HLA-B*57 Micropolymorphism shapes HLA allele-specific epitope immunogenicity, selection pressure, and HIV immune control

The genetic polymorphism that has the greatest impact on immune control of human immunodeficiency virus (HIV) infection is expression of HLA-B*57. Understanding of the mechanism for this strong effect remains incomplete. HLA-B*57 alleles and the closely related HLA-B*5801 are often grouped together because of their similar peptide-binding motifs and HIV disease outcome associations. However, we show here that the apparently small differences between HLA-B*57 alleles, termed HLA-B*57 micropolymorphisms, have a significant impact on immune control of HIV. In a study cohort of >2,000 HIV C-clade-infected subjects from southern Africa, HLA-B*5703 is associated with a lower viral-load set point than HLA-B*5702 and HLA-B*5801 (medians, 5,980, 15,190, and 19,000 HIV copies/ml plasma; P = 0.24 and P = 0.0005). In order to better understand these observed differences in HLA-B*57/5801-mediated immune control of HIV, we undertook, in a study of >1,000 C-clade-infected subjects, a comprehensive analysis of the epitopes presented by these 3 alleles and of the selection pressure imposed on HIV by each response. In contrast to previous studies, we show that each of these three HLA alleles is characterized both by unique CD8(+) T-cell specificities and by clear-cut differences in selection pressure imposed on the virus by those responses. These studies comprehensively define for the first time the CD8(+) T-cell responses and immune selection pressures for which these protective alleles are responsible. These findings are consistent with HLA class I alleles mediating effective immune control of HIV through the number of p24 Gag-specific CD8(+) T-cell responses generated that can drive significant selection pressure on the virus.     

Synthesis and structure–activity relationship of a novel series of heterocyclic sulfonamide γ-secretase inhibitors

γ-Secretase inhibitors have been shown to reduce the production of β-amyloid, a component of the plaques that are found in brains of patients with Alzheimer’s disease. A novel series of heterocyclic sulfonamide γ-secretase inhibitors that reduce β-amyloid levels in cells is reported. Several examples of compounds within this series demonstrate a higher propensity to inhibit the processing of amyloid precursor protein compared to Notch, an alternative γ-secretase substrate.     

(S)-N-(5-Chlorothiophene-2-sulfonyl)-β,β-diethylalaninol a Notch-1-sparing γ-secretase inhibitor

Accumulation of beta-amyloid (Aβ), produced by the proteolytic cleavage of amyloid precursor protein (APP) by β- and γ-secretase, is widely believed to be associated with Alzheimer’s disease (AD). Research around the high-throughput screening hit (S)-4-chlorophenylsulfonyl isoleucinol led to the identification of the Notch-1-sparing (9.5-fold) γ-secretase inhibitor (S)-N-(5-chlorothiophene-2-sulfonyl)-β,β-diethylalaninol 7.b.2 (Aβ _40/42 EC₅₀ = 28 nM), which is efficacious in reduction of Aβ production in vivo.     

Bovine viral diarrhea virus (BVDV) associated with single in vivo-derived and in vitro-produced preimplantation bovine embryos following artificial exposure

The objective was to determine the average amount of bovine viral diarrhea virus (BVDV) associated with single in vivo-derived and in vitro-produced bovine embryos following recommended processing procedures for embryos. In vivo-derived and in vitro-produced bovine embryos at 7 d post-fertilization were exposed (for 2 h) to 2 × 10^5-7 cell culture infective dose (CCID₅₀)/mL of SD-1 (a noncytopathic, Type 1a strain of BVDV), and then washed according to International Embryo Transfer Society (IETS) guidelines prior to testing. Of the 87 in vivo-derived embryos tested, 27% were positive for virus by quantitative polymerase chain reaction (qPCR). The range in amount of virus associated with 99% of the contaminated embryos was ≤6.62 ± 1.57 copies/5 μL; 90% of the contaminated embryos had ≤4.64 ± 1.57 viral copies/5 μL of embryo-associated virus, using tolerance intervals (P < 0.05). The SEM was 0.33 and the mean of averages was 1.12/5 μL. Of the 87 in vitro-produced embryos, 42% were positive for virus. The range in amount of virus associated with 99% of the contaminated embryos was ≤3.44 ± 0.89 copies/5 μL; 90% of the contaminated embryos had ≤2.40 ± 0.89 viral copies/5 μL of embryo-associated virus using tolerance intervals (P < 0.05; S.E.M. was 0.14 and the mean of averages was 0.55/5 μL). Therefore, although many embryos were positive for virus, there were limited numbers of copies, thereby posing doubt regarding their potential for contamination following embryo transfer.     

Washing and trypsin treatment of in vitro derived bovine embryos exposed to bovine viral diarrhea virus

Gametes, somatic cells and materials of animal origin in media are potential sources for introducing bovine viral diarrhea virus (BVDV) into systems for production of IVF bovine embryos. Further, the efficacy of washing and trypsin treatment for removal of BVDV from IVF embryos is questionable. Washing and trypsin treatments recommended by the International Embryo Transfer Society for in vivo-derived embryos were applied to in vitro-derived, virus-exposed, bovine embryos in this side-by-side comparison of treatments. Embryos for the study were produced in a virus-free system in which follicular oocytes were matured and fertilized in vitro and presumptive zygotes were co-cultured with bovine uterine tubal cells for 7 d. A total of 18 trials was performed, 9 using a noncytopathic BVDV and 9 using a cytopathic BVDV. In each trial, 4 equal groups of 10 or less, zona pellucida-intact embryos/ova were assembled, including 2 groups of morulae and blastocysts (M/B) and 2 groups of nonfertile or degenerated ova (NFD). Each group was prewashed and exposed to 10(4) to 10(6) TCID50/mL of either noncytopathic (SD-1) or cytopathic (NADL) BVDV for 2 h. Following in vitro viral exposure, one group of M/B and one group of NFD were washed. The other groups of M/B and NFD were trypsin-treated. Both treatments were consistent with IETS guidelines. After in vitro exposure to noncytopathic BVDV and washing, viral assays of 100% (9/9) and 78% (7/9) of the groups of M/B and NFD ova, respectively, were positive. After in vitro exposure to cytopathic BVDV and washing, viral assay of 33% (3/9) of the groups of both M/B and NFD ova were positive. After in vitro exposure to noncytopathic BVDV and trypsin treatment, viral assay of 44% (4/9) of groups of M/B and 67% (6/9) of groups of NFD ova were positive. Finally, after in vitro exposure to cytopathic BVDV and trypsin treatment, viral assay of 22% (2/9) of the groups of M/B and 44% (4/9) of the groups of NFD ova were positive. Contingency table analysis, in which data was stratified by embryo type and virus biotype, was used to compare results. While a difference existed between results of the 2 treatments of groups of M/B within the noncytopathic biotype (P = 0.01, Mantel Haenszel Chi-square), no difference was observed between comparison of treatment between all groups in both biotypes (P > 0.05).     

Structure, function, and motility of vacuoles in filamentous fungi

Current information on the structure and function of motile tubular vacuoles in Pisolithus tinctorius and other fungi is reviewed. The use of fluorochromes to label the vacuole lumen is evaluated and observations on the structure and motility of vacuoles in P. tinctorius are differentiated from possible artifacts. The styryl dyes FM4-64 and MDY-64, used in yeast to demonstrate endocytosis, show little or no labeling of internal membranes in undamaged P. tinctorius cells. This agrees with our data showing that other probes for endocytosis such as Lucifer yellow CH are not taken up by hyphal tip cells. Overall, the observations do not support endocytosis in hyphal tips. It has been suggested that tubular vacuole systems carry out longitudinal transport, and evidence in favor of this hypothesis is evaluated. New data are presented to show that many of the large vacuoles in subapical cells are attached to the plasma membrane and are relatively immobile, while video sequences show movement of fluorochrome in pulses along a series of several large vacuoles, all interconnected via tubules. Tubular vacuoles from thick sections of hyphae processed under anhydrous conditions are shown by X-ray microanalysis to contain relatively high levels of P and K, as seen previously in the larger vacuoles. These results provide further evidence for a role of the tubular vacuoles in longitudinal transport of P. Copyright 1998 Academic Press.     

Rapid and sensitive GC/MS characterization of glycolipid released Galalpha1,3Gal-terminated oligosaccharides from small organ specimens of a single pig

Pig to human xenotransplantation is considered a possible solution to the prevailing chronic lack of human donor organs for allotransplantation. The Galalpha1,3Gal determinant is the major porcine xenogeneic epitope causing hyperacute rejection following human antibody binding and complement activation. In order to characterize the tissue distribution of Galalpha1,3Gal-containing and blood group- type glycosphingolipids in pig, acid and nonacid glycosphingolipids were isolated from the kidney, small intestine, spleen, salivary gland, liver, and heart of a single pig obtained from a semi-inbred strain homozygous at the SLA locus. Glycolipids were analyzed by thin-layer immunostaining using monoclonal antibodies, and following ceramide glycanase cleavage as permethylated oligosaccharides by gas chromatography, gas chromatography-mass spectrometry, and matrix- assisted laser desorption/ionization mass spectrometry. The kidney contained large amounts of Galalpha1,3Gal-containing penta- and hexasaccharides having carbohydrate sequences consistent with the Galalpha1,3nLc4and Galalpha1,3Lexstructures, respectively. The former structure was tentatively identified in all organs by GC/MS. The presence of extended Galalpha1,3Gal-terminated structures in the kidney and heart was suggested by antibody binding, and GC/MS indicated the presence of a Galalpha1,3nLc6structure in the heart. The kidney, spleen, and heart contained blood group H pentaglycosylceramides based on type 1 (H-5-1) and type 2 (H-5-2) chains, and H hexaglycosylceramides based on the type 4 chain (H-6-4). In the intestine H-5-1 and H-6-4 were expressed, in the salivary gland H-5-1 and H-5-2, whereas only the H-5-1 structure was identified in the liver. Blood group A structures were identified in the salivary gland and the heart by antibody binding and GC/MS, indicating an organ- specific expression of blood group AH antigens in the pig.