Natural recombination event within the capsid genomic region leading to a chimeric strain of human enterovirus B

Recombination between two strains is a known phenomenon for enteroviruses replicating within a single cell. We describe a recombinant strain recovered from human stools, typed as coxsackievirus B4 (CV-B4) and CV-B3 after partial sequencing of the VP1 and VP2 coding regions, respectively. The strain was neutralized by a polyclonal CV-B3-specific antiserum but not by a CV-B4-specific antiserum. The nucleotide sequence analysis of the whole structural genomic region showed the occurrence of a recombination event at position 1950 within the VP3 capsid gene, in a region coding for the 2b antigenic site previously described for CV-B3. This observation evidences for the first time the occurrence of an interserotypic recombination within the VP2-VP3-VP1 capsid region between two nonpoliovirus enterovirus strains. The neutralization pattern suggests that the major antigenic site is located within the VP2 protein.     

Enhanced expression of VEGF-A in β cells increases endothelial cell number but impairs islet morphogenesis and β cell proliferation

There is a reciprocal interaction between pancreatic islet cells and vascular endothelial cells (EC) in which EC-derived signals promote islet cell differentiation and islet development while islet cell-derived angiogenic factors promote EC recruitment and extensive islet vascularization. To examine the role of angiogenic factors in the coordinated development of islets and their associated vessels, we used a "tet-on" inducible system (mice expressing rat insulin promoter-reverse tetracycline activator transgene and a tet-operon-angiogenic factor transgene) to increase the β cell production of vascular endothelial growth factor-A (VEGF-A), angiopoietin-1 (Ang1), or angiopoietin-2 (Ang2) during islet cell differentiation and islet development. In VEGF-A overexpressing embryos, ECs began to accumulate around epithelial tubes residing in the central region of the developing pancreas (associated with endocrine cells) as early as embryonic day 12.5 (E12.5) and increased dramatically by E16.5. While α and β cells formed islet cell clusters in control embryos at E16.5, the increased EC population perturbed endocrine cell differentiation and islet cell clustering in VEGF-A overexpressing embryos. With continued overexpression of VEGF-A, α and β cells became scattered, remained adjacent to ductal structures, and never coalesced into islets, resulting in a reduction in β cell proliferation and β cell mass at postnatal day 1. A similar impact on islet morphology was observed when VEGF-A was overexpressed in β cells during the postnatal period. In contrast, increased expression of Ang1 or Ang2 in β cells in developing or adult islets did not alter islet differentiation, development, or morphology, but altered islet EC ultrastructure. These data indicate that (1) increased EC number does not promote, but actually impairs β cell proliferation and islet formation; (2) the level of VEGF-A production by islet endocrine cells is critical for islet vascularization during development and postnatally; (3) angiopoietin-Tie2 signaling in endothelial cells does not have a crucial role in the development or maintenance of islet vascularization.     

Antibodies enhance the infection of phorbol-ester-differentiated human monocyte-like cells with coxsackievirus B4

Coxsackievirus B4 (CV-B4), in presence of antibodies and through a specific viral receptor CAR and Fcγ receptors II and III, can infect monocytes which results in interferon-α synthesis. The antibody-dependent enhancement of CV-B4 infection in the human monocytic-like THP-1 cell line has been investigated. The preincubation of CV-B4 with human plasma or human polyvalent immunoglobulins enhanced the infection of phorbol–myristate–acetate (PMA)-activated THP-1 cell cultures. CV-B4 replicated in these cells as demonstrated by the intracellular detection of infectious particles, viral protein VP1 (immunofluorescence), positive and negative viral RNA (RT-PCR). The viability of infected and control cell cultures was not different up to 20 days post-infection. Activated cell cultures inoculated with CV-B4 harbored intracellular RNA up to 14 days post-infection and produced IFNα that was detected by intracellular immunofluorescence staining as soon as 4 h post-infection with a maximum at 48 h post-infection and by RT-PCR all along the experiment. Together, these data demonstrate that PMA-activated THP-1 cells can be infected with CV-B4, can produce IFNα as a result of interactions between the virus, antibodies and specific receptors. This cellular model can be used to investigate further the mechanism and the result of the antibody-dependent enhancement of CV-B4 infection.     

NF-kappaB2 is required for the control of autoimmunity by regulating the development of medullary thymic epithelial cells

Medullary thymic epithelial cells function as antigen-presenting cells in negative selection of self-reactive T cell clones, a process essential for the establishment of central self-tolerance. These cells mirror peripheral tissues through promiscuous expression of a diverse set of tissue-restricted self-antigens. The genes and signaling pathways that regulate the development of medullary thymic epithelial cells are not fully understood. Here we show that mice deficient in NF-kappaB2, a member of the NF-kappaB family, display a marked reduction in the number of mature medullary thymic epithelial cells that express CD80 and bind the lectin Ulex europaeus agglutinin-1, leading to a significant decrease in the extent of promiscuous gene expression in the thymus of NF-kappaB2(-/-) mice. Moreover, NF-kappaB2(-/-) mice manifest autoimmunity characterized by multiorgan infiltration of activated T cells and high levels of autoantibodies to multiple organs. A subpopulation of the mice also develops immune complex glomerulonephritis. These findings identify a physiological function of NF-kappaB2 in the development of medullary thymic epithelial cells and, thus, the control of self-tolerance induction.     

Genetic analysis of type-1 insulin-like growth factor receptor signaling through insulin receptor substrate-1 and -2 in pancreatic beta cells

Signaling by receptor tyrosine kinases regulates pancreatic β cell function. Inactivation of insulin receptor (InsR), IGF1 receptor (Igf1r), or Irs1 in β cells impairs insulin secretion. Conversely, Irs2 ablation impairs β cell replication. In this study, we examined aspects of the Igf1r regulatory signaling cascade in β cells. To examine genetically the involvement of Irs1 and Irs2 in Igf1r signaling, we generated double mutant mice lacking Igf1r specifically in pancreatic β cells in an Irs1- or Irs2-null background. We show that Igf1r/Irs1 double mutants do not differ phenotypically from Irs1 single mutants and exhibit hyperinsulinemia, while maintaining normal β cell mass and glucose tolerance. In contrast, lack of Igf1r function in β cells aggravates the consequences of Irs2 ablation in double mutants and results in lethal diabetes by 6 weeks of age. This additivity of phenotypic manifestations indicates that Irs2 serves a pathway that is largely independent of Igf1r signaling. Consistent with the view that the latter is the InsR pathway, we show that combined β cell-specific knock-out of both Insr and Igf1r results in a phenocopy of double mutants lacking Igf1r and Irs2. We conclude that Igf1r signals primarily through Irs1 and affects insulin secretion, whereas β cell proliferation is mainly regulated by InsR using Irs2 as a downstream signaling effector. The insulin and IGF pathways appear to control β cell functions independently and selectively.     

Establishment of a mouse thymic epithelial cell line, IT-76MHC and a brief review on cultured thymic epithelial cells.

Interactions between thymocytes and thymic stromal cells are essential for thymocyte differentiation, but little evidence has been presented to directly show in vivo functions or interactions of the stromal cells. Among the stromal cells, the thymic epithelial cell has been considered to have profound effect on thymocyte differentiation and maturation. The calcium-depleted medium, originally developed for the culture of mouse epidermal cells, was applied for the culture of the mouse thymic epithelial cells, and successfully, an epithelial cell line, IT-76MHC was obtained from the mouse thymus. IT-76MHC cells were identified as distinct mouse thymic epithelial cells by 1/ mosaic-like arrangement, 2/ presence of well-developed desmosome and 3/ tonofilaments, 4/ positivity for cytokeratin, and 5/ induced expression of MHC class I and II by IFN-gamma treatment. IGF-1, IGF-2, oxytocin and vasopressin were also detected immunohistochemically in IT-76MHC cells. Furthermore, the IT-76MHC thymic epithelial cells, when injected intrathymically in the allogeneic mouse, prolonged the survival of skin graft from the same donor strain that IT-76MHC cells were derived. These results demonstrate that the thymic epithelial cell line IT-76MHC produces modest thymocyte survival factors as well as a growth suppressor, and that IT-76MHC cells have the ability to induce transplantation tolerance probably through their expression of MHC class I and II molecules. Taken altogether, the IT-76MHC thymic epithelial cells have been proved to be useful tools to better understand the in vivo functions of thymic epithelial cells, and to gain a deep insight into their involvement in the critical selection process of thymocytes which still remains obscure. Finally and additionally, literatures so far reported on thymic epithelial cells in culture, especially lines and clones, are reviewed and their identity as well as their functions are discussed.     

Induction of thymocyte proliferation by supernatants from a mouse thymic epithelial cell line

The thymic stroma plays a critical role in the generation of T lymphocytes by direct cell-to-cell contacts as well as by secreting growth factors or hormones. The thymic epithelial cells, responsible for thymic hormone secretion, include morphologically and antigenically distinct subpopulations that may exert different roles in thymocyte maturation. The recent development of thymic epithelial cell lines provided an interesting model for studying thymic epithelial influences on T cell differentiation. Treating mouse thymocytes by supernatants from one of TEC line (IT-76M1), we observed an induction of thymocyte proliferation and an increase in the percentages of CD4-/CD8- thymocytes. This proliferation was largely inhibited when thymocytes were incubated with IT-76M1 supernatants together with an anti-thymulin monoclonal antibody, but could be enhanced by pretreating growing epithelial cells by triiodothyronine. We suggest that among the target cells for thymulin within the thymus, some putative precursors of early phenotype might be included.     

Dendritic cell differentiation and immune tolerance to insulin-related peptides in Igf2-deficient mice

There is some evidence that insulin-like growth factor 2 (IGF-2) may intervene in the control of T cell differentiation. To further study the immunoregulatory function of this growth factor, we analyzed the immune system of Igf2-/- mice. Phenotypically, some immunological parameters such as lymphoid organ morphology and cellularity were unaltered in Igf2-/- mice, but an increase of CD8+ cells and a decrease of B220+ cells were observed in spleen. In vitro, the development of bone marrow-derived dendritic cells was affected by the absence of Igf2 expression. After maturation, a higher percentage of immature dendritic cells was observed in Igf2-/- population, together with a secondary decrease in allogenic T cell proliferation. Activation of T cells was also affected by the lack of expression of this growth factor. The profile of B cell response in mutant mice immunized with IGF-2 evidenced a T-dependent profile of anti-IGF-2 Abs that was absent in Igf2+/+ mice. The influence of IGF-2 upon tolerance to insulin was also assessed in this model, and this showed that IGF-2 also intervenes in tolerance to insulin. The presence of a T-dependent response in Igf2-deficient mice should allow cloning of specific "forbidden" T CD4+ lymphocytes directed against IGF-2, as well as further investigation of their possible pathogenic properties against insulin family.     

Tolerance to proinsulin-2 is due to radioresistant thymic cells

Proinsulin is a key Ag in type 1 diabetes, but the mechanisms regulating proinsulin immune tolerance are unknown. We have shown that preproinsulin-2 gene-deficient mice (proins-2(-/-)) are intolerant to proinsulin-2. In this study, we analyzed the mechanisms underlying T cell-mediated tolerance to proinsulin-2 in 129/Sv nonautoimmune mice. The expression of one proinsulin-2 allele, whatever its parental origin, was sufficient to maintain tolerance. The site of proinsulin-2 expression relevant to tolerance was evaluated in thymus and bone marrow chimeras. CD4+ T cell reactivity to proinsulin-2 was independent of proinsulin-2 expression in radiation-sensitive bone marrow-derived cells. A wt thymus restored tolerance in proins-2(-/-) mice. Conversely, the absence of the preproinsulin-2 gene in radioresistant thymic cells was sufficient to break tolerance. Although chimeric animals had proinsulin-2-reactive CD4+ T cells in their peripheral repertoire, they displayed no insulitis or insulin Abs, suggesting additional protective mechanisms. In a model involving transfer to immunodeficient (CD3epsilon(-/-)) mice, naive and proinsulin-2-primed CD4+ T cells were not activated, but could be activated by immunization regardless of whether the recipient mice expressed proinsulin-2. Furthermore, we could not identify a role for putative specific T cells regulating proinsulin-2-reactive CD4+ T in transfer experiments. Thus, proinsulin-2 gene expression by radioresistant thymic epithelial cells is involved in the induction of self-tolerance, and additional factors are required to induce islet abnormalities.     

Lymphokine-mediated induction of antigen-presenting ability in thymic stromal cells

We have established and characterized long term thymic stromal cultures from BALB/c (H-2d) and CBA/J (H-2k) mice. All cultures contained multiple adherent cell types, whereas some also contained thymic macrophages (TM). Culture supernatants from all cultures tested contained macrophage colony-stimulating factor activity, whereas only cultures with TM had soluble or membrane-associated interleukin (IL)-1. However, a thymic epithelial cell line (3D . 1), cloned from one of these cultures, produced IL-1 bioactivity. Further analysis confirmed the production of IL-1 alpha mRNA by the epithelial cell. No IL-2 or IL-4 (formerly called B cell stimulatory factor 1) activity was detected in any of the cultures. Antigen-presenting (AP) ability was determined using the chicken ovalbumin (OVA)-specific, I-Ad-restricted T cell hybridoma 3DO-18.3. Harvested TM exhibited antigen-specific, Ia-restricted AP ability which was enhanced by IL-4 as well as interferon-gamma (IFN-gamma). In contrast, AP ability was detected in non-macrophage stromal cell cultures (NMSC) only after preincubation with IFN-gamma. AP by preinduced NMSC was also Ia-restricted and could be blocked by anti-I-Ad antibodies. Since the T cell receptor of 3DO-18.3 is known to recognize a peptide produced by CNBr degradation of OVA, these observations suggest that both TM and NMSC can process OVA to produce this peptide. Glutaraldehyde-fixation experiments confirmed that NMSC must process native OVA into antigenic peptides for successful AP. Assays using several cloned stromal cell lines of different lineages suggested that only epithelial cells could be induced with IFN-gamma to exhibit competent AP. Given the possible role for IFN-gamma in the maintenance of Ia in the thymus, we investigated whether IFN-gamma production could be ascribed to a subpopulation of thymocytes. Culture supernatants from calcium ionophore and phorbol ester-stimulated peanut agglutinin-negative, but not peanut agglutinin-positive, thymocytes induced AP ability in NMSC. Thus, some thymocytes can produce an Ia-inducing lymphokine (most likely IFN-gamma) which may play an important role in T cell ontogeny through its effects on both thymic macrophages and thymic epithelial cells.     

Neuroendocrinology of the thymus

The neuropeptides oxytocin (OT) and vasopressin (VP) are synthesized in the human thymus in a similar way as in the hypothalamo-neurophypophyseal system. Immunocytochemistry with polyclonal and monoclonal antibodies revealed that immunoreactive OT- and VP-producing cells are localized in the subcapsular cortex and medulla of human and murine thymuses. The epithelial nature of the neuroendocrine thymic cells is demonstrated by their immunostaining with a monoclonal antibody against cytokeratin. An original example of a neuroendocrine-immune microenvironment is given by the thymic nurse cells which are composed of a large neuroendocrine epithelial cell enclosing numerous mitotic immature thymocytes. These observations and the previously reported mitogenic and immunomodulatory properties of VP and OT upon mature T cells and thymocytes strongly support the existence of a neuroendocrine thymo-lymphoid axis and an active role of thymic VP and OT in T cell differentiation and activation.     

Measles Virus Infection Induces Terminal Differentiation of Human Thymic Epithelial Cells

Measles virus infection induces a profound immunosuppression that may lead to serious secondary infections and mortality. In this report, we show that the human cortical thymic epithelial cell line is highly susceptible to measles virus infection in vitro, resulting in infectious viral particle production and syncytium formation. Measles virus inhibits thymic epithelial cell growth and induces an arrest in the G₀/G₁ phases of the cell cycle. Moreover, we show that measles virus induces a progressive thymic epithelial cell differentiation process: attached measles virus-infected epithelial cells correspond to an intermediate state of differentiation while floating cells, recovered from cell culture supernatants, are fully differentiated. Measles virus-induced thymic epithelial cell differentiation is characterized by morphological and phenotypic changes. Measles virus-infected attached cells present fusiform and stellate shapes followed by a loss of cell-cell contacts and a shift from low- to high-molecular-weight keratin expression. Measles virus infection induces thymic epithelial cell apoptosis in terminally differentiated cells, revealed by the condensation and degradation of DNA in measles virus-infected floating thymic epithelial cells. Because thymic epithelial cells are required for the generation of immunocompetent T lymphocytes, our results suggest that measles virus-induced terminal differentiation of thymic epithelial cells may contribute to immunosuppression, particularly in children, in whom the thymic microenvironment is of critical importance for the development and maturation of a functional immune system.     

Transplantation tolerance and autoimmunity after xenogeneic thymus transplantation

Successful grafting of vascularized xenografts (Xgs) depends on the ability to reliably induce both T cell-independent and -dependent immune tolerance. After temporary NK cell depletion, B cell suppression, and pretransplant infusion of donor Ags, athymic rats simultaneously transplanted with hamster heart and thymus Xgs developed immunocompetent rat-derived T cells that tolerated the hamster Xgs but provoked multiple-organ autoimmunity. The autoimmune syndrome was probably due to an insufficient development of tolerance for some rat organs; for example, it led to thyroiditis in the recipient rat thyroid, but not in simultaneously transplanted donor hamster thyroid. Moreover, grafting a mixed hamster/rat thymic epithelial cell graft could prevent the autoimmune syndrome. These experiments indicate that host-type thymic epithelial cells may be essential for the establishment of complete self-tolerance and that mixed host/donor thymus grafts may induce T cell xenotolerance while maintaining self-tolerance in the recipient.     

Establishment of a pancreatic β cell proliferation model in vitro and a platform for diabetes drug screening

Diabetes, a disease resulting from loss of functional β cells, is globally an increasingly important condition. Based on the islet-differentiation ability of ductal epithelial cells and stimulating β cell proliferation ability of the Reg Iα gene, we aimed to establish an in vitro pancreatic β cell proliferation model for screening therapeutic drugs of diabetes in the future. Pancreatic ductal epithelial cells were isolated from male Wistar rats, and induced to differentiate into pancreatic β cells. Immunofluorescence staining assay, western blot, RT-PCR analysis, and dithizone staining were used to characterize the cells. Rat Reg Iα protein was transiently expressed in vitro by transfection of HEK 293 cells with the PCMV6-entry-REG Ia plasmid, and expression was verified by RT-PCR analysis, proliferation assay, and apoptosis assay. The pancreatic β cell proliferation model was further validated by a proliferation assay using differentiated pancreatic β cells treated with transfection supernatant. Finally, we have successfully established an in vitro pancreatic β cells proliferation model using transiently expressed rat Reg Iα protein and differentiated pancreatic β cells from pancreatic ductal epithelial cells. This model could be used as a platform to screen new drugs for islet neogenesis to cure diabetes, especially Chinese herbal drugs in the future.     

Medullary epithelial cell lines from murine thymus constitutively secrete IL-1 and hematopoietic growth factors and express class II antigens in response to recombinant interferon-gamma

In this report, we describe the generation of two cloned epithelial cell lines, TE-71 and TE-75, from murine thymus. These cell lines resemble medullary thymic epithelium by a number of criteria, including reactivity with the monoclonal antibodies A2B5 and ER-TR5, the fucose-specific lectin derived from Ulex europeus, and the expression of keratins normally expressed by medullary thymic epithelial cells in situ. Constitutive Class II antigen expression by these cells is not detectable at the light or electron microscopic level or with flow cytometry. Following exposure to recombinant interferon-gamma or supernatants from mitogen-stimulated spleen cells, expression of Class II antigens by these thymic epithelial cell lines is increased, although less than the levels expressed by spleen cells. Medium conditioned by TE-71 and TE-75 cells exhibited colony-stimulating activity for bone marrow cells. In addition, TE-71-conditioned medium exhibited IL-1-like activity which could be neutralized with anti-IL-1 antibodies.