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.

Micronuclei to detect in vivo chemotherapy damage in a p53 mutated solid tumour.

Apoptosis induction and micronuclei formation were compared following cytotoxic treatments in two rat glioma differing in p53 integrity. In vitro, micronuclei emergence but not apoptosis was linked to the p53 mutated status. In vivo, micronuclei assays were more sensitive to evaluate DNA damage induced by chemotherapy in a p53-mutated solid tumour.

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.

[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.

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.

The P2Y11 receptor mediates the ATP-induced maturation of human monocyte-derived dendritic cells.

Recently, it has been shown that ATP and TNF-alpha synergize in the activation and maturation of human dendritic cells (DC); the effect of ATP was reproduced by hydrolysis-resistant derivatives of ATP and was blocked by suramin, suggesting the involvement of a P2 receptor, but the particular subtype involved was not identified. In this report we confirm that ATP and various derivatives synergize with TNF-alpha and LPS to induce the maturation of human monocyte-derived DC, as revealed by up-regulation of the CD83 marker and the secretion of IL-12. The rank order of potency of various analogs (AR-C67085 > adenosine 5'-O-(3-thiotriphosphate) = 2'- and 3'-O-(4-benzoyl-benzoyl) ATP > ATP > 2-methylthio-ATP) was close to that of the recombinant human P2Y11 receptor. Furthermore, these compounds activated cAMP production in DC, in a xanthine-insensitive way, consistent with the involvement of the P2Y11 receptor, which among P2Y subtypes has the unique feature of being dually coupled to phospholipase C and adenylyl cyclase activation. The involvement of the P2Y11/cAMP/protein kinase A signaling pathway in the nucleotide-induced maturation of DC is supported by the inhibitory effect of H89, a protein kinase A inhibitor. Taken together, our results demonstrate that ATP activates DC through stimulation of the P2Y11 receptor and subsequent increase in intracellular cAMP.

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.

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.

The two SH2-domain-containing inositol 5-phosphatases SHIP1 and SHIP2 are coexpressed in human T lymphocytes.

The activation of many hematopoietic cells via cytokine receptors, as well as B and T cell receptors, leads to the tyrosine phosphorylation of Shc and its association with both Grb2-Sos1 complexes and with a 145 kDa protein referred to as the SH2 containing inositol 5-phosphatase (SHIP1). In a search of putative 5-phosphatase isoenzymes, we have isolated a second SH2 domain containing inositol 5-phosphatase, referred to as (SHIP2). Both SHIP1 and SHIP2 are coexpressed in human T lymphocytes. This was shown at the protein level by Western blot analysis in transformed T cell lines and in peripheral blood T lymphocytes either unstimulated or after in vitro activation through TCR-CD3 complex. SHIP1 protein level was not modulated after activation of T lymphocytes, in contrast to SHIP2, which was increased after long-term stimulation. SHIP1 was tyrosine phosphorylated in resting naive T cells. This was not observed in the transformed T cell lines. T lymphocyte is therefore a model of coexpression of the two SH2-containing inositol 5-phosphatases SHIP1 and SHIP2.

Cellular vaccines.

This project is devoted to the development of novel cellular vaccines designed to treat cancer patients. These cellular vaccines present and enhance immunogens, which will elicit a potent immune response. The goal is to achieve safe and effective immune reaction against the patient's own tumour. (1) Autologous cellular vaccines are prepared by processing circulating blood mononuclear cells outside of the patient's body (ex vivo) to differentiate them into antigen-presenting cells (APCs). Monocyte-derived APCs (MD-APCs) are then grown in the presence of exogenous target antigens (tumour cell debris, or apoptotic bodies) to become fully mature APCs. (2) Functionality for antigen presentation to T cells of ex vivo MD-APCs is evaluated in vivo. (3) Cellular vaccines are tested in selected rodent animal models. Efficiency and immune response are monitored in pertinent experimental systems for cancer. Pharmacological data are generated for clinical investigation. Tolerance and biologic effects are documented in primates. (4) The first clinical trials on cancer patients are taking place in 1998 on melanoma and prostate cancer to validate the concept. Specialized cell processors with dedicated software and standardized controls are being developed and used for the preparation of cellular vaccines. (5) The evaluation of new non-viral vectors and the validation of new non-viral transfection methods of mononuclear cells with marker genes is in progress and will lead to the ex vivo transfection of genes coding for immunostimulating cytokines or for tumour antigens in MD-APCs. Efficiency will be validated in vitro and in animal models. The ex vivo and animal model studies validate the clinical relevance of this new cellular immunotechnology. Clinical validation of individual autologous cellular vaccines in specific indications for which no treatment is presently available will allow the development of cellular and gene immunotherapy for other types of cancers.

Methylprednisolone differentially regulates IL-10 and tumour necrosis factor (TNF) production during murine endotoxaemia.

IL-10 is an endogenous antiinflammatory cytokine that inhibits TNF biosynthesis and protects mice from lipopolysaccharide (LPS)-induced lethality. As synthetic glucocorticoids are widely used as antiinflammatory agents, we analysed the effects of methylprednisolone administration on IL-10 biosynthesis during murine endotoxaemia. We found that low doses of methylprednisolone (2-10 mg/kg) markedly inhibited TNF production but did not affect serum levels of IL-10, while a high methylprednisolone dose (50 mg/kg) increased LPS-induced IL-10 levels. In parallel, we observed that LPS-induced IL-10 production is TNF-independent in this experimental setting. Experiments conducted in vitro indicated that methylprednisolone (from 0.01 to 100 micrograms/ml) also increased the biosynthesis of IL-10 by LPS-activated mouse peritoneal macrophages. We conclude that methylprednisolone differentially regulates IL-10 and TNF production induced by LPS both in vivo and in vitro at the macrophage level.

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.

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.

IFN-gamma prevents Th2 cell-mediated pathology after neonatal injection of semiallogenic spleen cells in mice.

BALB/c mice injected at birth with 10(8) (A/J X BALB/c)F1 hybrid spleen cells develop an autoimmune host-vs-graft (HVG) disease as a result of activation of donor B cells by host CD4+ cells. The antidonor CD4+ cells seem to be Th2-like cells, inasmuch as they are profoundly deficient in IL-2 and IFN-gamma production, but secrete high levels of IL-4 and IL-10. As IFN-gamma is known to inhibit the development of TH2 cells, we attempted to modulate HVG disease by injecting rIFN-gamma. First, we found that 10 micrograms of rIFN-gamma given on days 1 and 3 after birth reduced the serum hyper-IgE of HVG mice by 90% and the serum hyper-IgG1, by 70%. In addition, rIFN-gamma administration significantly decreased the anti-DNA IgG1 titers and prevented the occurrence of anti-glomerular basement membrane and anti-laminin IgG1 Abs as well as the formation of immune deposits in renal glomeruli. These effects were not caused by the abrogation of chimerism, as indicated by the persistence of donor-type B cells in lymph nodes and of Igs bearing donor allotype in serum. MLC experiments indicated that the major effect of early rIFN-gamma administration was to restore the production of IL-2 and IFN-gamma by donor-specific T cells while these cells still secreted significant amounts of IL-4 and IL-10. Unresponsiveness of antidonor cytolytic T cells was not influenced by rIFN-gamma. We conclude that rIFN-gamma prevents the TH2-type response induced by the neonatal injection of semiallogeneic spleen cells and the associated pathology.

Interleukin-10 controls interferon-gamma and tumor necrosis factor production during experimental endotoxemia.

Interleukin-10 (IL-10) is a potent inhibitor of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) production and has been shown to protect mice from endotoxin shock. As IFN-gamma is another important mediator of LPS toxicity, we studied the effects of IL-10 on LPS-induced IFN-gamma synthesis in vitro and in vivo. First, we found that the addition of recombinant human IL-10 (rhIL-10) (10 U/ml) to human whole blood markedly suppressed LPS-induced IFN-gamma release while neutralization of endogenously synthesized IL-10 resulted in increased IFN-gamma levels. The ability of rIL-10 to inhibit LPS-induced IFN-gamma synthesis was also observed in vivo in mice. Indeed, administration of 1000 U recombinant mouse IL-10 (rmIL-10) 30 min before and 3 h after challenge of BALB/c mice with 100 micrograms LPS resulted in a threefold decrease in peak IFN-gamma serum levels. We then examined the production and the role of IL-10 during murine endotoxemia. We found that LPS injection causes the rapid release of IL-10, peak IL-10 serum levels being observed 90 min after LPS challenge. Neutralization of endogenously produced IL-10 by administration of 2 mg JES5-2A5 anti-IL-10 monoclonal antibody (mAb) 2 h before LPS challenge resulted in a marked increase in both TNF and IFN-gamma serum levels while irrelevant isotype-matched mAb had no effect. The enhanced production of inflammatory cytokines in anti-IL-10 mAb-treated mice was associated with a 60% lethality after injection of 500 micrograms LPS, while all mice pretreated with control mAb survived. We conclude that the rapid release of IL-10 during endotoxemia is a natural antiinflammatory response controlling cytokine production and LPS toxicity.