MHC class I A loci polymorphism and diversity in three Southeast Asian populations of cynomolgus macaque

Cynomolgus macaques (Macaca fascicularis, Mafa) have emerged as important animal models for biomedical research, necessitating a more extensive characterization of their major histocompatibility complex polymorphic regions. The current information on the polymorphism or diversity of the polygenetic Mafa class I A loci is limited in comparison to the more commonly studied rhesus macaque Mafa class I A loci. Therefore, in this paper, to better elucidate the degree and types of polymorphisms and genetic differences of Mafa-A1 among three native Southeast Asian populations (Indonesian, Vietnamese, and Filipino) and to investigate how the allele differences between macaques and humans might have evolved to affect their respective immune responses, we identified 83 Mafa-A loci-derived alleles by DNA sequencing of which 66 are newly described. Most alleles are unique to each population, but seven of the most frequent alleles were identical in sequence to some alleles in other macaque species. We also revealed (1) the large and dynamic genetic and structural differences and similarities in allelic variation by analyzing the population allele frequencies, Hardy-Weinberg’s equilibrium, heterozygosity, nucleotide diversity profiles, and phylogeny, (2) the difference in genetic structure of populations by Wright’s FST statistic and hierarchical analysis of molecular variance, and (3) the different demographic and selection pressures on the three populations by performing Tajima’s D test of neutrality. The large level of diversity and polymorphism at the Mafa-A1 was less evident in the Filipino than in the Vietnam or the Indonesian populations, which may have important implications in animal capture, selection, and breeding for medical research.     

HLA-B57/B*5801 Human Immunodeficiency Virus Type 1 Elite Controllers Select for Rare Gag Variants Associated with Reduced Viral Replication Capacity and Strong Cytotoxic T-Lymphotye Recognition

Human immunodeficiency virus type 1 (HIV-1) elite controllers (EC) maintain viremia below the limit of commercial assay detection (<50 RNA copies/ml) in the absence of antiviral therapy, but the mechanisms of control remain unclear. HLA-B57 and the closely related allele B*5801 are particularly associated with enhanced control and recognize the same Gag_240-249 TW10 epitope. The typical escape mutation (T242N) within this epitope diminishes viral replication capacity in chronically infected persons; however, little is known about TW10 epitope sequences in residual replicating viruses in B57/B*5801 EC and the extent to which mutations within this epitope may influence steady-state viremia. Here we analyzed TW10 in a total of 50 B57/B*5801-positive subjects (23 EC and 27 viremic subjects). Autologous plasma viral sequences from both EC and viremic subjects frequently harbored the typical cytotoxic T-lymphocyte (CTL)-selected mutation T242N (15/23 sequences [65.2%] versus 23/27 sequences [85.1%], respectively; P = 0.18). However, other unique mutants were identified in HIV controllers, both within and flanking TW10, that were associated with an even greater reduction in viral replication capacity in vitro. In addition, strong CTL responses to many of these unique TW10 variants were detected by gamma interferon-specific enzyme-linked immunospot assay. These data suggest a dual mechanism for durable control of HIV replication, consisting of viral fitness loss resulting from CTL escape mutations together with strong CD8 T-cell immune responses to the arising variant epitopes.A subset of human immunodeficiency virus type 1 (HIV-1)-infected persons who control viremia to below the limit of detection (<50 RNA copies/ml plasma) without antiviral therapy has been termed elite controllers/suppressors (EC) (2, 3, 6, 13, 32). Some of these individuals have been infected in excess of 30 years, indicating prolonged containment of HIV replication, but the mechanisms associated with this extreme viremia control remain elusive (13). Among EC, certain HLA class I alleles are overrepresented, in particular HLA-B57, strongly suggesting that HIV-1-specific cytotoxic T-lymphocyte (CTL) responses restricted by these alleles may be crucial for viremia control (16, 29, 32). However, to date, there has been no clear explanation as to why some subjects can control viremia but others cannot, even when carrying the same allegedly protective HLA alleles. Moreover, the characteristics of virus-specific immune responses as well as the impact of viral escape mutations on in vitro replicative fitness in persons with different disease outcomes remain unclear.Growing numbers of studies suggest that CTL targeting Gag, particularly the p24 capsid protein, play an important role in controlling viremia (7, 15, 22, 26, 32, 33, 38). Indeed, the most protective HLA class I allele, B57, which is present in over 40% of EC (32), restricts four immunodominant CTL epitopes in the p24 capsid protein. Previous studies have failed to find differences in the recognition of Gag epitopes or in gamma interferon (IFN-γ) responses to HIV proteins between B57-positive (B57⁺) long-term nonprogressors and B57⁺ progressors (28). Other studies have shown differences in the frequency of polyfunctional CD8⁺ T cells between B57⁺ EC and B57⁺ progressors (5); likewise, differences in the frequency of IFN-γ/interleukin-2-producing CD8⁺ T cells between controllers and progressors with protective HLA alleles were reported (16). Recently, Bailey et al. reported that plasma viruses in B57⁺ EC can harbor CTL escape mutations in the Gag protein, and in some cases these autologous variants were recognized by CTL (3). However, since there were no comparisons to progressors, it is unclear whether the viral variants that were detected or the apparent de novo CTL responses to the variant viruses are characteristic features among B57⁺ persons who maintain persistent control.Of the four immunodominant Gag CTL epitopes restricted by HLA-B57, TW10 (TSTLQEQIGW [Gag residues 240 to 249]) is known to be the earliest target in acute infection (1, 11, 36), therefore likely playing an important role in defining the plasma viral load set point. This epitope is also known to be presented by the closely related B*5801 allele, which is also associated with viral control (21). One of the most frequently detected mutations within this epitope, T242N, is known to occur rapidly and almost universally after acute infection in persons expressing HLA-B57/B*5801 (11, 17, 23). The same mutation has been shown to have a negative impact on viral replication capacity (VRC) by both clinical observation and in vitro experiments (8, 23, 25). Moreover, as plasma viral load increases, compensatory mutations accumulate, restoring VRC to some extent (8). Additional studies, predominantly with children, indicated that some TW10 escape variants may be targeted by specific immune responses (17). Together, these data suggest a hypothesis to explain the diverse disease courses among B57⁺ subjects, namely, that a combination of fitness cost by CTL escape from the TW10 response, variable accumulation of compensatory mutations, and variable generation of specific CTL responses to the new variant influence plasma viral loads.In this study, we investigated plasma viral sequences and IFN-γ-specific enzyme-linked immunospot (ELISPOT) assay responses to autologous Gag TW10 sequences in HLA-B57/B*5801-positive EC and compared these data to those obtained from persons with detectable viremia. Our results indicate that the TW10 T242N mutation does not differentiate HLA-B57/B*5801 EC from those with viremia and that CTL responses to this variant epitope are frequently detected in both viremic and aviremic subjects. However, some rare variants within and flanking this epitope were observed exclusively in HIV controllers, most of which not only reduced VRC but also were recognized by specific CTL at a high magnitude. These data suggest that the additive effects of both CTL-mediated selection for less fit viral variants and CD8 T-cell responses to the variant viruses contribute to strict viremia control in HLA-B57/B*5801-positive controllers.     

HLA-associated alterations in replication capacity of chimeric NL4-3 viruses carrying gag-protease from elite controllers of human immunodeficiency virus type 1

Human immunodeficiency virus type 1 (HIV-1)-infected persons who maintain plasma viral loads of <50 copies RNA/ml without treatment have been termed elite controllers (EC). Factors contributing to durable control of HIV in EC are unknown, but an HLA-dependent mechanism is suggested by overrepresentation of "protective" class I alleles, such as B*27, B*51, and B*57. Here we investigated the relative replication capacity of viruses (VRC) obtained from EC (n = 54) compared to those from chronic progressors (CP; n = 41) by constructing chimeric viruses using patient-derived gag-protease sequences amplified from plasma HIV RNA and inserted into an NL4-3 backbone. The chimeric viruses generated from EC displayed lower VRC than did viruses from CP (P < 0.0001). HLA-B*57 was associated with lower VRC (P = 0.0002) than were other alleles in both EC and CP groups. Chimeric viruses from B*57(+) EC (n = 18) demonstrated lower VRC than did viruses from B*57(+) CP (n = 8, P = 0.0245). Differences in VRC between EC and CP were also observed for viruses obtained from individuals expressing no described "protective" alleles (P = 0.0065). Intriguingly, two common HLA alleles, A*02 and B*07, were associated with higher VRC (P = 0.0140 and 0.0097, respectively), and there was no difference in VRC between EC and CP sharing these common HLA alleles. These findings indicate that cytotoxic T-lymphocyte (CTL) selection pressure on gag-protease alters VRC, and HIV-specific CTLs inducing escape mutations with fitness costs in this region may be important for strict viremia control in EC of HIV.     

Specific induction of a 72-kDa heat shock protein protects esophageal mucosa from reflux esophagitis

AimsThe aim of this study is to investigate the expression and cytoprotective function of a 72-kDa heat shock protein (HSP72) using a reflux esophagitis model in rats.Main methodsExpression of HSP60, HSP72, and HSP90 in rat esophageal mucosa was evaluated by Western blot analysis before and after hyperthermia (42.5 °C, 20 min). Rats received the operation to produce reflux esophagitis with or without pretreatment with hyperthermia to induce HSPs. The esophageal mucosal damage was evaluated 12 h after the operation.Key findingsExpression of HSP72 was significantly increased by hyperthermia in rat esophageal mucosa. Reflux esophagitis was dramatically prevented when HSP72 was preinduced by hyperthermia. Furthermore, activation of TNF-α and IL-1β in esophageal mucosa was also suppressed.SignificanceThese results suggested that hyperthermia protects the esophageal mucosa in reflux esophagitis model by inducing HSP72 and suppressing proinflammatory cytokine activation. These findings might suggest that HSP-inducing therapy could be a novel and unique therapy for reflux esophagitis.     

A novel and potent VLA-4 antagonist based on trans-4-substituted cyclohexanecarboxylic acid

During the course of our study, it was revealed that the poor pharmacokinetic properties of a series of benzoic acid derivatives such as 1 should be attributed to the diphenylurea moiety. Thus, we replaced the diphenylurea moiety in 1 with a 2-(2-methylphenylamino)benzoxazole moiety which mimics the diphenylurea structure. However, this modification resulted in a significant decrease (3, IC₅₀ = 19 nM) in VLA-4 inhibitory activity compared to 1 (IC₅₀ = 1.6 nM). To address this discrepancy, we worked on optimization of the carboxylic acid moiety in compound 3. As a result, our efforts have led to the discovery of trans-4-substituted cyclohexanecarboxylic acid derivative 11b (IC₅₀ = 2.8 nM) as a novel and potent VLA-4 antagonist. In addition, compound 11b exhibited favorable pharmacokinetic properties (CL = 3.3 ml/min/kg, F = 51%) in rats.     

Inhibitory effects of sesquiterpene lactones isolated from Eupatorium chinense L. on IgE-mediated degranulation in rat basophilic leukemia RBL-2H3 cells and passive cutaneous anaphylaxis reaction in mice

Sesquiterpene lactones (SQTLs) have been shown to suppress the degranulation as inferred by histamine release in rat basophilic leukemia RBL-2H3 cells. In this study, we isolated the 9 kinds of SQTLs from Eupatorium chinense L. and examined the effects of these SQTLs on the degranulation in RBL-2H3 cells. The chemical structures of two novel compounds (SQTL-3 and 8) were determined. All the SQTLs suppressed the degranulation from Ag-stimulated RBL-2H3 cells. To disclose the inhibitory mechanism of degranulation by SQTLs, we examined the activation of intracellular signaling molecules such as Lyn, Syk, and PLCγs and intracellular free Ca²⁺ concentration ([Ca²⁺]i). None of these SQTLs showed the activation of Syk and PLCγs. The intracellular free Ca²⁺ concentration ([Ca²⁺]i) was elevated by FcεRI activation, but SQTLs treatment reduced the elevation of [Ca²⁺]i by suppressing Ca²⁺ influx. Thus, it was suggested that the suppression of Ag-stimulated degranulation by these SQTLs is mainly due to the decreased Ca²⁺ influx.Furthermore, in order to clarify the in vivo effect of SQTL-rich extract, we administered SQTL-rich extract to the type I allergic model mice and measured the passive cutaneous anaphylaxis (PCA) reaction induced by IgE-antigen complex. The SQTLs remarkably suppressed PCA reaction in a dose-dependent manner. Thus, it was suggested that SQTLs would be a candidate as an anti-allergic agent.     

Varp Is a Novel Rab32/38-binding Protein That Regulates Tyrp1 Trafficking in Melanocytes

Two small GTPase Rabs, Rab32 and Rab38, have recently been proposed to regulate trafficking of melanogenic enzymes to melanosomes in mammalian epidermal melanocytes; however, the exact molecular mechanism of Rab32/38-mediated transport of melanogenic enzymes has never been clarified, because no Rab32/38-specific effector has ever been identified. In this study, we screened for a Rab32/38-specific effector by a yeast two-hybrid assay using a guanosine triphosphate (GTP)-locked Rab32/38 as bait and found that VPS9-ankyrin-repeat protein (Varp)/Ankrd27, characterized previously as a guanine nucleotide exchange factor (GEF) for Rab21, functions as a specific Rab32/38-binding protein in mouse melanocyte cell line melan-a. Deletion analysis showed that the first ankyrin-repeat (ANKR1) domain functions as a GTP-dependent Rab32/38-binding domain, but that the N-terminal VPS9 domain (i.e., Rab21-GEF domain) does not. Small interfering RNA-mediated knockdown of endogenous Varp in melan-a cells caused a dramatic reduction in Tyrp1 (tyrosinase-related protein 1) signals from melanosomes but did not cause any reduction in Pmel17 signals. Furthermore, expression of the ANKR1 domain in melan-a cells also caused a dramatic reduction of Tyrp1 signals, whereas the VPS9 domain had no effect. Based on these findings, we propose that Varp functions as the Rab32/38 effector that controls trafficking of Tyrp1 in melanocytes.     

Dimethylfumarate inhibits tumor cell invasion and metastasis by suppressing the expression and activities of matrix metalloproteinases in melanoma cells

NF-κB acts as a signal transducer during tumor progression, cell invasion, and metastasis. Dimethylfumarate (DMF) is reported to inhibit tumor necrosis factor-α-induced nuclear entry of NF-κB/p65. However, only a few reports suggest that DMF inhibits tumor metastasis; also the molecular mechanisms underlying the inhibition of metastasis are poorly understood. We investigated the inhibition of tumor invasion and metastasis by DMF in a melanoma cell line, B16BL6. DMF inhibited B16BL6 cell invasion and metastasis by suppressing the expression and activities of MMPs. DMF also inhibited the nuclear entry of NF-κB/p65, thus inhibiting B16BL6 cell invasion and metastasis. These results suggest that DMF is potentially useful as an anti-metastatic agent for the treatment of malignant melanoma.     

CERT-mediated trafficking of ceramide

The transport and sorting of lipids from the sites of their synthesis to their appropriate destinations are fundamental for membrane biogenesis. In the synthesis of sphingolipids in mammalian cells, ceramide is newly produced at the endoplasmic reticulum (ER), and transported from the ER to the trans Golgi regions, where it is converted to sphingomyelin. CERT mediates the ER-to-Golgi trafficking of ceramide. CERT contains several functional domains and motifs including i) a START domain capable of catalyzing inter-membrane transfer of ceramide, ii) a pleckstrin homology domain, which serves to target the Golgi apparatus, iii) a FFAT motif which interacts with the ER-resident membrane protein VAP, and iv) a serine-repeat motif, of which hyperphosphorylation down-regulates CERT activity. It has been suggested that CERT extracts ceramide from the ER and carries it to the Golgi apparatus in a non-vesicular manner and that efficient CERT-mediated trafficking of ceramide occurs at membrane contact sites between the ER and the Golgi apparatus.