Successful immunotherapy of autoimmune cholangitis by adoptive transfer of forkhead box protein 3(+) regulatory T cells.

Treatment of primary biliary cirrhosis (PBC) has lagged behind that of other autoimmune diseases. In this study we have addressed the potential utility of immunotherapy using regulatory T cells (Treg ) to treat murine autoimmune cholangitis. In particular, we have taken advantage of our ability to produce portal inflammation and bile duct cell loss by transfer of CD8(+) T cells from the dominant negative form of transforming growth factor beta receptor type II (dnTGF-ßRII) mice to recombination-activating gene (Rag)1(-/-) recipients. We then used this robust established adoptive transfer system and co-transferred CD8(+) T cells from dnTGF-ßRII mice with either C57BL/6 or dnTGF-ßRII forkhead box protein 3 (FoxP3(+) ) T cells. Recipient mice were monitored for histology, including portal inflammation and intralobular biliary cell damage, and also included a study of the phenotypical changes in recipient lymphoid populations and local and systemic cytokine production. Importantly, we report herein that adoptive transfer of Treg from C57BL/6 but not dnTGF-ßRII mice significantly reduced the pathology of autoimmune cholangitis, including decreased portal inflammation and bile duct damage as well as down-regulation of the secondary inflammatory response. Further, to define the mechanism of action that explains the differential ability of C57BL/6 Treg versus dnTGF-ßRII Treg on the ability to down-regulate autoimmune cholangitis, we noted significant differential expression of glycoprotein A repetitions predominant (GARP), CD73, CD101 and CD103 and a functionally significant increase in interleukin (IL)-10 in Treg from C57BL/6 compared to dnTGF-ßRII mice. Our data reflect the therapeutic potential of wild-type CD4(+) FoxP3(+) Treg in reducing the excessive T cell responses of autoimmune cholangitis, which has significance for the potential immunotherapy of PBC.

Enhanced luminescence of CaMoO4:Eu by core@shell formation and its hyperthermia study after hybrid formation with Fe3O4: cytotoxicity assessment on human liver cancer cells and mesenchymal stem cells.

Highly water dispersible Eu³âº doped CaMoO4 nanoparticles (core) covered by CaMoO4 (shell) have been prepared using the polyol method. Significant enhancement in luminescence intensity by core@shell formation is observed due to the decrease of non-radiative rate arising from surface/defect of particles. Effect of 266 nm laser excitation (Mo-O charge transfer band) on the asymmetric ratio (A21 = intensity ratio of electric to magnetic dipole transitions) has been studied and compared with a xenon lamp source. Luminescence intensity increases with the increase of power at 532 nm laser excitation. In order to explore materials, which can show dual functionalities such as luminescence as well as magnetic properties (magnetization of ∼14.2 emu g⁻¹), water dispersible Fe3O4-CaMoO4:Eu hybrid magnetic nanoparticles (MN) have been prepared. This shows good heating ability up to ∼42 °C (hyperthermia) and luminescence in the red region (∼612 nm), which is in a biological window (optical imaging). Biocompatibility of the synthesized Fe3O4-CaMoO4:Eu hybrid magnetic nanoparticles has been evaluated in vitro by assessing their cytotoxicity on human liver cancer cells (HepG2 cells) and hTERT cells using the MTT assay and fluorescent microscopy studies.

Anti-CD40 ligand monoclonal antibody delays the progression of murine autoimmune cholangitis.

While there have been significant advances in our understanding of the autoimmune responses and the molecular nature of the target autoantigens in primary biliary cirrhosis (PBC), unfortunately these data have yet to be translated into new therapeutic agents. We have taken advantage of a unique murine model of autoimmune cholangitis in which mice expressing a dominant negative form of transforming growth factor ß receptor II (dnTGFßRII), under the control of the CD4 promoter, develop an intense autoimmune cholangitis associated with serological features similar to human PBC. CD40-CD40 ligand (CD40L) is a major receptor-ligand pair that provides key signals between cells of the adaptive immune system, prompting us to determine the therapeutic potential of treating autoimmune cholangitis with anti-CD40L antibody (anti-CD40L; MR-1). Four-week-old dnTGFßRII mice were injected intraperitoneally with either anti-CD40L or control immunoglobulin (Ig)G at days 0, 2, 4 and 7 and then weekly until 12 or 24 weeks of age and monitored for the progress of serological and histological features of PBC, including rigorous definition of liver cellular infiltrates and cytokine production. Administration of anti-CD40L reduced liver inflammation significantly to 12 weeks of age. In addition, anti-CD40L initially lowered the levels of anti-mitochondrial autoantibodies (AMA), but these reductions were not sustained. These data indicate that anti-CD40L delays autoimmune cholangitis, but the effect wanes over time. Further dissection of the mechanisms involved, and defining the events that lead to the reduction in therapeutic effectiveness will be critical to determining whether such efforts can be applied to PBC.

Human intrahepatic biliary epithelial cells engulf blebs from their apoptotic peers.

The phagocytic clearance of apoptotic cells is critical for tissue homeostasis; a number of non-professional phagocytic cells, including epithelial cells, can both take up and process apoptotic bodies, including the release of anti-inflammatory mediators. These observations are particularly important in the case of human intrahepatic biliary cells (HiBEC), because such cells are themselves a target of destruction in primary biliary cirrhosis, the human autoimmune disease. To address the apoptotic ability of HiBECs, we have focused on their ability to phagocytize apoptotic blebs from autologous HiBECs. In this study we report that HiBEC cells demonstrate phagocytic function from autologous HiBEC peers accompanied by up-regulation of the chemokines CCL2 [monocyte chemotactic protein-1 (MCP-1)] and CXCL8 [interleukin (IL)-8]. In particular, HiBEC cells express the phagocytosis-related receptor phosphatidylserine receptors (PSR), implying that HiBECs function through the 'eat-me' signal phosphatidylserine expressed by apoptotic cells. Indeed, although HiBEC cells acquire antigen-presenting cell (APC) function, they do not change the expression of classic APC function surface markers after engulfment of blebs, both with and without the presence of Toll-like receptor (TLR) stimulation. These results are important not only for understanding of the normal physiological function of HiBECs, but also explain the inflammatory potential and reduced clearance of HiBEC cells following the inflammatory cascade in primary biliary cirrhosis.

Epitope-specific anti-nuclear antibodies are expressed in a mouse model of primary biliary cirrhosis and are cytokine-dependent.

Although the hallmark of primary biliary cirrhosis (PBC) is the presence of anti-mitochondrial antibodies (AMA), a significant number of patients have anti-nuclear antibodies (ANA) directed primarily against two nuclear proteins, gp210 and sp100. In PBC, there are considerable data on the specificity of these anti-nuclear antibodies as well as suggestive evidence that antibodies to gp210 predict a poor outcome. However, a further understanding of the significance of these autoantibodies has been hampered by limitations in accessing human subjects in a preclinical or early asymptomatic stage. To overcome this limitation, we have taken advantage of transgenic mice with abrogated transforming growth factor-ß signalling in T cells (dnTGF-ßRII) that develop histological features of PBC as well as the same AMA specificity. We studied these mice for serum ANA, including specific autoantibodies against gp210 and sp100. We further examined sera from dnTGF-ßRII mice with concurrent deletions of the genes encoding interleukin (IL)-12p35, IL-12p40, IL-23p19, IL-17, IL-6, interferon (IFN)-γ or tumour necrosis factor (TNF)-α. Sera from all the dnTGF-ßRII mouse lines contained antibodies against gp210 and sp100. Of significance, mice with germline deletions of the genes encoding IL-12p40, IL-23p19, IL-17, IL-6 and TNF-α had significantly lower titres of anti-gp210 antibodies. These results provide a platform to dissect the mechanisms of gp210 and sp100 autoantibody production in dnTGF-ßRII mice as well as to study the possible role of ANA in the pathophysiology of PBC.

Induction of autoimmune cholangitis in non-obese diabetic (NOD).1101 mice following a chemical xenobiotic immunization.

Our laboratory has suggested that loss of tolerance to pyruvate dehydrogenase (PDC-E2) leads to an anti-mitochondrial antibody response and autoimmune cholangitis, similar to human primary biliary cirrhosis (PBC). We have suggested that this loss of tolerance can be induced either via chemical xenobiotic immunization or exposure to select bacteria. Our work has also highlighted the importance of genetic susceptibility. Using the non-obese diabetic (NOD) congenic strain 1101 (hereafter referred to as NOD.1101 mice), which has chromosome 3 regions from B6 introgressed onto a NOD background, we exposed animals to 2-octynoic acid (2OA) coupled to bovine serum albumin (BSA). 2OA has been demonstrated previously by a quantitative structural activity relationship to react as well as or better than lipoic acid to anti-mitochondrial antibodies. We demonstrate herein that NOD.1101 mice immunized with 2OA-BSA, but not with BSA alone, develop high titre anti-mitochondrial antibodies and histological features, including portal infiltrates enriched in CD8(+) cells and liver granulomas, similar to human PBC. We believe this model will allow the rigorous dissection of early immunogenetic cause of biliary damage.

Generation of functionally distinct B lymphocytes from common myeloid progenitors.

Current models of adult haematopoiesis propose that haematopoietic stem cells (HSCs) differentiate into common lymphoid (CLP) and common myeloid (CMP) progenitors and establish an early separation between myeloid and lymphoid lineages. Nevertheless, the developmental potential of CMP-associated B cells suggests the existence of alternate pathways for B lymphopoesis. The aim of this study was to compare the developmental and functional properties of CMP- and CLP-derived B cells. While both populations matured through pro-B cell and transitional B cell intermediates in the bone marrow and spleen, respectively, following transfer into irradiated mice, mature CMP- and CLP-derived B cells exhibit distinct functional responses. Specifically, CMP-derived B cells did not respond to mitogenic stimulation to the same degree as their CLP-derived counterparts and secrete lower levels of IgM and the inflammatory cytokines such as interleukin (IL)-6 and IL-10. Together, these data suggest the existence of multiple pathways for generating functionally distinct B cells from bone marrow precursors.

The role of CD11c(+) hepatic dendritic cells in the induction of innate immune responses.

The role of the liver in the initiation and maintenance of tolerance is a critical immune function that involves multiple lineages of immune cells. Included within these populations are liver dendritic cells (DCs). Although there has been significant work on the phenotypic and functional roles of splenic and bone marrow dendritic cells, as well as their subsets, comparable studies in liver have often been difficult. To address this issue we have isolated, from C57BL/6 mice, relatively pure populations of DCs and compared phenotype and function to the data from spleen using flow cytometry, cell sorter assisted purification and culture, morphology by cytospin and May-Giemsa staining, cell cycle progression, antigen uptake, cytokine production and allo-activation potential. natural killer (NK)1.1(-)CD11c(+) liver DC subsets (conventional DCs, T cell receptor (TcR)beta(-)NK1.1(-)CD11c(+)B220(-) and plasmacytoid DCs, TcRbeta(-)NK1.1(-)CD11c(+)B220(+)) efficiently endocytose dextran and produce significant levels of tumour necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-12 p40 in response to Toll-like receptor (TLR) ligands, with responses higher than splenic DCs. There is also a differential capability of hepatic DCs to respond to innate signals. Indeed, CD11c(+) hepatic DCs have a greater capacity to respond to innate stimulation but are less capable of inducing CpG activated-allogeneic T cells. These data suggest that hepatic dendritic cells function as a critical bridge between innate and adaptive immunity and are capable of inducing stronger innate responses with a lower capacity for allo-stimulation than splenic dendritic cells. These properties of liver dendritic cells contribute to their unique role in the induction of tolerance.

Protection by SIV VLP DNA prime/protein boost following mucosal SIV challenge is markedly enhanced by IL-12/GM-CSF co-administration.

The ever increasing number of people infected by human immunodeficiency virus (HIV) throughout the world renders the development of effective vaccines an urgent priority. Herein, we report on an attempt to induce and enhance antiviral responses using a deoxyribonucleic acid (DNA) prime/virus-like particles (VLP) protein boost strategy adjuvanted with interleukin (IL)-12/GM-CSF in rhesus macaques challenged with simian immunodeficiency virus (SIV). Thus, groups of monkeys were administered three consecutive doses of pVecB7 a plasmid expressing VLP with or without plasmids expressing IL-12 and GM-CSF at weeks 0, 13 and 26. The VLP boost was administered at week 39 with or without IL-12. All monkeys were challenged intrarectally with SIVsmE660 2 months following the protein boost. All except one immunized monkey became infected. While all immunized monkeys showed a marked reduction of acute viral peaks, reduction of viral load set points was only achieved in groups whose prime-boost immunizations were supplemented with IL-12/GM-CSF (prime) and/or with IL-12 (boost). Control of viremia correlated with lack of disease progression and survival. Detection of virus in rectal washes at 1 year post-challenge was only successful in monkeys whose immunizations did not include cytokine adjuvant, but these loads did not correlate with plasma viral loads. In summary, use of IL-12 and/or GM-CSF was shown to provide significant differences in the outcome of SIV challenge of prime/boost immunized monkeys.

Genomic analysis of differentially expressed genes in liver and biliary epithelial cells of patients with primary biliary cirrhosis.

The characterization of differentially expressed genes provides a powerful tool for identifying molecules that may be involved in the pathogenesis of disease. We have used two independent techniques to identify overexpressed transcripts in bile duct cells and in liver from patients with primary biliary cirrhosis (PBC). In the first method, we used suppressive subtractive hybridization to compare mRNA from isolated PBC bile duct epithelial cells (BECs) to normal BECs and identified 71 clones as transcribed at higher levels in PBC-BECs. Amongst these clones, 62/71 had matches in a non-redundant nucleotide database and 9/71 had matches in an EST database. Of the 62 clones, 51/62 include a complexity of genes involved in cell proliferation, signal transduction, transcription regulation, RNA processing, carbohydrate metabolism and hypothetical/unknown proteins; 4/62 were identified as interstitial collagenase and collagenase precursors, 4/62 as ribosomal proteins, 3/62 as mitochondrial DNA. The mitochondrial cDNA sequences included cytochrome c oxidase, Wnt-13, and the pHL gene, a c-myc oncogene containing coxIII sequence. In the second method, we constructed cDNA libraries from three different PBC livers and sequenced a total of 12,324 independent clones. These 12,324 clones underwent virtual subtraction with 2,814,148 independent clones from Incyte LifeSeq libraries. Twenty one sequences were identified as unique to PBC liver. Collectively, these approaches identified a number of genes involved in signalling, RNA processing, mitochondrial function, inflammation, and fibrosis. Interestingly, both Wnt-13 and Notch transcripts are overexpressed in PBC liver. Further studies are needed to focus on the significance of these genes during the natural history of disease.

Cloning, sequencing, and homology analysis of nonhuman primate Fas/Fas-ligand and co-stimulatory molecules.

The finding that a single administration of select recombinant human cytokines to nonhuman primates leads to potent cytokine-neutralizing antibody responses in the heterologous host despite >95% homology at the nucleotide and protein level prompted our laboratory to clone, sequence, and prepare recombinant nonhuman primate cytokines, chemokines, growth factors, and other immunoregulatory molecules. In the present report, we present findings on the gene sequences encoding the nonhuman primate homologues of human CD80, CD86, their ligands CD28 and CD152, CD154, CD95, and CD95-L from rhesus macaques and for phylogenetic analysis from pig-tailed macaques, African sooty mangabey monkeys, baboons, and vervets as well as select molecules from the New World aotus and marmoset monkeys. With the exception of CD95, the homology between nonhuman primate and human co-stimulatory molecules was above 95%. In contrast, CD95 was only 89.2% homologous to human CD95, but the differences were essentially found in the transmembrane and intracellular (death) domains. The extracellular portion of CD95 was more homologous which was in accordance with approximately 98% homology between Old World monkey and human CD95-L. In general, sequences from the New World monkey species appeared equidistant to sequences from Old World species and humans in terms of homology suggesting distinct evolutionary patterns. Of interest was the isolation of various splice variants of monkey CD86, CD152 (CTLA-4), CD154, and CD95 transcripts. This is also the first report documenting the occurrence of natural CD86 variants with deleted transmembrane domains, found both in sooty mangabeys and baboon RNA samples. Monkey CD95 showed various deletions and addition of residues in the transmembrane and intracytoplasmic domains compared with human CD95 and between Old and New World species. Subcloning of rhesus CD154 into an expression vector demonstrated expression of a functional protein in cell culture. The other genes are being cloned into expression vectors for the preparation and biological characterization of the nonhuman primate molecules. These investigations will provide novel reagents for in vivo use as immunomodulatory reagents in nonhuman primates in studies which may provide a rationale for their use in humans.

Hantavirus infection induces the expression of RANTES and IP-10 without causing increased permeability in human lung microvascular endothelial cells.

Sin Nombre virus (SNV) and Hantaan virus (HTN) infect endothelial cells and are associated with different patterns of increased vascular permeability during human disease. It is thought that such patterns of increased vascular permeability are a consequence of endothelial activation and subsequent dysfunction mediated by differential immune responses to hantavirus infection. In this study, the ability of hantavirus to directly induce activation of human lung microvascular endothelial cells (HMVEC-Ls) was examined. No virus-specific modulation in the constitutive or cytokine-induced expression of cellular adhesion molecules (CD40, CD54, CD61, CD62E, CD62P, CD106, and major histocompatibility complex classes I and II) or in cytokines and chemokines (eotaxin, tumor necrosis factor alpha, interleukin 1beta [IL-1beta], IL-6, IL-8, MCP-1, MIP-1alpha, and MIP-1beta) was detected at either the protein or message level in hantavirus-infected HMVEC-Ls. Furthermore, no virus-specific enhancement of paracellular or transcellular permeability or changes in the organization and distribution of endothelial intercellular junctional proteins was observed. However, infection with either HTN or SNV resulted in detectable levels of the chemokines RANTES and IP-10 (the 10-kDa interferon-inducible protein) in HMVEC-Ls within 72 h and was associated with nuclear translocation of interferon regulatory factor 3 (IRF-3) and IRF-7. Gamma interferon (IFN-gamma)-induced expression of RANTES and IP-10 could also be detected in uninfected HMVEC-Ls and was associated with nuclear translocation of IRF-1 and IRF-3. Treatment of hantavirus-infected HMVEC-Ls with IFN-gamma for 24 h resulted in a synergistic enhancement in the expression of both RANTES and IP-10 and was associated with nuclear translocation of IRF-1, IRF-3, IRF-7, and NF-kappaB p65. These results reveal a possible mechanism by which hantavirus infection and a TH1 immune response can cooperate to synergistically enhance chemokine expression by HMVEC-Ls and trigger immune-mediated increases in vascular permeability.

Endocrine-immune interaction: alteractions in immune function resulting from neonatal treatment with a GnRH antagonist and seasonality in male primates.

PROBLEM: The effect of neonatal gonadotropin releasing hormone (GnRH) antagonist (Ant) treatment and seasonality on immune system development and function was investigated in male primates. METHOD OF STUDY: Neonatal male rhesus monkeys and marmosets were treated with Ant, and its effect on immune system morphology, circulating lymphocyte subsets, and cell- and humorally-mediated immune responses was assessed during development. In adult rhesus monkeys, we correlated seasonal changes in immune function with circannual fluctuations in immunoactive hormones. RESULTS: In neonatal marmosets, Ant reduced the number of B cells and T cells in the thymic medulla and T cells in the periarterial lymphatic sheaths (PALS) of the spleen. Ant also altered the development of, but did not permanently impair, the proliferative index (PI) of blood lymphocytes to mitogens. In vitro treatment of control lymphocytes with GnRH analogues altered their response to these proliferative agents. In neonatal rhesus monkeys, Ant treatment increased the frequency of clinical problems, lowered circulating levels of lymphocytes, total T cells, CD8+ T cells and B cells, and altered the PI of lymphocytes to mitogens. As adults, the cell- and humorally-mediated immune responses remained impaired. We also documented seasonal fluctuations in the prevalence of diseases, circulating immune cells and immune function in rhesus monkeys. The number of cases of campylobacteriosis and shigellosis was lowest in the winter and highest in the spring. Circulating numbers of white blood cells (WBC) and neutrophils and the PI of lymphocytes to mitogens were higher in the winter than in the summer. Natural killer cell activity also varied with season. Cortisol and leptin secretion exhibited circannual rhythms, rising in concert with decreasing photoperiod and increasing testicular activity in the fall. Conversely, prolactin levels declined with decreasing photoperiod and then rose in the spring. CONCLUSION: Neonatal exposure of male primates to Ant appears to alter early postnatal programming of immune function. In the rhesus monkey, immune function shows seasonal fluctuations that may be driven by circannual changes in the secretion of immunoactive hormones.

Hematopoietic response to lineage-non-specific (rrIL-3) and lineage-specific (rhG-CSF, rhEpo, rhTpo) cytokine administration in SIV-infected rhesus macaques is related to stage of infection.

The present study reports the hematopoietic response to the exogenous administration of recombinant rhesus interleukin-3 (rrIL-3) or a combination of recombinant human granulocyte colony-stimulating factor (rhG-CSF)/erythropoietin (Epo)/thrombopoietin (Tpo) at two different stages of SIV infection: Early-stage (n = 6, CD4 + > 1000/microl and mild splenomegaly) and late-stage (n = 6, CD4 + < 500/microl, progressive hepatosplenomegaly and/or weight loss). SIV-infected animals exhibited significantly impaired bone marrow (BM) and peripheral blood (PB) responses to both rrIL-3 and rhG-CSF/Epo/Tpo administration, as compared to historic controls. In addition, compared to early-stage SIV-infected animals, late-stage SIV-infected macaques demonstrated a more marked dysfunction, as assessed by PB and BM CD34 + content and clonogenic progenitors (colony-forming unit). Neither rrIL-3 nor rhG-CSF/Epo/Tpo administration during either early-stage or late-stage SIV infection increased the viral load, as assessed by bDNA assay. These data suggest that hematopoietic reserve and the response to various cytokines is decreased even in early-stage SIV infection, with the hematopoietic dysfunction progressing in parallel to SIV infection.

A novel role for tumor necrosis factor-alpha in regulating susceptibility of activated CD4+ T cells from human and nonhuman primates for distinct coreceptor using lentiviruses.

Although CD4+ T-cell activation has long been shown to promote infection and replication of simian immunodeficiency virus (SIV) and HIV, recent studies have documented that not all activated CD4+ T cells from human and nonhuman primates are susceptible to infection with HIV/SIV, respectively. Activation of CD4+ T cells with anti-CD3 + anti-CD28 conjugated beads led to induction of a state of anti-viral resistance to infection with strains of viruses that primarily use CCR5 as a coreceptor. The studies reported herein were designed to address the mechanism by which anti-CD3 + anti-CD28-induced stimulation in turn induced antiviral resistance. Results of these studies show that the anti-viral resistance induced by activation of CD4+ T cells with anti-CD3 + anti-CD28 is primarily conferred by the synthesis of tumor necrosis factor-alpha (TNF-alpha), and highlight a unique regulatory role for TNF-alpha in regulating synthesis of MIP-1alpha, MIP-1beta, and regulated-on-activation normal T-expressed and secreted cells, which contributes to this state of antiviral resistance to R5-tropic strains of HIV/SIV. However, while TNF-alpha has a protective role in antiviral resistance of activated CD4+ T cells to R5-tropic viruses, it enhances CXCR4 expression of CD4+ T cells and mediates increased susceptibility to infection with X4-tropic strains of HIV and recombinant SIVs. The results of the studies reported herein also suggest that it is not the Th1 v/s Th2 cytokine profile but the mode of CD4+ T-cell activation that dictates the synthesis of distinct cytokines which regulate the expression of chemokines and chemokine receptors which in turn regulate and confer susceptibility/resistance to R5 v/s X4-tropic HIV and SIV.

Suppression of acute viremia by short-term postexposure prophylaxis of simian/human immunodeficiency virus SHIV-RT-infected monkeys with a novel reverse transcriptase inhibitor (GW420867) allows for development of potent antiviral immune responses resulting in efficient containment of infection.

A nonnucleoside reverse transcriptase (RT) inhibitor, GW420867, was tested for postexposure prophylaxis (PEP) in rhesus macaques experimentally infected with 100 50% tissue culture infective doses of a chimeric simian/human immunodeficiency virus (SHIV) containing the RT gene of HIV-1 (SHIV-RT). Animals were either mock treated, or treated for 4 weeks starting at 8 or 24 h postinfection (p.i.) with GW420867. While such therapy led to undetectable plasma viremia in three of six monkeys, a transient plasma viremia was noted in the other three treated animals at 2 to 4 weeks following cessation of therapy. Following this transient viremia all drug-treated animals showed low or undetectable levels of plasma viremia up to the last sample examined at 90 weeks p.i. Despite low and/or undetectable viremia, virus-specific cytotoxic T lymphocyte and viral Env-specific proliferative responses were seen in the peripheral blood mononuclear cells of both mock- and drug-treated animals as early as 3 weeks p.i. Such virus-specific cellular responses, however, were better maintained in the drug-treated animals than the mock-treated animals. In contrast to the virus-specific cellular response, the magnitude and kinetics of virus specific humoral responses appeared to correlate with the detection of viremia. These data support the view that a short-term PEP with GW420867 permits the generation and maintenance of long-lasting virus-specific cell-mediated immune responses while markedly reducing viral loads to undetectable levels for a prolonged period of time (90 weeks) and leads to long-term disease protection. This model provides a unique means to define mechanisms and correlates of disease protection.

Primary biliary cirrhosis: an orchestrated immune response against epithelial cells.

Primary biliary cirrhosis (PBC) is an organ-specific autoimmune disease that predominantly affects women and is characterized by chronic progressive destruction of small intrahepatic bile ducts with portal inflammation and ultimately fibrosis. The serologic hallmark of PBC is the presence of antibodies to mitochondria, especially to the E2 component of the pyruvate dehydrogenase complex. The mechanisms by which (and if) such antibodies produce liver tissue injury are unknown. However, the presence of these antibodies has allowed detailed immunological definition of the antigenic epitopes, the nature of reactive autoantibodies and the characterization of T-cell responses. Several mechanisms may now be proposed regarding the immune-mediated bile duct damage in PBC, including the possible role of T-cell-mediated cytotoxicity and intracellular interaction between the IgA class of antimitochondrial antibodies and mitochondrial autoantigens. There are major questions which remain unanswered, including, of course, etiology, but also the reasons for female predominance, the absence of PBC in children, the relative ineffectiveness of immunosuppressive drugs, and the specific role of mitochondrial antigens. The data so far provide suggestive evidence that PBC is a mucosal disease; this thesis provides a basis for discussion of etiology via the enterohepatic circulation of toxins and/or infection.

In vitro and in vivo responses to interleukin 12 are maintained until the late SIV infection stage but lost during AIDS.

The in vitro proliferative responses of macaque peripheral blood mononuclear cells (PBMCs) to IL-12 appeared similar before and early after SIV infection, whereas macaque PBMCs sampled during symptomatic stages of SIV infection showed markedly decreased responses. IL-12 was administered to SIVmac239-infected rhesus macaques either during the asymptomatic or the AIDS stage of infection in efforts to evaluate the effect of this cytokine on immune responses, viral loads, and hematopoietic functions in vivo. IFN-gamma secretion levels induced during the asymptomatic or early symptomatic phase were similar to preinfection induced levels, whereas in later AIDS stages this response was lost. The constitutive levels of other measured cytokines were not affected by IL-12 administration in vivo. The frequency and activity of circulating NK cells were markedly enhanced at early stages but not at symptomatic stages of SIV infection. pCTL frequencies were enhanced at early symptomatic stages but not at late AIDS stages. Despite its immunomodulatory effect, IL-12 did not seem to exacerbate or inhibit the replication of SIV in vivo, or the frequency of circulating infected lymphocytes. IL-12 administration was associated with a significant yet subclinical and transient decrease in hematocrit and hemoglobin levels without evidence of hemolysis, hemodilution, or reduction in the frequency of colony-forming unit potential of bone marrow CD34+ cells. This phenomenon may be explained by a functional inhibition of differentiation rather than an altered generation of bone marrow precursors. Thus, these results suggest that IL-12 may benefit HIV-1-infected patients only as long as their immune system retains its capability to respond to cytokine stimulation.

Identification and cloning of a complementary DNA encoding a vicilin-like proprotein, jug r 2, from english walnut kernel (Juglans regia), a major food allergen.

BACKGROUND: Walnuts and other tree nuts are important food-allergen sources that have the potential to be associated with life-threatening, IgE-mediated systemic reactions in some individuals. OBJECTIVE: The purpose of this study was to characterize a complementary (c)DNA clone encoding one of the walnut food allergens. METHODS: A cDNA expression library prepared from walnut somatic embryo was screened for IgE reactivity with patient serum. A reactive clone of 2060 bp, which encoded a protein of 593 amino acids in length, was subcloned by excision into the pGEX expression vector. IgE-binding inhibition experiments were performed. RESULTS: A recombinant fusion protein was induced and shown to bind serum IgE from 9 of 15 patients tested, thus identifying a major allergen. This clone, named Jug r 2, exhibited significant homology with genes encoding the vicilin group of seed proteins. An IgE-binding inhibition experiment suggested that the encoded protein undergoes posttranslational modification into at least one major polypeptide (47 kd) and possibly several others, which is similar to the vicilin-like proteins characterized in cocoa bean (Theobroma cacao) and cottonseed (Gossypium hirsutum). N-terminal sequencing of the 47-kd band, Jug r 2, identified it as a mature protein obtained from the precursor. A second IgE-binding inhibition experiment showed that there is minimal or no cross-reactivity between Jug r 2 and pea vicilin, peanut proteins, or cacao proteins. CONCLUSION: Jug r 2 is the third vicilin food allergen identified in addition to vicilins from soy and peanut. The availability of recombinant food allergens should help advance studies on the immunopathogenesis and possible treatment of IgE-mediated food hypersensitivity.

Abnormal thymic expression of epithelial cell adhesion molecule (EP-CAM) in New Zealand Black (NZB) mice.

New Zealand Black (NZB) mice have been well documented to have a variety of thymic epithelial cell microenvironmental abnormalities, including disruption of corticoepithelial cell networks and medullary cell clusters. These abnormalities of the thymic stromal network are particularly important because similar observations have been noted in other models of murine lupus. Thymic epithelial cells, a key component of the microenvironment, play an important role in selection of the mature T cell receptor repertoire. Recently, a homotypic calcium-independent human and murine epithelial cell adhesion molecule, Ep-CAM, has been described which is located at the thymocyto-cortical cell junction. The function of Ep-CAM is still unclear but its unique location within the thymus suggests that it is critical in the process of providing maturation signals. Consequently, we examined the thymic expression of Ep-CAM in a series of autoimmune prone mice by thymic distribution of Ep-CAM in NZB, NZW, NZB/W, BXSB-Yaa, MRL- lpr/lpr, C3H- gld/gld and the control strains BALB/c, C57BL6, C3H and MRL(+/+), by immunohistology and flow cytometry. Interestingly, NZB mice are similar to control mice from day 4 to 2 weeks of age, having a very low expression of Ep-CAM at the thymocyto-cortical junction. In control strains, there is a marked increased in expression of Ep-CAM beginning at 5 weeks of age. In contrast, NZB mice fail to show significant expression of Ep-CAM even well into adulthood. This abnormality of NZB mice was also noted in NZB/W F1 and BXSB mice, but not MRL- lpr/lpr or C3H- gld/gld mice. Given the potential importance of Ep-CAM in thymic selection, this study provides important evidence that a defective stromal microenvironment is likely to be of etiological significance in the susceptibility of NZB to autoimmune disease.