cells gradually became shorter as temperature and moisture content were increased. At 150°C, desiccation-adapted Salmonella cells survived for 50 min in chicken litter with a 20% moisture content, whereas control cells were detectable by enrichment for only 10 min. Our results demonstrated that the thermal resistance of Salmonella in aged chicken litter was increased significantly when the cells were adapted to desiccation. This study also validated the effectiveness of thermal processing being used for producing chicken litter free of Salmonella contamination.     

Monocarboxylate Transporter 8 Modulates the Viability and Invasive Capacity of Human Placental Cells and Fetoplacental Growth in Mice

Monocarboxylate transporter 8 (MCT8) is a well-established thyroid hormone (TH) transporter. In humans, MCT8 mutations result in changes in circulating TH concentrations and X-linked severe global neurodevelopmental delay. MCT8 is expressed in the human placenta throughout gestation, with increased expression in trophoblast cells from growth-restricted pregnancies. We postulate that MCT8 plays an important role in placental development and transplacental TH transport. We investigated the effect of altering MCT8 expression in human trophoblast in vitro and in a Mct8 knockout mouse model. Silencing of endogenous MCT8 reduced T3 uptake into human extravillous trophoblast-like cells (SGHPL-4; 40%, P<0.05) and primary cytotrophoblast (15%, P<0.05). MCT8 over-expression transiently increased T3 uptake (SGHPL-4∶30%, P<0.05; cytotrophoblast: 15%, P<0.05). Silencing MCT8 did not significantly affect SGHPL-4 invasion, but with MCT8 over-expression T3 treatment promoted invasion compared with no T3 (3.3-fold; P<0.05). Furthermore, MCT8 silencing increased cytotrophoblast viability (∼20%, P<0.05) and MCT8 over-expression reduced cytotrophoblast viability independently of T3 (∼20%, P<0.05). In vivo, Mct8 knockout reduced fetal:placental weight ratios compared with wild-type controls at gestational day 18 (25%, P<0.05) but absolute fetal and placental weights were not significantly different. The volume fraction of the labyrinthine zone of the placenta, which facilitates maternal-fetal exchange, was reduced in Mct8 knockout placentae (10%, P<0.05). However, there was no effect on mouse placental cell proliferation in vivo. We conclude that MCT8 makes a significant contribution to T3 uptake into human trophoblast cells and has a role in modulating human trophoblast cell invasion and viability. In mice, Mct8 knockout has subtle effects upon fetoplacental growth and does not significantly affect placental cell viability probably due to compensatory mechanisms in vivo.     

Decreased memory T-cell response and function in human immunodeficiency virus-infected patients with tegumentary leishmaniasis

The effects of human immunodeficiency virus (HIV) on the immune response in patients with cutaneous leishmaniasis have not yet been fully delineated. This study quantified and evaluated the function of memory T-cell subsets in response to soluble Leishmania antigens (SLA) from patients coinfected with HIV and Leishmania with tegumentary leishmaniasis (TL). Eight TL/HIV coinfected subjects and 10 HIV seronegative subjects with TL were evaluated. The proliferative response of CD4+and CD8+T-cells and naïve, central memory (CM) and effector memory (EM) CD4+T-cells in response to SLA were quantified using flow cytometry. The median cell division indices for CD4+and CD8+T-cells of coinfected patients in response to SLA were significantly lower than those in patients with Leishmania monoinfection (p < 0.05). The proportions of CM and EM CD4+T-cells in response to SLA were similar between the coinfected patients and patients with Leishmania monoinfection. However, the median CM and EM CD4+T-cell counts from coinfected patients were significantly lower (p < 0.05). The reduction in the lymphoproliferative response to Leishmania antigens coincides with the decrease in the absolute numbers of both EM and CM CD4+T-cells in response to Leishmania antigens in patients coinfected with HIV/Leishmania.     

HLA Class-II Associated HIV Polymorphisms Predict Escape from CD4+ T Cell Responses

Antiretroviral therapy, antibody and CD8⁺ T cell-mediated responses targeting human immunodeficiency virus-1 (HIV-1) exert selection pressure on the virus necessitating escape; however, the ability of CD4⁺ T cells to exert selective pressure remains unclear. Using a computational approach on HIV gag/pol/nef sequences and HLA-II allelic data, we identified 29 HLA-II associated HIV sequence polymorphisms or adaptations (HLA-AP) in an African cohort of chronically HIV-infected individuals. Epitopes encompassing the predicted adaptation (AE) or its non-adapted (NAE) version were evaluated for immunogenicity. Using a CD8-depleted IFN-γ ELISpot assay, we determined that the magnitude of CD4⁺ T cell responses to the predicted epitopes in controllers was higher compared to non-controllers (p<0.0001). However, regardless of the group, the magnitude of responses to AE was lower as compared to NAE (p<0.0001). CD4⁺ T cell responses in patients with acute HIV infection (AHI) demonstrated poor immunogenicity towards AE as compared to NAE encoded by their transmitted founder virus. Longitudinal data in AHI off antiretroviral therapy demonstrated sequence changes that were biologically confirmed to represent CD4⁺ escape mutations. These data demonstrate an innovative application of HLA-associated polymorphisms to identify biologically relevant CD4⁺ epitopes and suggests CD4⁺ T cells are active participants in driving HIV evolution.