ICAM-1 and VCAM-1 are differentially expressed on blood-retinal barrier cells during experimental autoimmune uveitis.

Adhesion molecules play a central role in leukocyte adhesion to the blood-retinal barrier (BRB) during uveitis. VCAM-1 expression on the BRB has been already described but although structurally similar, ICAM-1 has shown in various autoimmunity models to have distinct role and expression. Here, we induced uveitis in C57Bl/6 mice by adoptive transfer of semi-purified T cells from IRBP1-20-immunized mice. Using Flow cytometry analysis on transferred cells and immunofluorescence staining on retina we have studied the comparative ocular expression of both ICAM-1 and VCAM-1 and their ligands LFA-1 and VLA-4 at the surface of uveitogenic cells. Our results showed that LFA-1 and VLA-4 are expressed on both T and non T cells, VLA-4 sparsely and LFA-1 ubiquitously. Considering retinal expression, ICAM-1 is faintly present and VCAM-1 is absent in naive eyes. Only ICAM-1 is present on infiltrating cells in the retina and vitreous, while only VCAM-1 extends to perivascular glial cells and all along the internal limiting membrane. Finally, ICAM-1 is strongly expressed on the RPE, where VCAM-1 expression is much weaker. VCAM-1 seems most strongly expressed on the internal BRB while ICAM-1 predominates on the external BRB. Those major differences in the expression pattern could represent differential entry pathways for inflammatory cells to penetrate the eye.

Characterization of retinal expression of vascular cell adhesion molecule (VCAM-1) during experimental autoimmune uveitis.

Leukocyte adhesion to the blood retinal barrier is a critical step in the pathogenesis of non-infectious uveitis and is mediated in part through the induction of adhesion molecules on retinal cells. Here, we have investigated the retinal expression of Vascular Cell Adhesion Molecule 1 (VCAM-1) in mouse experimental models of non-infectious uveitis. For each eyes, a histological score was given, and the expression of VCAM-1 analyzed by immunohistology. Co-labellings for GFAP, endoglin, aquaporin 4 and recoverin were also performed in order to determine which cell type expressed VCAM-1. In low grade uveitis, obtained after adoptive transfer of semi-purified autoreactive lymphocytes, VCAM-1 was only punctually expressed in the internal limiting membrane and epithelial cells of the ciliary body. Using the same adoptive transfer protocol, we found that, in correlation with disease severity, the staining extended to all internal limiting membranes, vasculitis lesions, Müller cell extensions, outer limiting membranes and RPE cells. VCAM-1 expression in the inner limiting membrane and Müller cell extensions co-stained with GFAP expression. In vasculitis lesions, VCAM-1 co-localized with either GFAP and endoglin expression. The labeling in the outer limiting membrane, did not exactly co-stained with AQ4 (Müller cells marker) or recoverin (photoreceptor marker) and the nature of this expression remained unexplained. Finally, VCAM-1 expression was also analyzed in classical experimental autoimmune uveitis eyes, and a similar pattern of expression was found. In conclusion VCAM-1 is expressed on all blood retinal barrier cells during experimental non-infectious uveitis and might thus play an important role in inflammatory cell recruitment during disease development.

Interleukin 10 reduces the release of tumor necrosis factor and prevents lethality in experimental endotoxemia.

Because of its ability to efficiently inhibit in vitro cytokine production by activated macrophages, we hypothesized that interleukin (IL) 10 might be of particular interest in preventing endotoxin-induced toxicity. We therefore examined the effects of IL-10 administration before lipopolysaccharide (LPS) challenge in mice. A marked reduction in the amounts of LPS-induced tumor necrosis factor (TNF) release in the circulation was observed after IL-10 pretreatment at doses at low as 10 U. IL-10 also efficiently prevented the hypothermia generated by the injection of 100 micrograms LPS. Finally, pretreatment with a single injection of 1,000 U IL-10 completely prevented the mortality consecutive to the challenge with 500 micrograms LPS, a dose that was lethal in 50% of the control mice. We conclude that IL-10 inhibits in vivo TNF secretion and protects against the lethality of endotoxin in a murine model of septic shock.

Transient expansion of peptide-specific lymphocytes producing IFN-gamma after vaccination with dendritic cells pulsed with MAGE peptides in patients with mage-A1/A3-positive tumors.

Assessment of T-cell activation is pivotal for evaluation of cancer immunotherapy. We initiated a clinical trial in patients with MAGE-A1 and/or -A3 tumors using autologous DC pulsed with MAGE peptides aimed at analyzing T-cell-derived, IFN-gamma secretion by cytokine flow cytometry and ELISPOT. We also tested whether further KLH addition could influence this response favorably. Monocyte-derived DC were generated from leukapheresis products. They were pulsed with the relevant MAGE peptide(s) alone in group A (n=10 pts) and additionally with KLH in group B (n=16 pts). A specific but transient increase in the number of peripheral blood T lymphocytes secreting IFN-gamma in response to the vaccine peptide(s) was observed in 6/8 patients of group A and in 6/16 patients of group B. We conclude that anti-tumor vaccination using DC pulsed with MAGE peptides induces a potent but transient anti-MAGE, IFN-gamma secretion that is not influenced by the additional delivery of a nonspecific, T-cell help.

Loss of tumorigenicity and increased immunogenicity induced by interleukin-10 gene transfer in B16 melanoma cells.

Because interleukin-10 (IL-10) has potent immunosuppressive and anti-inflammatory properties and is produced by some cancers, we hypothesized that its production might play a role in carcinogenesis by inhibiting adequate antitumoral immune responses. To test this hypothesis, retroviral vectors containing the IL-10 cDNA were generated and used to infect B16F1 melanoma cells that were injected subcutaneously in syngeneic mice. Surprisingly, IL-10 gene transfer resulted in a loss of tumorigenicity that was proportional to the amount of IL-10 secreted. Histological analysis showed massive area of necrosis of these tumor cells, with infiltration of polymorphic inflammatory cells. Parental cells simultaneously implanted had decreased tumorigenicity only when mixed with IL10-producing cells, but not when injected contralaterally, suggesting that their eradication is mediated mostly by a local phenomenon. Host T lymphocytes and natural killer (NK) cells were involved in this eradication because IL-10-producing cells grew in nude mice and in CD8+ or NK-depleted mice. Finally, mice injected with IL-10-secreting cells developed an antitumoral systemic immune response able to protect them against a subsequent challenge with parental cells. These results demonstrate that, in some settings, IL10 may have in vivo immunostimulating and proinflammatory properties that need to be considered in its therapeutic development.

Chimerism and cytotoxic T lymphocyte unresponsiveness after neonatal injection of spleen cells in mice. Effects of T cell depletion and of a semiallogeneic or fully allogeneic inoculum.

Newborn Balb/c mice received a single neonatal injection of either (A/J x Balb/c) F1 hybrid spleen cells, T-cell-depleted (A/J x Balb/c) F1 hybrid spleen cells, or T-cell-depleted fully allogeneic A/J spleen cells. Chimerism was followed longitudinally during the life span by the detection of circulating donor allotype. At sacrifice, the percentage of donor cells in the spleen was measured, and cytotoxic T lymphocyte (CTL) reactivity to the tolerogen was tested. We found that T cell depletion of the semiallogeneic inoculum did not modify its capacity to generate persistent chimerism and CTL tolerance, while T-cell-depleted allogeneic cells were intrinsically deficient both in the induction and in the long-term maintenance of chimerism and CTL unresponsiveness.

Critical role of interleukin 4 in the induction of neonatal transplantation tolerance.

Neonatal injection of semiallogeneic cells is known to promote differentiation of donor-specific CD4+ T cells into TH2-like cells in the peripheral lymphoid organs. We reasoned that the propensity of neonatal T cells to synthesize high levels of IL-4 might be involved in this polarization of the alloreactive response and thereby in the development of neonatal transplantation tolerance. First, analysis of cytokine gene expression in lymph nodes after neonatal injection of 10(7) (A/J x BALB/c)F1 cells in BALB/c mice indicated that IL-4 but not IL-2 is rapidly produced by CD4+ cells after allogeneic challenge in vivo. To determine whether the early production of IL-4 was involved in the establishment of allotolerance, BALB/c mice neonatally injected with (A/J x BALB/c)F1 spleen cells received on days 1 and 3 after birth 1 mg of anti-IL-4 mAb (11B11) or the same amount of control mAb. When grafted with A/J skin at 4 weeks, 88% of mice treated with control mAb retained their graft for more than 50 days, whereas rejection occurred within 30 days in 93% of mice treated with anti-IL-4 mAb. Analysis of T cell functions after in vitro restimulation with A/J spleen cells indicated that early IL-4 neutralization did not prevent donor-specific CTL unresponsiveness but allowed the emergence of alloreactive T cells secreting increased levels of IL-2 and IFN-gamma. We conclude that early production of IL-4 is critical for the establishment of neonatal transplantation tolerance in this strain combination, which has disparities across the entire H-2 region.

Role and expression of CD40 on human retinal pigment epithelial cells.

PURPOSE: To examine the CD40 costimulatory molecule expression on normal resting or activated adult human retinal pigment epithelium (hRPE) cells and to evaluate its role as an activation molecule considering the potential antigen presentation functions of hRPE cells. METHODS: Expression of HLA-DR and costimulatory (CD40, B7.1, B7.2, CD54, and CD58) molecules on hRPE cells was analyzed by flow cytometry. CD40 triggering was performed using soluble CD40L or cocultures with CD40L transfected fibroblasts. Interleukin (IL)-6, -8, -10, and -12 secretions were measured by enzyme-linked immunosorbent assay. Antigen presentation function of hRPE cells was assessed by coculturing hRPE cells with allogeneic T cells. T-cell proliferation was measured by [(3)H]-thymidine incorporation, and T-cell apoptosis by measurement of caspase-3 activity. RESULTS: Interferon (IFN)gamma-activated hRPE cells expressed CD40, but not B7.1 or B7.2. Although interferongamma enhanced IL-6 and IL-8 production, CD40 triggering of IFNgamma-activated hRPE cells did not induce IL-12 secretion. hRPE cells did not stimulate allogeneic resting T cells and downregulated phytohemagglutinin-activated allogeneic T cells via a cell-to-cell contact-dependent mechanism. Some induction of apoptosis was detected. CONCLUSIONS: CD40 is expressed on IFNgamma-activated hRPE cells. Its ligation leads to an increased production of IL-6 and IL-8 but fails to induce B7.1 or B7. 2 expression, or to induce IL-12 secretion. Accordingly, hRPE cells do not activate allogenic T cells but inhibit T-cell proliferation, partly through induction of apoptosis. These results suggest that hRPE cells could be implicated more in a deviant antigen presentation. If the exact molecular mechanisms are unclear, it is likely that CD40-CD40L interaction could play a role in this process.

CXCR4 expression in vitreoretinal membranes.

BACKGROUND/AIM: Proliferative vitreoretinopathy (PVR) and macular pucker (MP) vitreoretinal membranes are caused by abnormal cell migration. By their role in chemotactism, chemokine receptors represent good candidates to sustain this process. The authors thus investigated the expression of one of them, CXCR4, in these pathologies. METHODS: Three PVR and four MP membranes were surgically removed and processed for immunochemical studies with antibodies for CXCR4, cytokeratins or smooth muscle actin. RESULTS: CXCR4 expression was found in all membranes. There was no relation between severity of PVR or MP and presence of CXCR4. In addition, there was no difference in CXCR4 expression between MP and PVR. CONCLUSION: CXCR4 is expressed in PVR and MP. Further experiments are needed to test if CXCR4 and other chemokine receptors are implicated in vitreoretinal membrane formation.

Retinal pigment epithelial cells phagocytosis of T lymphocytes: possible implication in the immune privilege of the eye.

AIM: To investigate the capability of retinal pigment epithelium (RPE) cells to phagocytose T lymphocytes and to further analyse the immunobiological consequences of this phagocytosis. METHODS: Human RPE cells pretreated or not by cytochalasin, a phagocytosis inhibitor, were co-cultured with T lymphocytes for different time points. Phagocytosis was investigated by optic microscopy, electron microscopy, and flow cytometry. T cell proliferation was measured by (3)H thymidine incorporation. RPE interleukin 1beta mRNA expression was quantified by real time PCR. RESULTS: RPE cells phagocytose apoptotic and non-apoptotic T lymphocytes, in a time dependent manner. This is an active process mediated through actin polymerisation, blocked by cytochalasin E treatment. Inhibition of RPE cell phagocytosis capabilities within RPE-T cell co-cultures led to an increase of lectin induced T cell proliferation and an upregulation of interleukin 1beta mRNA expression in RPE cells. CONCLUSIONS: It is postulated that T lymphocyte phagocytosis by RPE cells might, by decreasing the total number of T lymphocytes, removing apoptotic lymphocytes, and downregulating the expression of IL-1beta, participate in vivo in the induction and maintenance of the immune privilege of the eye, preventing the development of intraocular inflammation.

Detection of rotavirus in faecal specimens with a monoclonal antibody enzyme-linked immunosorbent assay: comparison with polyclonal antibody enzyme immuno-assays and a latex agglutination test.

Monoclonal antibodies have been produced against the 81/36F strain of rotavirus. One of them, was chosen as diagnostic reagent: it showed high ELISA reactivity with all the bovine, human and porcine rotavirus strains tested and reacted with VP6, structural protein product known to support the common rotavirus antigen. A sandwich ELISA procedure using the chosen monoclonal as "capture and detecting" antibody was performed to detect rotavirus in faecal samples from experimentally inoculated newborn calves: it always gave a negative response with meconium and a positive response for the stool specimens which rotavirus have been isolated. This assay was compared with Enzygnost and Slidex Rota Kit tests and with a non-commercial sandwich ELISA test using polyclonal antibodies: it showed more sensitivity than the agglutination test and was as sensitive as the other two tests to detect rotavirus in routine diagnostic material. The test evaluated showed no equivocal results.

[Allogenic interactions in immunopathology]. / Interactions allogéniques en immunopathologie.

Allogeneic interactions associated with experimental graft-versus-host reaction are sometimes responsible for the development of autoantibodies, immune complex lesions and malignant lymphocytic proliferation. Hypergammaglobulinaemia reflects the activation of B cells, and T cell-associated responses are deeply depressed. Some of these abnormalities are also found in chimera mice following injection of semi-allogeneic cells. The place of allogeneic interactions in human pathology has not yet been determined, but they might intervene in the pathogenesis of some autoimmune and lymphoproliferative diseases and in the acquired immune deficiency syndrome.

A virtual environment for simulated rat dissection.

Animal dissection for the scientific examination of organ subsystems is a delicate procedure. Performing this procedure under the complex environment of microgravity presents additional challenges because of the limited training opportunities available that can recreate the altered gravity environment. Traditional crew training often occurs several months in advance of experimentation, provides limited realism, and involves complicated logistics. We have developed an interactive virtual environment that can simulate several common tasks performed during animal dissection. In this paper, we describe the imaging modality used to reconstruct the rat in virtual space, provide an overview of the simulation environment and briefly discuss some of the techniques used to manipulate the virtual rat.

[The medical oncology clinic]. / La Clinique d'Oncologie Médicale.

The clinic of medical oncology is mainly devoted to the development of new anticancer treatments based on molecular biology and immunology. The clinic was the first in Belgium to start a protocol of gene therapy. Scientific contributions deal with the role of various oncogens in cell transformation, the interaction between cancer and the immune system and, new tools for the molecular diagnosis of cancers. Focus was particularly put on the development of new vectors for gene therapy and antitumor cell vaccines for cell therapy.