Therapeutic effect of cytotoxic T lymphocyte antigen 4/immunoglobulin on a murine model of primary biliary cirrhosis.

UNLABELLED: Collectively, the data in both humans and murine models of human primary biliary cirrhosis (PBC) suggest that activated T cells, particularly CD8 T cells, play a critical role in biliary cell destruction. Under physiological conditions, T-cell activation involves two critical signals that involve the major histocompatibility complex and a set of costimulatory molecules, which include a receptor on T cells termed cytotoxic T lymphocyte antigen 4 (CTLA-4). Germane to the studies reported herein, signaling by CTLA-4 has the potential to modulate costimulation and induce inhibitory signals. In this study, we have taken advantage of our well-defined murine model of PBC, in which mice are immunized with 2-octynoic acid coupled to bovine serum albumin (2OA-BSA), leading to the production of high-titer antimitochondrial autoantibodies (AMAs) and portal cellular infiltrates. To investigate the potential of CTLA-4-Ig (immunoglobulin) as an immunotherapeutic agent, we treated mice both before and after induction of autoimmune cholangitis. First, we demonstrate that CTLA-4-Ig treatment, begun 1 day before 2OA-BSA immunization, completely inhibits the manifestations of cholangitis, including AMA production, intrahepatic T-cell infiltrates, and bile duct damage. However, and more critically, treatment with CTLA-4-Ig, initiated after the development of autoimmune cholangitis in previously immunized mice, also resulted in significant therapeutic benefit, including reduced intrahepatic T-cell infiltrates and biliary cell damage, although AMA levels were not altered. CONCLUSION: These data suggest that an optimized regimen with CTLA-4-Ig has the potential to serve as an investigative therapeutic tool in patients with PBC.

Animal models of primary biliary cirrhosis: materials and methods.

Primary biliary cirrhosis (PBC) is a female-predominant autoimmune disease of the liver characterized by immune-mediated destruction of the intrahepatic bile ducts and the presence of antimitochondrial antibodies (AMAs). There have been limited advances in understanding the molecular pathogenesis of the disease because of the difficulty in accessing human tissues and the absence of appropriate animal models. Recently, several unique murine models that manifest the serological, biochemical, and histological features similar to human PBC have been described. In this article, we discuss the current data on three spontaneous and two induced murine models of PBC. The spontaneous models are: (a) NOD.c3c4, (b) dominant negative TGF-ß receptor II (dnTGFßRII), and (c) IL-2Rα(-/-) mouse line models. The two induced models are: (a) xenobiotic and (b) Novosphingobium aromaticivorans immunized mice. These animal models provide various important platforms to further investigate the etiology and mechanisms of pathogenesis in PBC. Laboratory methodologies and the protocols that are used in evaluating these animal models are described. Finally, we stress the importance of realizing the strengths and limitations of the animal models are essential in data analysis and their application in therapeutic studies.

B cell depletion therapy exacerbates murine primary biliary cirrhosis.

UNLABELLED: Primary biliary cirrhosis (PBC) is considered a model autoimmune disease due to the clinical homogeneity of patients and the classic hallmark of antimitochondrial antibodies (AMAs). Indeed, the presence of AMAs represents the most highly directed and specific autoantibody in autoimmune diseases. However, the contribution of B cells to the pathogenesis of PBC is unclear. Therefore, although AMAs appear to interact with the biliary cell apotope and contribute to biliary pathology, there is no correlation of disease severity and titer of AMAs. The recent development of well-characterized monoclonal antibodies specific for the B cell populations, anti-CD20 and anti-CD79, and the development of a well-defined xenobiotic-induced model of autoimmune cholangitis prompted us to use these reagents and the model to address the contribution of B cells in the pathogenesis of murine PBC. Prior to the induction of autoimmune cholangitis, mice were treated with either anti-CD20, anti-CD79, or isotype-matched control monoclonal antibody and followed for B cell development, the appearance of AMAs, liver pathology, and cytokine production. Results of the studies reported herein show that the in vivo depletion of B cells using either anti-CD20 or anti-CD79 led to the development of a more severe form of cholangitis than observed in control mice, which is in contrast with results from several other autoimmune models that have documented an important therapeutic role of B cell-specific depletion. Anti-CD20/CD79-treated mice had increased liver T cell infiltrates and higher levels of proinflammatory cytokines. CONCLUSION: Our results reflect a novel disease-protective role of B cells in PBC and suggest that B cell depletion therapy in humans with PBC should be approached with caution.

Gene therapy in autoimmune diseases: challenges and opportunities.

Clinical treatment of autoimmune disorders presents a special challenge. For decades, most clinical regimens in autoimmunity has been largely symptomatic and non-disease specific. Although data from vigorous research has lead to accumulating knowledge on the pathogenic and immunological mechanisms of many autoimmune diseases, their direct clinical applications have been sparse. Advances in biotechnology have laid the groundwork for potent and specific molecular targeting therapies by gene therapy, and have just begun to be investigated in the treatment of autoimmune disorders. Such work has been largely based on the availability of well-established animal models of common autoimmune disorders, and the efficacy of strategic approaches initially investigated and validated in these models. Although these preclinical animal model studies have provided the proof-of-concept for multiple potential applications, human clinical trials on gene therapy in autoimmunity are still at its infancy. The recent success of Phase I/II clinical trials of gene therapy in rheumatoid arthritis and multiple sclerosis, development of cutting edge technology in target identification, as well as gene delivery systems have now set the stage for a more thorough and vigorous pace in the near future to advance this exciting field.

The immunological potential of galectin-1 and -3.

A family of beta-galactosides-binding proteins, called galectins, have recently emerged as novel molecules with immunoregulatory functions. These proteins are expressed in both inflammatory and non-inflammatory cells including monocytes, macrophages, dendritic cells, mast cells, and B and T cells, giving a broad spectrum of involvement in the immune response. Galectins are uniquely capable of acting both intracellularly and extracellularly, affecting such processes as cell adhesion, signaling, proliferation, differentiation, and apoptosis. Different members of this family have been shown to modulate several pathological processes such as allergic reactions, autoimmunity, and tumor invasion. Therefore, understanding the role of galectins in achieving appropriate proliferative and effector responses to antigens will yield important insights to autoimmune diseases and delineate novel strategies for disease intervention.

Rapid and quantitative DNA analysis of genetic mutations for polycystic kidney disease (PKD) using magnetic/luminescent nanoparticles.

Rapid and accurate detection of genetic mutations based on nanotechnology would provide substantial advances in detection of polycystic kidney disease (PKD), a disease whose current methods of detection are cumbersome due to the large size and duplication of the mutated gene. In this study, a nanotechnology-based DNA assay was developed for detection of SNPs (single nucleotide polymorphisms) in a feline autosomal dominant PKD (ADPKD) model which can readily be adapted to diagnosis of human ADPKD type 1. Europium and terbium phosphors were doped into gadolinium crystal hosts with a magnetic core, providing stable luminescence and the possibility of magnetic manipulations in a solution-based assay. A hybridization-in-solution DNA assay was optimized for feline PKD gene SNP detection using genomic DNA extracted from feline kidney tissue and blood. This assay showed a substantial differentiation between PKD and control specimens. The nanotechnology-based DNA assay is attractive from the viewpoint of rapid availability, simple methodology, and cost reduction for clinical use to detect mutations involved in human ADPKD and other genetic diseases.

Pathway analysis of kidney cancer using proteomics and metabolic profiling.

BACKGROUND: Renal cell carcinoma (RCC) is the sixth leading cause of cancer death and is responsible for 11,000 deaths per year in the US. Approximately one-third of patients present with disease which is already metastatic and for which there is currently no adequate treatment, and no biofluid screening tests exist for RCC. In this study, we have undertaken a comprehensive proteomic analysis and subsequently a pathway and network approach to identify biological processes involved in clear cell RCC (ccRCC). We have used these data to investigate urinary markers of RCC which could be applied to high-risk patients, or to those being followed for recurrence, for early diagnosis and treatment, thereby substantially reducing mortality of this disease. RESULTS: Using 2-dimensional electrophoresis and mass spectrometric analysis, we identified 31 proteins which were differentially expressed with a high degree of significance in ccRCC as compared to adjacent non-malignant tissue, and we confirmed some of these by immunoblotting, immunohistochemistry, and comparison to published transcriptomic data. When evaluated by several pathway and biological process analysis programs, these proteins are demonstrated to be involved with a high degree of confidence (p values < 2.0 E-05) in glycolysis, propanoate metabolism, pyruvate metabolism, urea cycle and arginine/proline metabolism, as well as in the non-metabolic p53 and FAS pathways. In a pilot study using random urine samples from both ccRCC and control patients, we performed metabolic profiling and found that only sorbitol, a component of an alternative glycolysis pathway, is significantly elevated at 5.4-fold in RCC patients as compared to controls. CONCLUSION: Extensive pathway and network analysis allowed for the discovery of highly significant pathways from a set of clear cell RCC samples. Knowledge of activation of these processes will lead to novel assays identifying their proteomic and/or metabolomic signatures in biofluids of patient at high risk for this disease; we provide pilot data for such a urinary bioassay. Furthermore, we demonstrate how the knowledge of networks, processes, and pathways altered in kidney cancer may be used to influence the choice of optimal therapy.

TNF-alpha-mediated apoptosis in vascular smooth muscle cells requires p73.

Atherosclerosis, now considered an inflammatory process, is the leading cause of death in the Western world and is manifested by a variety of diseases in multiple organ systems. Because of its prevalence and associated morbidity, novel therapies directed at arresting this progressive process are urgently needed. The inflammatory mediator TNF-alpha, which is known to contribute to apoptosis in vascular smooth muscle cells, has been shown to be intimately involved in the atherosclerotic process, being present at elevated levels in human atheroma as well as possibly being responsible for plaque rupture, a clinically devastating event. In light of our earlier finding that p73 is a proapoptotic protein in vascular smooth muscle cells, which are involved in plaque progression as well as rupture, we asked whether TNF-alpha mediates apoptosis in these cells through p73. We now show that p73 is present in spindle-shaped cells within human atheroma, and p73beta, an isoform that is pivotal in both apoptosis and growth suppression, is induced in vascular smooth muscle cells in vitro by serum but not by PDGF-BB. In addition, TNF-alpha, when added to these cells in the presence of serum-containing media, increases p73beta expression and causes apoptosis in both rat and human vascular smooth muscle cells. Inhibition of p73 activity with a dominant inhibitory NH2-terminally deleted p73 plasmid results in markedly decreased TNF-alpha-induced apoptosis. Thus p73beta is likely a mediator of the apoptotic effect of TNF-alpha in the vasculature, such that future targeting of the p73 isoforms may ultimately prove useful in novel atherosclerosis therapies.