Systemic overexpression of IL-10 induces CD4+CD25+ cell populations in vivo and ameliorates type 1 diabetes in nonobese diabetic mice in a dose-dependent fashion

Early systemic treatment of nonobese diabetic mice with high doses of recombinant adeno-associated virus (rAAV) vector expressing murine IL-10 prevents type 1 diabetes. To determine the therapeutic parameters and immunological mechanisms underlying this observation, female nonobese diabetic mice at 4, 8, and 12 wk of age were given a single i.m. injection of rAAV-murine IL-10 (10(4), 10(6), 10(8), and 10(9) infectious units (IU)), rAAV-vector expressing truncated murine IL-10 fragment (10(9) IU), or saline. Transduction with rAAV-IL-10 at 10(9) IU completely prevented diabetes in all animals injected at all time points, including, surprisingly, 12-wk-old animals. Treatment with 10(8) IU provided no protection in the 12-wk-old injected mice, partial prevention in 8-wk-old mice, and full protection in all animals injected at 4 wk of age. All other treatment groups developed diabetes at a similar rate. The rAAV-IL-10 therapy attenuated pancreatic insulitis, decreased MHC II expression on CD11b+ cells, increased the population of CD11b+ cells, and modulated insulin autoantibody production. Interestingly, rAAV-IL-10 therapy dramatically increased the percentage of CD4+CD25+ regulatory T cells. Adoptive transfer studies suggest that rAAV-IL-10 treatment alters the capacity of splenocytes to impart type 1 diabetes in recipient animals. This study indicates the potential for immunomodulatory gene therapy to prevent autoimmune diseases, including type 1 diabetes, and implicates IL-10 as a molecule capable of increasing the percentages of regulatory cells in vivo.     

Entry into the thymic microenvironment triggers Notch activation in the earliest migrant T cell progenitors

Interactions between T cell precursors and thymic stromal cells are essential during thymocyte development. However, the role of the thymus in initial commitment of lymphoid progenitors to the T lineage remains controversial, with data providing evidence for both extra- and intrathymic commitment mechanisms. In this context, it is clear that Notch1 is an important mediator during initiation of T cell development. Here we have analyzed the mechanisms regulating Notch activation in lymphoid precursors at extrathymic sites and in the thymus, including stages representing the first wave of embryonic thymus colonization on embryonic day 12 of gestation. We show that Notch activation in migrant lymphoid precursors requires entry into the thymic microenvironment where they are exposed to Notch ligands expressed by immature thymic epithelial cells. Moreover, continued Notch signaling in such precursors requires sustained interactions with Notch ligands. Collectively, these findings suggest a role for Notch in an intrathymic mechanism of T cell lineage commitment involving sustained interactions with Notch ligand bearing thymic epithelium.     

Human catecholamine sulfotransferase (SULT1A3) pharmacogenetics: functional genetic polymorphism

Sulfotransferase (SULT) 1A3 catalyzes the sulfate conjugation of catecholamines and structurally related drugs. As a step toward studies of the possible contribution of inherited variation in SULT1A3 to the pathophysiology of human disease and/or variation in response to drugs related to catecholamines, we have resequenced all seven coding exons, three upstream non-coding exons, exon-intron splice junctions and the 5'-flanking region of SULT1A3 using DNA samples from 60 African-American (AA) and 60 Caucasian-American (CA) subjects. Eight single nucleotide polymorphisms (SNPs) were observed in AA and five in CA subjects, including one non-synonymous cSNP (Lys234Asn) that was observed only in AA subjects with an allele frequency of 4.2%. This change in amino acid sequence resulted in only 28 +/- 4.5% (mean +/- SEM) of the enzyme activity of the wild-type (WT) sequence after transient expression in COS-1 cells, with a parallel decrease (54 +/- 2.2% of WT) in level of SULT1A3 immunoreactive protein. Substrate kinetic studies failed to show significant differences in apparent Km values of the two allozymes for either dopamine (10.5 versus 10.2 micro m for WT and variant, respectively) or the cosubstrate 3'-phosphoadenosine 5'-phosphosulfate (0.114 versus 0.122 micro m, respectively). The decrease in level of immunoreactive protein in response to this single change in amino acid sequence was due, at least in part, to accelerated SULT1A3 degradation through a proteasome-mediated process. These observations raise the possibility of ethnic-specific inherited alterations in catecholamine sulfation in humans.     

First report of a natural hybrid between Schistosoma mansoni and S. rodhaini

Experimental crosses between Schistosoma mansoni and S. rodhaini have shown that hybrid offspring are viable, yet, until now, no naturally occurring hybrid has been identified. A collection of freshwater snails from Nyamlebi-Ngoma, Ukerewe Island, Lake Victoria, Tanzania, yielded a mixed infection within a single Biomphalaria sudanica of S. mansoni females and S. mansoni-S. rodhaini hybrid males. The hybrids were identified using deoxyribonucleic acid (DNA) sequences. Mitochondrial DNA 16S and 12S sequences of the hybrids match those of S. mansoni, whereas their nuclear ribosomal DNA ITS1 and ITS2 sequences match those of S. rodhaini. The identification of hybrids in Tanzania highlights the possibility that the genetic identity of either parasite species might be modified by introgression.     

Induction of antigen-specific regulatory T cells following overexpression of a Notch ligand by human B lymphocytes

In mice, activation of the Notch pathway in T cells by antigen-presenting cells overexpressing Notch ligands favors differentiation of regulatory T lymphocytes responsible for antigen-specific tolerance. To determine whether this mechanism operates in human T cells, we used Epstein-Barr virus-positive lymphoblastoid cell lines (EBV-LCL) as our (viral) antigen-presenting cells and overexpressed the Notch ligand Jagged-1 (EBV-LCL J1) by adenoviral transduction. The EBV-LCL J1s were cocultured with autologous T cells, and the proliferative and cytotoxic responses to EBV antigens were measured. Transduction had no effect on EBV-LCL expression of major histocompatibility complex (MHC) antigens or of costimulatory molecules CD80, CD86, and CD40. However, we observed a 35% inhibition of proliferation and a >65% reduction in cytotoxic-T-cell activity, and interleukin 10 production was increased ninefold. These EBV-LCL J1-stimulated T lymphocytes act as antigen-specific regulatory cells, since their addition to fresh autologous T cells cultured with autologous nontransduced EBV-LCL cells significantly inhibited both proliferation and cytotoxic effector function. Within the inhibitory population, CD4(+)CD25(+) and CD8(+)CD25(-) T cells had the greatest activity. This inhibition appears to be antigen-specific, since responses to Candida and cytomegalovirus antigens were unaffected. Hence, transgenic expression of Jagged-1 by antigen-presenting cells can induce antigen-specific regulatory T cells in humans and modify immune responses to viral antigens.     

Identification of a new simian immunodeficiency virus lineage with a vpu gene present among different cercopithecus monkeys (C. mona, C. cephus, and C. nictitans) from Cameroon

During a large serosurvey of wild-caught primates from Cameroon, we found 2 mona monkeys (Cercopithecus mona) out of 8 and 47 mustached monkeys (Cercopithecus cephus) out of 302 with human immunodeficiency virus (HIV)-simian immunodeficiency virus (SIV) cross-reactive antibodies. In this report, we describe the full-length genome sequences of two novel SIVs, designated SIVmon-99CMCML1 and SIVmus-01CM1085, isolated from one mona (CML1) and one mustached (1085) monkey, respectively. Interestingly, these viruses displayed the same genetic organization (i.e., presence of a vpu homologue) as members of the SIVcpz-HIV type 1 lineage and SIVgsn isolated from greater spot-nosed monkeys (Cercopithecus nictitans). Phylogenetic analyses of SIVmon and SIVmus revealed that these viruses were genetically distinct from other known primate lentiviruses but were more closely related to SIVgsn all across their genomes, thus forming a monophyletic lineage within the primate lentivirus family, which we designated the SIVgsn lineage. Interestingly, mona, mustached, and greater spot-nosed monkeys are phylogenetically related species belonging to three different groups of the genus Cercopithecus, the C. mona, C. cephus, and Cercopithecus mitis groups, respectively. The presence of new viruses closely related to SIVgsn in two other species reinforces the hypothesis that a recombination event between ancestral SIVs from the family Cercopithecinae is the origin of the present SIVcpz that is widespread among the chimpanzee population.     

Enhanced detection of human immunodeficiency virus type 1-specific T-cell responses to highly variable regions by using peptides based on autologous virus sequences

The antigenic diversity of human immunodeficiency virus type 1 (HIV-1) represents a significant challenge for vaccine design as well as the comprehensive assessment of HIV-1-specific immune responses in infected persons. In this study we assessed the impact of antigen variability on the characterization of HIV-1-specific T-cell responses by using an HIV-1 database to determine the sequence variability at each position in all expressed HIV-1 proteins and a comprehensive data set of CD8 T-cell responses to a reference strain of HIV-1 in infected persons. Gamma interferon Elispot analysis of HIV-1 clade B-specific T-cell responses to 504 overlapping peptides spanning the entire expressed HIV-1 genome derived from 57 infected subjects demonstrated that the average amino acid variability within a peptide (entropy) was inversely correlated to the measured frequency at which the peptide was recognized (P = 6 x 10(-7)). Subsequent studies in six persons to assess T-cell responses against p24 Gag, Tat, and Vpr peptides based on autologous virus sequences demonstrated that 29% (12 of 42) of targeted peptides were only detected with peptides representing the autologous virus strain compared to the HIV-1 clade B consensus sequence. The use of autologous peptides also allowed the detection of significantly stronger HIV-1-specific T-cell responses in the more variable regulatory and accessory HIV-1 proteins Tat and Vpr (P = 0.007). Taken together, these data indicate that accurate assessment of T-cell responses directed against the more variable regulatory and accessory HIV-1 proteins requires reagents based on autologous virus sequences. They also demonstrate that CD8 T-cell responses to the variable HIV-1 proteins are more common than previously reported.     

Binding of adenovirus capsid to dipalmitoyl phosphatidylcholine provides a novel pathway for virus entry

Adenovirus (Ad) is an airborne, nonenveloped virus infecting respiratory epithelium. To study the mechanism of Ad entry, we used alveolar adenocarcinoma A549 cells, which have retained the ability of alveolar epithelial type II cells to synthesize the major component of pulmonary surfactant, disaturated phosphatidylcholine. Stimulation of phosphatidylcholine secretion by calcium ionophore or phorbol ester augmented the susceptibility of these cells to Ad. Both Ad infection and recombinant-Ad-mediated transfection increased in the presence of dipalmitoyl phosphatidylcholine (DPPC) liposomes in culture medium. Importantly, in the presence of DPPC liposomes, virus penetrates the cells independently of virus-specific protein receptors. DPPC vesicles bind Ad and are efficiently incorporated by A549 lung cells, serving as a virus vehicle during Ad penetration. To identify the viral protein(s) mediating Ad binding, a flotation of liposomes preincubated with structural viral proteins was employed, showing that the only Ad protein bound to DPPC vesicles was a hexon. The hexon preserved its phospholipid-binding properties upon purification, confirming its involvement in virus binding to the phospholipid. Given that disaturated phosphatidylcholine not only covers the inner surface of alveoli in the lungs but also reenters alveolar epithelium during lung surfactant turnover, Ad binding to this phospholipid may provide a pathway for virus entry into alveolar epithelium in vivo.     

Persistent replication of hepatitis C virus replicons expressing the beta-lactamase reporter in subpopulations of highly permissive Huh7 cells

Progress toward development of better therapies for the treatment of hepatitis C virus (HCV) infection has been hampered by poor understanding of HCV biology and the lack of biological assays suitable for drug screening. Here we describe a powerful HCV replication system that employs HCV replicons expressing the beta-lactamase reporter (bla replicons) and subpopulations of Huh7 cells that are more permissive (or "enhanced") to HCV replication than na?ve Huh7 cells. Enhanced cells represent a small fraction of permissive cells present among na?ve Huh7 cells that is enriched during selection with replicons expressing the neomycin phosphotransferase gene (neo replicons). The level of permissiveness of cell lines harboring neo replicons can vary greatly, and the enhanced phenotype is usually revealed upon removal of the neo replicon with inhibitors of HCV replication. Replicon removal is responsible for increased permissiveness, since this effect could be reproduced either with alpha interferon or with an HCV NS5B inhibitor. Moreover, adaptive mutations present in the replicon genome used during selection do not influence the permissiveness of the resulting enhanced-cell population, suggesting that the mechanisms governing the permissiveness of enhanced cells are independent from viral adaptation. Because the beta-lactamase reporter allows simultaneous quantitation of replicon-harboring cells and reporter activity, it was possible to investigate the relationship between genome replication activity and the frequency with which transfected genomes can establish persistent replication. Our study demonstrates that differences in the replication potential of the viral genome are manifested primarily in the frequency with which persistent replication is established but modestly affect the number of replicons observed per replicon-harboring cell. Replicon copy number was found to vary over a narrow range that may be defined by a minimal number required for persistent maintenance and a maximum that is limited by the availability of essential host factors.     

Partial molecular characterization of two simian immunodeficiency viruses (SIV) from African colobids: SIVwrc from Western red colobus (Piliocolobus badius) and SIVolc from olive colobus (Procolobus verus)

In order to study primate lentivirus evolution in the Colobinae subfamily, in which only one simian immunodeficiency virus (SIV) has been described to date, we screened additional species from the three different genera of African colobus monkeys for SIV infection. Blood was obtained from 13 West African colobids, and HIV cross-reactive antibodies were observed in 5 of 10 Piliocolobus badius, 1 of 2 Procolobus verus, and 0 of 1 Colobus polykomos specimens. Phylogenetic analyses of partial pol sequences revealed that the new SIVs were more closely related to each other than to the other SIVs and especially did not cluster with the previously described SIVcol from Colobus guereza. This study presents evidence that the three genera of African colobus monkeys are naturally infected with an SIV and indicates also that there was no coevolution between virus and hosts at the level of the Colobinae subfamily.