技术资料

Molecular mimicry between microbial and self-components is postulated as the mechanism that accounts for the antigen and tissue specificity of immune responses in postinfectious autoimmune diseases. Little direct evidence exists,and research in this area has focused principally on T cell-mediated,antipeptide responses,rather than on humoral responses to carbohydrate structures. Guillain-Barré syndrome,the most frequent cause of acute neuromuscular paralysis,occurs 1-2 wk after various infections,in particular,Campylobacter jejuni enteritis. Carbohydrate mimicry [Galbeta1-3GalNAcbeta1-4(NeuAcalpha2-3)Galbeta1-] between the bacterial lipooligosaccharide and human GM1 ganglioside is seen as having relevance to the pathogenesis of Guillain-Barré syndrome,and conclusive evidence is reported here. On sensitization with C. jejuni lipooligosaccharide,rabbits developed anti-GM1 IgG antibody and flaccid limb weakness. Paralyzed rabbits had pathological changes in their peripheral nerves identical with those present in Guillain-Barré syndrome. Immunization of mice with the lipooligosaccharide generated a mAb that reacted with GM1 and bound to human peripheral nerves. The mAb and anti-GM1 IgG from patients with Guillain-Barré syndrome did not induce paralysis but blocked muscle action potentials in a muscle-spinal cord coculture,indicating that anti-GM1 antibody can cause muscle weakness. These findings show that carbohydrate mimicry is an important cause of autoimmune neuropathy. View Publication

Using whole-cell patch-clamp,fluorescence microscopy and flow cytometry,we demonstrate a switch in potassium channel expression during differentiation of human B cells from naive to memory cells. Naive and IgD(+)CD27(+) memory B cells express small numbers of the voltage-gated Kv1.3 and the Ca(2+)-activated intermediate-conductance IKCa1 channel when quiescent,and increase IKCa1 expression 45-fold upon activation with no change in Kv1.3 levels. In contrast,quiescent class-switched memory B cells express high levels of Kv1.3 ( approximately 2000 channels/cell) and maintain their Kv1.3(high) expression after activation. Consistent with their channel phenotypes,proliferation of naive and IgD(+)CD27(+) memory B cells is suppressed by the specific IKCa1 inhibitor TRAM-34 but not by the potent Kv1.3 blocker Stichodactyla helianthus toxin,whereas the proliferation of class-switched memory B cells is suppressed by Stichodactyla helianthus toxin but not TRAM-34. These changes parallel those reported for T cells. Therefore,specific Kv1.3 and IKCa1 inhibitors may have use in therapeutic manipulation of selective lymphocyte subsets in immunological disorders. View Publication

The human peripheral B-cell compartment displays a large population of immunoglobulin M-positive,immunoglobulin D-positive CD27(+) (IgM(+)IgD(+)CD27(+)) memory" B cells carrying a mutated immunoglobulin receptor. By means of phenotypic analysis� View Publication

Like their human counterparts,mouse plasmacytoid dendritic cells (pDCs) play a central role in innate immunity against viral infections,but their capacity to prime T cells in vivo remains unknown. We show here that virus-activated pDCs differentiate into antigen-presenting cells able to induce effector/memory CD8(+) T-cell responses in vivo against both epitopic peptides and endogenous antigen,whereas pDCs activated by synthetic oligodeoxynucleotides containing unmethylated cytosine-guanine motifs (CpG) acquire only the ability to recall antigen-experienced T-cell responses. We also show that immature pDCs are unable to induce effector or regulatory CD8(+) T-cell responses. Thus,murine pDCs take part in both innate and adaptive immune responses by directly priming naive CD8(+) T cells during viral infection. View Publication

Amphoterin (HMGB1) is a 30-kD heparin-binding protein involved in process extension and migration of cells by a mechanism involving the receptor for advanced glycation end products (RAGE). High levels of amphoterin are released to serum during septic shock. We have studied the expression of amphoterin in monocytes and the role of amphoterin and RAGE in monocyte transendothelial migration. Un-activated monocytes in suspension did not reveal amphoterin on their surface,but adherent monocytes exported amphoterin to the cell surface. Immunohistochemical staining of arterial thrombi in vivo revealed amphoterin in mononuclear cells and in surrounding extracellular matrix. Amphoterin was secreted from phorbol ester and interferon-gamma (IFN-gamma)-activated macrophages,and the secretion was inhibited by blocking the adenosine 5'-triphosphate (ATP)-binding cassette transporter-1,a member of the multidrug resistance protein family. Amphoterin was specifically adhesive for monocytes in peripheral blood leukocyte adhesion assay. Adhesion caused an extensive spreading of cells,which was inhibited by the dominant-negative RAGE receptor (soluble ectodomain of RAGE),and adhesion up-regulated chromogranin expression in monocytes,also suggesting a RAGE-dependent interaction. Monocyte transendothelial migration was efficiently inhibited by anti-amphoterin and anti-RAGE antibodies and by the soluble RAGE. We suggest that amphoterin is an autocrine/paracrine regulator of monocyte invasion through the endothelium. View Publication

The galactose-alpha-1,3-galactose (alphaGal) carbohydrate epitope is expressed on porcine,but not human cells,and therefore represents a major target for preformed human anti-pig natural Abs (NAb). Based on results from pig-to-primate animal models,NAb binding to porcine endothelial cells will likely induce complement activation,lysis,and hyperacute rejection in pig-to-human xenotransplantation. Human NK cells may also contribute to innate immune responses against xenografts,either by direct recognition of activating molecules on target cells or by FcgammaRIII-mediated xenogeneic Ab-dependent cellular cytotoxicity (ADCC). The present study addressed the question as to whether the lack of alphaGal protects porcine endothelial cells from NAb/complement-induced lysis,direct xenogeneic NK lysis,NAb-dependent ADCC,and adhesion of human NK cells under shear stress. Homologous recombination,panning,and limiting dilution cloning were used to generate an alphaGal-negative porcine endothelial cell line,PED2*3.51. NAb/complement-induced xenogeneic lysis of PED2*3.51 was reduced by an average of 86% compared with the alphaGal-positive phenotype. PED2*3.51 resisted NK cell-mediated ADCC with a reduction of lysis ranging from 30 to 70%. However,direct xenogeneic lysis of PED2*3.51,mediated either by freshly isolated or IL-2-activated human NK cells or the NK cell line NK92,was not reduced. Furthermore,adhesion of IL-2-activated human NK cells did not rely on alphaGal expression. In conclusion,removal of alphaGal leads to a clear reduction in complement-induced lysis and ADCC,but does not resolve adhesion of NK cells and direct anti-porcine NK cytotoxicity,indicating that alphaGal is not a dominant target for direct human NK cytotoxicity against porcine cells. View Publication

Malignant pleural mesothelioma is an uncommon tumor largely confined to the thoracic cavity,which is resistant to conventional therapies,therefore prompting an intensive search for effective treatment alternatives. This study focuses on dendritic cell (DC) vaccination for malignant pleural mesothelioma and evaluates the in vitro efficacy of antigen-loaded DC-based vaccines for the induction of major histocompatibility complex Class I-restricted antimesothelioma cytotoxic T lymphocyte responses. The source of tumor-associated antigens for HLA-A2(+) DCs from healthy donors was apoptotic HLA-A2(-) mesothelioma cells either lacking or expressing heat shock protein 70 according to whether tumor cells were heat shocked or not before ultraviolet-mediated apoptosis. Our results show that both apoptotic preparations were equivalent regarding the responsiveness of DCs to combined treatment with tumor necrosis factor-alpha and poly(inosinic-cytidylic) acid,as determined by similar increased expression of costimulatory molecules and interleukin-12 production. However,only DCs loaded with apoptotic heat shock protein 70-expressing cells were found to be potent in vitro inducers of cytotoxic T lymphocyte activity against HLA-A2(+) mesothelioma cells. Such elicited cytotoxic T lymphocytes also exhibit cytotoxic activity against an HLA-A2(+) melanoma cell line,suggesting recognition of shared antigens. These findings therefore carry the potential of offering an alternative,promising approach for the therapy of patients with malignant pleural mesothelioma. View Publication

Langerhans cells (LCs) constitute a subset of DCs that initiate immune responses in skin. Using leprosy as a model,we investigated whether expression of CD1a and langerin,an LC-specific C-type lectin,imparts a specific functional role to LCs. LC-like DCs and freshly isolated epidermal LCs presented nonpeptide antigens of Mycobacterium leprae to T cell clones derived from a leprosy patient in a CD1a-restricted and langerin-dependent manner. LC-like DCs were more efficient at CD1a-restricted antigen presentation than monocyte-derived DCs. LCs in leprosy lesions coexpress CD1a and langerin,placing LCs in position to efficiently present a subset of antigens to T cells as part of the host response to human infectious disease. View Publication

Primitive hematopoietic cells from several species are known to efflux both Hoechst 33342 and Rhodamine-123. We now show that murine hematopoietic stem cells (HSCs) defined by long-term multilineage repopulation assays efflux both dyes variably according to their developmental or activation status. In day 14.5 murine fetal liver,very few HSCs efflux Hoechst 33342 efficiently,and they are thus not detected as side population" (SP) cells. HSCs in mouse fetal liver also fail to efflux Rhodamine-123. Both of these features are retained by most of the HSCs present until 4 weeks after birth but are reversed by 8 weeks of age or after a new HSC population is regenerated in adult mice that receive transplants with murine fetal liver cells. Activation of adult HSCs in vivo following 5-fluorouracil treatment� View Publication

Human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) is correlated with increased monocyte migration to the brain,and the incidence of HAD among otherwise asymptomatic subjects appears to be lower in India than in the United States and Europe (1 to 2% versus 15 to 30%). Because of the genetic differences between HIV-1 strains circulating in these regions,we sought to identify viral determinants associated with this difference. We targeted Tat protein for these studies in view of its association with monocyte chemotactic function. Analyses of Tat sequences representing nine subtypes revealed that at least six amino acid residues are differentially conserved in subtype C Tat (C-Tat). Of these,cysteine (at position 31) was highly (textgreater99%) conserved in non-subtype C viruses and more than 90% of subtype C viruses encoded a serine. We hypothesized a compromised chemotactic function of C-Tat due to the disruption of CC motif and tested it with the wild type C-Tat (CS) and its two isogenic variants (CC and SC) derived by site-directed mutagenesis. We found that the CS natural variant was defective for monocyte chemotactic activity without a loss in the transactivation property. While the CC mutant is functionally competent for both the functions,in contrast,the SC mutant was defective in both. Therefore,the loss of the C-Tat chemotactic property may underlie the reduced incidence of HAD; although not presenting conclusive evidence,this study provides the first evidence for a potential epidemiologic phenomenon associated with biological differences in the subtype C viruses. View Publication