Gerhild Wildner

Cells of the immune system and their cytokines in epiretinal membranes and in the vitreous of patients with proliferative diabetic retinopathy.

In the present study, we specifically investigated the presence of the main immune cells, namely T helper/inducer lymphocytes, T suppressor/cytotoxic lymphocytes, B lymphocytes and macrophages, for HLA-DR antigen expression and the cytokines IL-1 alpha and IL-2 in epiretinal membranes of proliferative diabetic retinopathy (PDR) using immunohistochemical staining. The levels of the two cytokines (IL-1 alpha and IL-2) in the vitreous were measured by ELISA. The results show that 18 out of the 32 epiretinal specimens reacted positively with monoclonal anti-CD4 (marker for T helper/inducer cell subset) antibody (56%), 21 reacted with monoclonal anti-CD8 (marker for T suppressor/cytotoxic subset) antibody (66%), 15 reacted with monoclonal anti-CD22 (marker for B lymphocytes) antibody (47%), and 25 (78%) were positive for monoclonal anti-macrophage and anti-HLA-DR antibodies. IL-1 alpha and IL-2 were detected in the epiretinal membranes in 8 out of 13 tested cases (62%). However, in the vitreous, IL-1 alpha was detected in only 3 out of 13 cases (70, 75 and 80 pg/ml, respectively), while IL-2 was not detected in any of the vitreous samples. The results clearly demonstrate that immune cells and their cytokines were found in about half of the epiretinal membrane specimens. The invasion of the immunocompetent cells in the membranes seems to be a secondary event. They would not participate in the pathogenesis of PDR, but probably induce unspecific reactions and thus complicate the disease.

Latency and reactivation of a precore mutant hepatitis B virus in a chronically infected patient

In human patients infected with hepatitis B virus (HBV) seroconversion from HBe to anti-HBe often signals virus elimination. Occasionally, a second viremic phase is observed which may be due to superinfection with a variant or reactivation of a latent virus. To differentiate between these two possibilities we investigated the nucleotide sequences of virus populations from sera of a chronically infected patient who had two distinct viremic phases, one e-antigen positive and one anti-HBe antibody positive. By direct sequencing of amplified HBV C- and pre-S gene sequences we found that the viruses in the two populations differed by only two point mutations, one of which prevents expression of precore derived e-antigen. In the nonviremic phase viral DNA and antigen were found in the liver but nucleocapsid protein expression appeared drastically downregulated. These data demonstrate that HBV can enter a latent phase with low expression of core protein and selection for HBV variants which cannot synthesize e-antigen. This suggests that e-antigen expression can be a critical target for virus elimination and that loss of its expression can prolong chronicity and provoke latency of HBV infection.

Rationally Evolving MCP-1/CCL2 into a Decoy Protein with Potent Anti-inflammatory Activity in Vivo

Leukocyte recruitment from the blood into injured tissues during inflammatory diseases is the result of sequential events involving chemokines binding to their GPC receptors as well as to their glycosaminoglycan (GAG) co-receptors. The induction and the crucial role of MCP-1/CCL2 in the course of diseases that feature monocyte-rich infiltrates have been validated in many animal models, and several MCP-1/CCL2 as well as CCR2 antagonists have since been generated. However, despite some of them being shown to be efficacious in a number of animal models, many failed in clinical trials, and therapeutically interfering with the activity of this chemokine is not yet possible. We have therefore generated novel MCP-1/CCL2 mutants with increased GAG binding affinity and knocked out CCR2 activity, which were designed to interrupt the MCP-1/CCL2-related signaling cascade. We provide evidence that our lead mutant MCP-1(Y13A/S21K/Q23R) exhibits a 4-fold higher affinity toward the natural MCP-1 GAG ligand heparan sulfate and that it shows a complete deficiency in activating CCR2 on THP-1 cells. Furthermore, a significantly longer residual time on GAG ligands was observed by surface plasmon resonance. Finally, we were able to show that MCP-1(Y13A/S21K/Q23R) had a mild ameliorating effect on experimental autoimmune uveitis and that a marginal effect on oral tolerance in the group co-fed with Met-MCP-1(Y13A/S21K/Q23R) plus immunogenic peptide PDSAg was observed. These results suggest that disrupting wild type chemokine-GAG interactions by a chemokine-based antagonist can result in anti-inflammatory activity that could have potential therapeutic implications.

Mechanism, kinetics, and role of duck hepatitis B virus e-antigen expression in vivo

No duck hepatitis B virus (DHBV) pre-C transcript has been identified so far, and neither the interrelationship of e-antigen (DHBeAg) with the expression of other viral antigens or virus replication nor its function is known. In this study we identified in infected livers a minor transcript from which the precursor protein of DHBeAg could be synthesized. Mutation of the first AUG on this transcript abolished expression of DHBeAg. DHBV genomes containing this mutation were infectious in Pekin ducks, the kinetics of pre-S envelope protein expression and virus secretion were not significantly different from wild-type, and the mutant genomes did not revert to wild-type to a detectable level after several passages. In contrast to pre-S protein, the level of DHBeAg in the serum was independent of the level of viremia, accumulated gradually to a high and constant level after a lag phase, and was also easily detectable in a mixed infection containing less than 0.1% of wild-type in a pre-C mutant virus containing inoculum. These data indicate that precore protein is synthesized from a minor pre-C mRNA with translation initiation at the pre-C AUG codon, and leads to high levels of DHBeAg rather late in infection. High levels of DHBeAg can even be produced efficiently by a very small subpopulation of wild-type virus in a mixed infection with predominantly pre-C mutant virus. Lack of DHBeAg appears to have no effect on DHBV viability and kinetics of virus secretion into the bloodstream when ducklings are infected with the pre-C AUG mutant virus a few days after birth.

Dynamics of Intraocular IFN-γ, IL-17 and IL-10-Producing Cell Populations during Relapsing and Monophasic Rat Experimental Autoimmune Uveitis

A major limitation of most animal models of autoimmune diseases is that they do not reproduce the chronic or relapsing-remitting pattern characteristic of many human autoimmune diseases. This problem has been overcome in our rat models of experimentally induced monophasic or relapsing-remitting autoimmune uveitis (EAU), which depend on the inducing antigen peptides from retinal S-Antigen (monophasic EAU) or interphotoreceptor retinoid-binding protein (relapsing EAU). These models enable us to compare autoreactive and regulatory T cell populations. Intraocular, but not peripheral T cells differ in their cytokine profiles (IFN-γ, IL-17 and IL-10) at distinct time points during monophasic or relapsing EAU. Only intraocular T cells concomitantly produced IFN-γ, IL-17 and/or IL-10. Monophasic EAU presented rising numbers of cells expressing IFN-γ and IL-17 (Th1/Th17) and cells expressing IL-10 or Foxp3. During relapsing uveitis an increase of intraocular IFN-γ+ cells and a concomitant decrease of IL-17+ cells was detected, while IL-10+ populations remained stable. Foxp3+ cells and cells expressing IL-10, even in combination with IFN-γ or IL-17, increased during the resolution of monophasic EAU, suggesting a regulatory role for these T cells. In general, cells producing multiple cytokines increased in monophasic and decreased in relapsing EAU. The distinct appearance of certain intraocular populations with characteristics of regulatory cells points to a differential influence of the ocular environment on T cells that induce acute and monophasic or relapsing disease. Here we provide evidence that different autoantigens can elicit distinct and differently regulated immune responses. IFN-γ, but not IL-17 seems to be the key player in relapsing-remitting uveitis, as shown by increased, synchronized relapses after intraocular application of IFN-γ. We demonstrated dynamic changes of the cytokine pattern during monophasic and relapsing-remitting disease with strongly increasing IL-10 expression in intraocular T cells during monophasic uveitis.

Characterization of infectious and defective cloned avian hepadnavirus genomes

The infectivity in vivo, replication competence in vitro, and expression of viral genes of several molecularly cloned duck hepatitis B virus (DHBV) genomes were investigated. In addition, replication competence, core protein expression, and secretion of viral proteins were investigated for a grey heron hepatitis B virus genome. Except two, all DHBV isolates tested induced a systemic infection in Pekin ducks when injected as cloned viral DNA into the liver. After transfection of chicken hepatoma cells, both defective DHBV genomes expressed intracellular nucleocapsid and pre-S envelope proteins and secreted DHBs/pre-S particles into the medium. One of the defective DHBV genomes and HHBV produced within the cells replicative intermediates encapsidated in core particles and secreted virions, whereas the other defective DHBV genome did not and was unable to efficiently encapsidate the RNA pregenome. Comparative sequence analysis was performed to identify potential amino acid changes in viral proteins of both defective DHBV genomes. The data obtained demonstrate that most cloned avian hepadnaviruses are infectious or replication competent and suggest defects in envelope, polymerase or encapsidation function, respectively, in two cloned DHBV genomes.

Spontaneously relapsing-remitting experimental autoimmune uveitis in rats allows successful therapeutic oral tolerance induction in ongoing disease.

Antigen-specific tolerance induction is a desired therapy for uveitis patients. Our relapsing-remitting rat model of experimental autoimmune uveitis (EAU) induced with IRBP peptide R14 enables us to test the effect of oral tolerance on the prevention of relapsing uveitis. We investigated several peptides overlapping the sequence of R14 for prevention and different doses of R14 for therapy to determine the tolerogenic epitope and the most effective therapeutic regimen for uveitis. Lewis rats were immunized with R14-CFA to induce EAU. Oral tolerance was induced prior to immunization (prevention) or after onset of EAU to prevent relapses (therapy). Therapeutic feeding was performed with high and/or low doses of oral antigen for clonal deletion of effector and induction of regulatory T cells. Uveitis was determined clinically and histologically; mesenteric lymph node (mLN) cells of tolerized rats were tested for surface markers, cytokines and Foxp3 expression. Preventive feeding of R14 and its major epitope R16, but none of the overlapping peptides significantly suppressed EAU and also prevented relapses, irrespective of their pathogenicity. Therapeutic feeding with R14 dramatically reduced relapses, while only the consecutive feeding of high and low-dose R14 had an ameliorating effect on the first course of disease. IL-10-producing T cells from mLN decreased after oral tolerization, and with R14-stimulation in vitro the TCRαβ+/Foxp3+ population increased in the low-dose fed group. No mLN population could be clearly assigned to successful oral tolerance induction during active autoimmune uveitis.

The immunopathogenesis of chronic and relapsing autoimmune uveitis – Lessons from experimental rat models

ABSTRACT Autoimmune diseases usually follow a relapsing‐remitting or a chronic progressive course. To understand the underlying immunopathogenesis we investigated experimental Lewis rat models displaying both disease types, which were only dependent on the autoantigen peptide used for immunization. Retinal S‐Antigen‐peptide PDSAg induces chronic, monophasic disease, whilst interphotoreceptor retinoid‐binding protein (IRBP)‐peptide R14 causes a spontaneously relapsing‐remitting course. R14‐mediated uveitis can be re‐induced by immunization; PDSAg‐induced disease is even preventable by prior CFA‐injection. T cells with different antigen specificities preferentially infiltrate the eyes from different sites, e.g. choroid or retinal vessels, they remain in the retina after resolution of inflammation for many weeks. The major inflammatory cell populations in the eyes during rat uveitis are CD4+ or CD8+ monocytes/macrophages. Chemokine mutants only suppress PDSAg‐mediated EAU, while IFN‐&agr;‐treatment ameliorated R14‐, but worsened PDSAg‐induced disease. Comparison of T cells revealed upregulated expression of 26 genes related to various signal transduction pathways upstream and downstream of IFN‐&ggr; only in T cells causing relapsing EAU. Intraocular injection of IFN‐&ggr; induces synchronized relapses in R14‐mediated uveitis, while VEGF‐expression of PDSAg‐specific T cells causing chronic disease induced chorioretinal neovascularization that is suppressed by anti‐CD146 antibody. Intraocular T cells from rat eyes during EAU express IL‐17, IFN‐&ggr; or IL‐10, with dynamic changes of the cell populations during the disease course, differing in both disease types. Immunization of animals with a mixture of both antigens suppressed relapses, indicating a dominance of the monophasic disease. Understanding the exact pathogenesis of both disease courses is key to developing novel therapies for autoimmune diseases. HIGHLIGHTSEAU in Lewis rats represents monophasic/chronic or relapsing uveitis in patients.Similar clinical pictures but different responses to same therapeutic approaches.Autoreactive T cells coproducing inflammatory cytokines and IL‐10 found only in eyes.CD4+ autoreactive T cells of both diseases differ in gene/protein expression.Chronic EAU develops neovascularization and dominates over the relapsing disease.