HLA-B63 presents HLA-B57/B58-restricted cytotoxic T-lymphocyte epitopes and is associated with low human immunodeficiency virus load

Several HLA class I alleles have been associated with slow human immunodeficiency virus (HIV) disease progression, supporting the important role HLA class I-restricted cytotoxic T lymphocytes (CTL) play in controlling HIV infection. HLA-B63, the serological marker for the closely related HLA-B*1516 and HLA-B*1517 alleles, shares the epitope binding motif of HLA-B57 and HLA-B58, two alleles that have been associated with slow HIV disease progression. We investigated whether HIV-infected individuals who express HLA-B63 generate CTL responses that are comparable in breadth and specificity to those of HLA-B57/58-positive subjects and whether HLA-B63-positive individuals would also present with lower viral set points than the general population. The data show that HLA-B63-positive individuals indeed mounted responses to previously identified HLA-B57-restricted epitopes as well as towards novel, HLA-B63-restricted CTL targets that, in turn, can be presented by HLA-B57 and HLA-B58. HLA-B63-positive subjects generated these responses early in acute HIV infection and were able to control HIV replication in the absence of antiretroviral treatment with a median viral load of 3,280 RNA copies/ml. The data support an important role of the presented epitope in mediating relative control of HIV replication and help to better define immune correlates of controlled HIV infection.     

Regulation of N-methyl-D-aspartate receptors by calpain in cortical neurons

The N-methyl-D-aspartate (NMDA) receptor is a cation channel highly permeable to calcium and plays critical roles in governing normal and pathologic functions in neurons. Calcium entry through NMDA receptors (NMDARs) can lead to the activation of the Ca2+-dependent protease, calpain. Here we investigated the involvement of calpain in regulation of NMDAR channel function. After prolonged (5-min) treatment with NMDA or glutamate, the whole-cell NMDAR-mediated current was significantly reduced in both acutely dissociated and cultured cortical pyramidal neurons. The down-regulation of NMDAR current was blocked by bath application of selective calpain inhibitors. Intracellular injection of a specific calpain inhibitory peptide also eliminated the down-regulation of NMDAR current induced by prolonged NMDA treatment. In contrast, dynamin inhibitory peptide had no effect on the depression of NMDAR current, suggesting the lack of involvement of dynamin/clathrin-mediated NMDAR internalization in this process. Immunoblotting analysis showed that the NR2A and NR2B subunits of NMDARs were markedly degraded in cultured cortical neurons treated with glutamate, and the degradation of NR2 subunits was prevented by calpain inhibitors. Taken together, our results suggest that prolonged activation of NMDARs in neurons activates calpain, and activated calpain in turn down-regulates the function of NMDARs, which provides a neuroprotective mechanism against NMDAR overstimulation accompanying ischemia and stroke.     

Altered Memory T-Cell Responses to Bacillus Calmette-Guerin and Tetanus Toxoid Vaccination and Altered Cytokine Responses to Polyclonal Stimulation in HIV-Exposed Uninfected Kenyan Infants

Implementation of successful prevention of mother-to-child transmission of HIV strategies has resulted in an increased population of HIV-exposed uninfected (HEU) infants. HEU infants have higher rates of morbidity and mortality than HIV-unexposed (HU) infants. Numerous factors may contribute to poor health in HEU infants including immunological alterations. The present study assessed T-cell phenotype and function in HEU infants with a focus on memory Th1 responses to vaccination. We compared cross-sectionally selected parameters at 3 and 12 months of age in HIV-exposed (n = 42) and HU (n = 28) Kenyan infants. We measured ex vivo activated and bulk memory CD4 and CD8 T-cells and regulatory T-cells by flow cytometry. In addition, we measured the magnitude, quality and memory phenotype of antigen-specific T-cell responses to Bacillus Calmette-Guerin and Tetanus Toxoid vaccine antigens, and the magnitude and quality of the T cell response following polyclonal stimulation with staphylococcal enterotoxin B. Finally, the influence of maternal disease markers on the immunological parameters measured was assessed in HEU infants. Few perturbations were detected in ex vivo T-cell subsets, though amongst HEU infants maternal HIV viral load positively correlated with CD8 T cell immune activation at 12 months. Conversely, we observed age-dependent differences in the magnitude and polyfunctionality of IL-2 and TNF-α responses to vaccine antigens particularly in Th1 cells. These changes mirrored those seen following polyclonal stimulation, where at 3 months, cytokine responses were higher in HEU infants compared to HU infants, and at 12 months, HEU infant cytokine responses were consistently lower than those seen in HU infants. Finally, reduced effector memory Th1 responses to vaccine antigens were observed in HEU infants at 3 and 12 months and higher central memory Th1 responses to M. tuberculosis antigens were observed at 3 months only. Long-term monitoring of vaccine efficacy and T-cell immunity in this vulnerable population is warranted.     

Differential distribution of HP1 proteins after trichostatin a treatment influences chromosomal stability in HCT116 and WI-38 cells

Background

Heterochromatin protein 1 (HP1) is important in the establishment, propagation, and maintenance of constitutive heterochromatin, especially at the pericentromeric region. HP1 might participate in recruiting and directing Mis12 to the centromere during interphase, and HP1 disruption or abrogation might lead to the loss of Mis12 incorporation into the kinetochore. Therefore, the centromere structure and kinetochore relaxation that are promoted in the absence of Mis12 could further induce chromosome instability (CIN) by reducing the capacity of the kinetochore to anchor microtubules. The aim of this study was to determine whether alterations in the localization of HP1 proteins induced by trichostatin A (TSA) modify Mis12 and Centromere Protein A (CENP-A) recruitment to the centromere and whether changes in the expression of HP1 proteins and H3K9 methylation at centromeric chromatin increase CIN in HCT116 and WI-38 cells.

Methods

HCT116 and WI-38 cells were cultured and treated with TSA to evaluate CIN after 24 and 48 h of exposure. Immunofluorescence, Western blot, ChIP, and RT-PCR assays were performed in both cell lines to evaluate the localization and abundance of HP1α/β, Mis12, and CENP-A and to evaluate chromatin modifications during interphase and mitosis, as well as after 24 and 48 h of TSA treatment.

Results

Our results show that the TSA-induced reduction in heterochromatic histone marks on centromeric chromatin reduced HP1 at the centromere in the non-tumoral WI-38 cells and that this reduction was associated with cell cycle arrest and CIN. However, in HCT116 cells, HP1 proteins, together with MIS12 and CENP-A, relocated to centromeric chromatin in response to TSA treatment, even after H3K9me3 depletion in the centromeric nucleosomes. The enrichment of HP1 and the loss of H3K9me3 were associated with an increase in CIN, suggesting a response mechanism at centromeric and pericentromeric chromatin that augments the presence of HP1 proteins in those regions, possibly ensuring chromosome segregation despite serious CIN. Our results provide new insight into the epigenetic landscape of centromeric chromatin and the role of HP1 proteins in CIN.

     

Fetal Growth and Risk of Stillbirth: A Population-Based Case–Control Study

Background

Stillbirth is strongly related to impaired fetal growth. However, the relationship between fetal growth and stillbirth is difficult to determine because of uncertainty in the timing of death and confounding characteristics affecting normal fetal growth.

Methods and Findings

We conducted a population-based case–control study of all stillbirths and a representative sample of live births in 59 hospitals in five geographic areas in the US. Fetal growth abnormalities were categorized as small for gestational age (SGA) (<10th percentile) or large for gestational age (LGA) (>90th percentile) at death (stillbirth) or delivery (live birth) using population, ultrasound, and individualized norms. Gestational age at death was determined using an algorithm that considered the time-of-death interval, postmortem examination, and reliability of the gestational age estimate. Data were weighted to account for the sampling design and differential participation rates in various subgroups. Among 527 singleton stillbirths and 1,821 singleton live births studied, stillbirth was associated with SGA based on population, ultrasound, and individualized norms (odds ratio [OR] [95% CI]: 3.0 [2.2 to 4.0]; 4.7 [3.7 to 5.9]; 4.6 [3.6 to 5.9], respectively). LGA was also associated with increased risk of stillbirth using ultrasound and individualized norms (OR [95% CI]: 3.5 [2.4 to 5.0]; 2.3 [1.7 to 3.1], respectively), but not population norms (OR [95% CI]: 0.6 [0.4 to 1.0]). The associations were stronger with more severe SGA and LGA (<5th and >95th percentile). Analyses adjusted for stillbirth risk factors, subset analyses excluding potential confounders, and analyses in preterm and term pregnancies showed similar patterns of association. In this study 70% of cases and 63% of controls agreed to participate. Analysis weights accounted for differences between consenting and non-consenting women. Some of the characteristics used for individualized fetal growth estimates were missing and were replaced with reference values. However, a sensitivity analysis using individualized norms based on the subset of stillbirths and live births with non-missing variables showed similar findings.

Conclusions

Stillbirth is associated with both growth restriction and excessive fetal growth. These findings suggest that, contrary to current practices and recommendations, stillbirth prevention strategies should focus on both severe SGA and severe LGA pregnancies. Please see later in the article for the Editors'' Summary     

Reference Values for Clinical Laboratory Parameters in Young Adults in Maputo,Mozambique

Background

Clinical laboratory reference values from North American and European populations are currently used in most Africans countries due to the absence of locally derived reference ranges, despite previous studies reporting significant differences between populations. Our aim was to define reference ranges for both genders in 18 to 24 year-old Mozambicans in preparation for clinical vaccine trials.

Methods

A cross-sectional study including 257 volunteers (102 males and 155 females) between 18 and 24 years was performedat a youth clinic in Maputo, Mozambique. All volunteers were clinically healthy and human immunodeficiency virus, Hepatitis B virus and syphilis negative.Median and 95% reference ranges were calculated for immunological, hematological and chemistry parameters. Ranges were compared with those reported based on populations in other African countries and the US. The impact of applying US NIH Division of AIDS (DAIDS) toxicity tables was assessed.

Results

The immunology ranges were comparable to those reported for the US and western Kenya.There were significant gender differences in CD4⁺ T cell values 713 cells/µL in males versus 824 cells/µL in females (p<0.0001). Hematologic values differed from the US values but were similar to reports of populations in western Kenya and Uganda. The lower and upper limits of the ranges for hemoglobin, hematocrit, red blood cells, white blood cells and lymphocytes were somewhat lower than those from these African countries. The chemistry values were comparable to US values, with few exceptions. The upper limits for ALT, AST, bilirubin, cholesterol and triglycerides were higher than those from the US. DAIDStables for adverse events predicted 297 adverse events and 159 (62%) of the volunteers would have been excluded.

Conclusion

This study is the first to determine normal laboratory parameters in Mozambique. Our results underscore the necessity of establishing region-specific clinical reference ranges for proper patient management and safe conduct of clinical trials.     

Functional expression of human prostaglandin E2 receptor 4 (EP4) in E. coli and characterization of the binding property of EP4 with Gα proteins

Human prostaglandin E2 receptor 4 (EP4) is one of the four subtypes of prostaglandin E₂ (PGE₂) receptors and belongs to the rhodopsin-type G protein-coupled receptor (GPCR) family. Particularly, EP4 is expressed in various cancer cells and is involved in cancer-cell proliferation by a G protein signaling cascade. To prepare an active form of EP4 for biochemical characterization and pharmaceutical application, this study designed a recombinant protein comprising human EP4 fused to the P9 protein (a major envelope protein of phi6 phage) and overexpressed the P9-EP4 fusion protein in the membrane fraction of E. coli. The solubilized P9-EP4 with sarkosyl (a strong anionic detergent) was purified by affinity chromatography. The purified protein was stabilized with amphiphilic polymers derived from poly-γ-glutamate. The polymer-stabilized P9-EP4 showed specific interaction with the alpha subunits of G_s or G_i proteins, and a high content of α-helical structure by a circular dichroism spectroscopy. Furthermore, the polymer-stabilized P9-EP4 showed strong heat resistance compared with P9-EP4 in detergents. The functional preparation of EP4 and its stabilization with amphiphilic polymers could facilitate both the biochemical characterization and pharmacological applications targeting EP4.     

Sir2 deletion prevents lifespan extension in 32 long-lived mutants

Activation of Sir2 orthologs is proposed to increase lifespan downstream of dietary restriction. Here, we describe an examination of the effect of 32 different lifespan-extending mutations and four methods of DR on replicative lifespan (RLS) in the short-lived sir2Δ yeast strain. In every case, deletion of SIR2 prevented RLS extension; however, RLS extension was restored when both SIR2 and FOB1 were deleted in several cases, demonstrating that SIR2 is not directly required for RLS extension. These findings indicate that suppression of the sir2Δ lifespan defect is a rare phenotype among longevity interventions and suggest that sir2Δ cells senesce rapidly by a mechanism distinct from that of wild-type cells. They also demonstrate that failure to observe lifespan extension in a short-lived background, such as cells or animals lacking sirtuins, should be interpreted with caution.     

Complex of Fas-associated Factor 1 (FAF1) with Valosin-containing Protein (VCP)-Npl4-Ufd1 and Polyubiquitinated Proteins Promotes Endoplasmic Reticulum-associated Degradation (ERAD)

Fas-associated factor 1 (FAF1) is a ubiquitin receptor containing multiple ubiquitin-related domains including ubiquitin-associated (UBA), ubiquitin-like (UBL) 1, UBL2, and ubiquitin regulatory X (UBX). We previously showed that N-terminal UBA domain recognizes Lys⁴⁸-ubiquitin linkage to recruit polyubiquitinated proteins and that a C-terminal UBX domain interacts with valosin-containing protein (VCP). This study shows that FAF1 interacts only with VCP complexed with Npl4-Ufd1 heterodimer, a requirement for the recruitment of polyubiquitinated proteins to UBA domain. Intriguingly, VCP association to C-terminal UBX domain regulates ubiquitin binding to N-terminal UBA domain without direct interaction between UBA and UBX domains. These interactions are well characterized by structural and biochemical analysis. VCP-Npl4-Ufd1 complex is known as the machinery required for endoplasmic reticulum-associated degradation. We demonstrate here that FAF1 binds to VCP-Npl4-Ufd1 complex via UBX domain and polyubiquitinated proteins via UBA domain to promote endoplasmic reticulum-associated degradation.