Facilitating collaborative animal research: The development and implementation of a Master Reciprocal Institutional Agreement for Animal Care and Use.

Ensuring appropriate review, approval, and oversight of research involving animals becomes increasingly complex when researchers collaborate across multiple sites. In these situations, it is important that the division of responsibilities is clear and that all involved parties share a common understanding. The National Institutes of Health Office of Laboratory Animal Welfare and the United States Department of Agriculture Animal Plant Health Inspection Service require an Institutional Animal Care and Use Committee (IACUC) to review the care and use of animals in research, and both agree that it is acceptable for one IACUC to review the work taking place at multiple institutions. With this in mind, several Harvard-affiliated hospitals and academic centers developed the Master Reciprocal Institutional Agreement for Animal Care and Use (Master IACUC Agreement) to support collaboration, decrease administrative burden, increase efficiencies, reduce duplicative efforts, and ensure appropriate protections for animals used in research. Locally, the Master IACUC Agreement has fostered greater collaboration and exchange while ensuring appropriate review and oversight of research involving animals. As multisite animal protocols become more prevalent, this Agreement could provide a model for a distributed, national network of IACUC reliance.

Characterization of the anti-angiogenic properties of arresten, an alpha1beta1 integrin-dependent collagen-derived tumor suppressor.

Physiological and pathological turnover of basement membranes liberates biologically active cryptic molecules. Several collagen-derived fragments possess anti-angiogenic activity. Arresten is the 26-kDa non-collagenous domain of type IV collagen alpha1 chain. It functions as an efficient inhibitor of angiogenesis and tumor growth in mouse models, but its anti-angiogenic mechanism is not completely known. Here we show that arresten significantly increases apoptosis of endothelial cells in vitro by decreasing the amount of anti-apoptotic molecules of the Bcl-family; Bcl-2 and Bcl-xL. Although the pro-apoptotic effect of arresten is endothelial cell specific in vitro, in mouse tumors arresten induced apoptosis both in endothelial and tumor cells. The tumor cell apoptosis is likely an indirect effect due to the inhibition of blood vessel growth into the tumor. The active site of arresten was localized by deletion mutagenesis within the C-terminal half of the molecule. We have previously shown that arresten binds to alpha1beta1 integrin on human umbilical vein endothelial cells. However, the microvascular endothelial cells (MLECs) are more important in the context of tumor vasculature. We show here that arresten binds also to the microvascular endothelial cells via alpha1beta1 integrin. Furthermore, it has no effect on Matrigel neovascularization or the viability of integrin alpha1 null MLECs. Tumors implanted on integrin alpha1 deficient mice show no integrin alpha1 expression in the host-derived vascular endothelium, and thus arresten does not inhibit the tumor growth. Collectively, this data sheds more light into the anti-angiogenic mechanism of arresten.

Zebrafish to humans: evolution of the alpha3-chain of type IV collagen and emergence of the autoimmune epitopes associated with Goodpasture syndrome.

Goodpasture syndrome is an autoimmune vascular disease associated with kidney and lung failure, with pathogenic circulating autoantibodies targeted to a set of discontinuous epitope sequences within the noncollagenous domain-1 (NC1) of the alpha3 chain of type IV collagen (alpha3(IV)NC1), the Goodpasture autoantigen. We demonstrate that basement membrane extracted NC1 domain preparations from Caenorhabditis elegans, Drosophila melanogaster, and Danio rerio do not bind Goodpasture autoantibodies, while Xenopus laevis, chicken, mouse and human alpha3(IV)NC1 domains bind autoantibodies. The alpha3(IV) chain is not present in C elegans and Drosophila melanogaster, but is first detected in the Danio rerio. Interestingly, native Danio rerio alpha3(IV)NC1 does not bind Goodpasture autoantibodies. Next, we cloned, sequenced, and generated recombinant Danio rerio alpha3(IV)NC1 domain. In contrast to recombinant human alpha3(IV)NC1 domain, there was complete absence of autoantibody binding to recombinant Danio rerio alpha3(IV)NC1. Three-dimensional molecular modeling from existing x-ray coordinates of human NC1 domain suggest that evolutionary alteration of electrostatic charge and polarity due to the emergence of critical serine, aspartic acid, and lysine residues, accompanied by the loss of asparagine and glutamine, contributes to the emergence of the 2 major Goodpasture epitopes on the human alpha3(IV)NC1 domain, as it evolved from the Danio rerio over 450 million years.

Nasal administration of recombinant rat alpha3(IV)NC1 prevents the development of experimental autoimmune glomerulonephritis in the WKY rat.

Experimental autoimmune glomerulonephritis (EAG), an animal model of Goodpasture's disease, can be induced in Wistar Kyoto (WKY) rats by immunization with either collagenase-solubilized rat glomerular basement membrane (GBM) or the recombinant NC1 domain of the alpha3 chain of type IV collagen [alpha3(IV)NC1]. EAG is characterized by circulating and deposited anti-glomerular basement membrane antibodies, focal necrotizing glomerulonephritis with crescent formation, and glomerular infiltration by T cells and macrophages. Previous studies have demonstrated that oral administration of collagenase-solubilized GBM to WKY rats prevented the development of EAG. Nasal administration of specific autoantigens has been reported to be more effective than oral administration in other models of autoimmune disease. The main aim of this study was to investigate further the concept of mucosal tolerance in EAG by examining the effect of nasal administration of recombinant rat alpha3(IV)NC1. Groups of WKY rats with EAG, induced by immunization with recombinant rat alpha3(IV)NC1, were given alpha3(IV)NC1 nasally on 3 consecutive days before immunization, at total cumulative doses of 25, 100, or 250 microg per rat. A dose-dependent effect was observed on the development of EAG. A dose of 25 microg had no effect on disease; 100 microg resulted in a moderate reduction in the severity of nephritis; and 250 microg led to a marked reduction in circulating and deposited antibodies, albuminuria, severity of glomerular abnormalities, and numbers of glomerular CD8+ T cells and macrophages. In addition, there was a reduction in the proliferative response of splenocytes from rats in the high dose group (250 microg) to alpha3(IV)NC1 in vitro. The results from this study clearly demonstrate for the first time that mucosal tolerance in EAG can be induced by nasal administration of recombinant rat alpha3(IV)NC1 and that this approach is effective in the prevention of crescentic glomerulonephritis. Further work using new antigen-specific treatment strategies may provide a novel approach to the treatment of patients with anti-glomerular basement membrane disease.

Regulation by CD25+ lymphocytes of autoantigen-specific T-cell responses in Goodpasture's (anti-GBM) disease.

BACKGROUND: Goodpasture's, or anti-glomerular basement membrane (GBM), disease is unusual among autoimmune diseases in that it rarely follows a relapsing-remitting course. Moreover, untreated, autoantibodies disappear spontaneously after 1 to 3 years and, following treatment, autoreactive T cells diminish in frequency. This suggests that operational tolerance toward the autoantigen is reestablished. However, the mechanisms underlying this have remained unclear. Recent data have suggested that a population of regulatory T lymphocytes can suppress both autoimmune and alloimmune responses in animal models and are present in normal individuals. However, to date, they have not been demonstrated to play a role in human renal autoimmune disease. METHODS: We studied the role of regulatory CD25+ cells in suppressing T-cell responses to the Goodpasture autoantigen in nine patients with Goodpasture's disease. RESULTS: At the time of acute presentation, there was no evidence of a regulatory cell population. However, from 3 months onward a population emerged, capable of suppressing the response to the Goodpasture autoantigen. Following depletion of CD25+ cells, the frequencies of autoreactive-, GBM-, or collagen alpha 3(IV)NC1-specific T cells were significantly increased (P = 0.031 by paired t test), with five of seven (71%) convalescent patients and no acute patients demonstrating regulation. CONCLUSION: These data demonstrate that, in Goodpasture's disease, regulatory CD25+ T cells play a role in inhibiting the autoimmune response. Their emergence and persistence may underlie the "single hit" nature of this condition. Understanding the conditions required for the development and propagation of these cells would allow development of novel therapeutic strategies for inducing hyporesponsiveness in autoimmune disease.

Liver fibrosis: insights into migration of hepatic stellate cells in response to extracellular matrix and growth factors.

BACKGROUND & AIMS: In liver fibrosis, alterations within the space of Disse microenvironment occur and facilitate further progression of chronic liver disease. The normal basement membrane-like matrix present within the space of Disse converts to a matrix rich in fibril-forming collagens during fibrosis. METHODS: To further understand the pathogenesis of liver fibrosis, we modified an in vitro Boyden chamber system to partially mimic in vivo conditions of hepatic stellate cells (HSCs) during health and disease. RESULTS: Stimulation of HSCs with platelet-derived growth factor (PDGF)-BB, transforming growth factor (TGF)-beta1, and/or epithelial growth factor (EGF) resulted in an increase in their migratory capacity and up-regulated matrix metalloproteinase (MMP)-2 activity. Migration induced by PDGF-BB was associated with increased proliferation, whereas TGF-beta1/EGF-induced migration was proliferation independent. COL-3, an inhibitor of MMP-2 and MMP-9, inhibited migration of HSCs induced by direct activation of PDGF-BB or TGF-beta1 but had no effect on migration induced by chemotactic stimuli without direct contact, suggesting 2 distinct MMP-dependent and MMP-independent mechanisms of PDGF-BB- or TGF-beta1-induced migration. Additionally, we show that type I collagen by itself induced migration of HSCs. Migration induced by PDGF-BB, TGF-beta1, and collagen I could be inhibited by alpha(1)- and/or alpha(2)-integrin blocking antibodies, collectively suggesting an integrin-dependent, MMP-2-mediated migration of HSCs. CONCLUSIONS: Basement membrane matrix integrity, composition, and cell-matrix interactions play an important role in anchoring HSCs and preventing them from spreading within the space of Disse and potentially elsewhere in the liver. Additionally, our data provide strong evidence for MMPs in regulation of HSCs migration.