Membrane cofactor protein (MCP; CD46), a widely distributed regulator of complement activation, is a cofactor for the factor I-mediated degradation of C3b and C4b deposited on host cells. MCP possesses four extracellular, contiguous complement control protein modules (CCPs) important for this inhibitory activity. The goal of the present study was to delineate functional sites within these modules. We employed multiple approaches including mutagenesis, epitope mapping, and comparisons to primate MCP to make the following observations. First, functional sites were located to each of the four CCPs. Second, some residues were important for both C3b and C4b interactions while others were specific for one or the other. Third, while a reduction in ligand binding was invariably accompanied by a parallel reduction in cofactor activity (CA), other mutants lost or had reduced CA but retained ligand binding. Fourth, two C4b-regulatory domains overlapped measles virus interactive regions, indicating that the hemagglutinin docks to a site important for complement inhibition. Fifth, several MCP regulatory areas corresponded to functionally critical, homologous positions in other CCP-bearing C3b/C4b-binding proteins. Based on these data and the recently derived crystal structure of repeats one and two, computer modeling was employed to predict MCP structure and examine active sites.

Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival times. The efficacy of current systemic therapies for HCC is limited. In this study, we used xenograft tumor models to investigate the use of antibodies that block CD47 and inhibit HCC tumor growth. Immunostaining of tumor tissue and HCC cell lines demonstrated CD47 over-expression in HCC as compared to normal hepatocytes. Macrophage phagocytosis of HCC cells was increased after treatment with CD47 antibodies (CD47mAbs) that block CD47 binding to SIRPα. Further, CD47 blockade inhibited tumor growth in both heterotopic and orthotopic models of HCC, and promoted the migration of macrophages into the tumor mass. Our results demonstrate that targeting CD47 by specific antibodies has potential immunotherapeutic efficacy in human HCC.

Recent studies strongly suggest an increasing role for immune responses against self-antigens (Ags) which are not encoded by the major histocompatibility complex in the immunopathogenesis of allograft rejection. Although, improved surgical techniques coupled with improved methods to detect and avoid sensitization against donor human leukocyte antigen (HLA) have improved the immediate and short term function of transplanted organs. However, acute and chronic rejection still remains a vexing problem for the long term function of the transplanted organ. Immediately following organ transplantation, several factors both immune and non immune mechanisms lead to the development of local inflammatory milieu which sets the stage for allograft rejection.Traditionally, development of antibodies (Abs) against mismatched donor HLA have been implicated in the development of Ab mediated rejection. However, recent studies from our laboratory and others have demonstrated that development of humoral and cellular immune responses against non-HLA self-Ags may contribute in the pathogenesis of allograft rejection. There are reports demonstrating that immune responses to self-Ags especially Abs to the self-Ags as well as cellular immune responses especially through IL17 has significant pro-fibrotic properties leading to chronic allograft failure. This review summarizes recent studies demonstrating the role for immune responses to self-Ags in allograft immunity leading to rejection as well as present recent evidence suggesting there is interplay between allo-and autoimmunity leading to allograft dysfunction.

Immune responses against lung-associated self-antigens (self-Ags) are hypothesized to play a role in the development of chronic lung graft rejection. We determined whether immune responses to lung self-Ags, K-alpha-1-tubulin (Kα1T) and Collagen V (Col-V) in the absence of alloimmunity, could promote airway inflammation and fibrosis. Following syngeneic murine orthotopic lung transplantation (LTx) we administered antibodies (Abs) to either Kα1T or Col-V or in combination to both of these self-Ags. As compared to recipients of isotype control Abs Kα1T Abs and/or Col-V Abs-treated recipients had marked lung graft cellular infiltration and bronchiolar fibrosis, This inflammation was also associated the accumulation of Kα1T and Col-V specific IFN-γ+ and IL-17+ T cells. Notably, the administration of Abs to Kα1T led to cellular and humoral immune responses to Col-V prior to development of fibrosis, and vice versa, indicating that epitope spreading can occur rapidly in an alloantigen independent manner. Collectively, these data support a model of chronic lung transplant rejection where the progressive loss of self-tolerance through epitope spreading promotes airway fibrosis. Strategies that target autoreactive Abs may be useful to inhibit chronic rejection of lung grafts.

Liver disease due to hepatitis C virus (HCV) infection is an important health problem worldwide. HCV induced changes in microRNAs (miRNA) are shown to mediate inflammation leading to liver fibrosis. Gene expression analyses identified dysregulation of miRNA-449a in HCV patients but not in alcoholic and non-alcoholic liver diseases. By sequence analysis of the promoter for YKL40, an inflammatory marker upregulated in patients with chronic liver diseases with fibrosis, adjacent binding sites for nuclear factor of Kappa B/P65 and CCAAT/enhancer-binding protein alpha (CEBPα) were identified. P65 interacted with CEBPα to co-operatively activate YKL40 expression through sequence specific DNA binding. In vitro analysis demonstrated that tumor necrosis factor alpha (TNFα) mediated YKL40 expression is regulated by miRNA-449a and its target NOTCH1 in human hepatocytes.NOTCH1 facilitated nuclear localization of P65 in response to TNFα. Further, HCV patients demonstrated upregulation of NOTCH1 along with downregulation of miRNA-449a. Taken together it is demonstrated that miRNA-449a plays an important role in modulating expression of YKL40 through targeting the components of the NOTCH signaling pathway following HCV infection. Therefore, defining transcriptional regulatory mechanisms which control inflammatory responses and fibrosis will be important towards developing strategies to prevent hepatic fibrosis especially following HCV recurrence in liver transplant recipients.

The goal of this study was to identify the site(s) in CR1 that mediate the dissociation of the C3 and C5 convertases. To that end, truncated derivatives of CR1 whose extracellular part is composed of 30 tandem repeating modules, termed complement control protein repeats (CCPs), were generated. Site 1 (CCPs 1-3) alone mediated the decay acceleration of the classical and alternative pathway C3 convertases. Site 2 (CCPs 8 -10 or the nearly identical CCPs 15-17) had one-fifth the activity of site 1. In contrast, for the C5 convertase, site 1 had only 0.5% of the decay accelerating activity, while site 2 had no detectable activity. Efficient C5 decay accelerating activity was detected in recombinants that carried both site 1 and site 2. The activity was reduced if the intervening repeats between site 1 and site 2 were deleted. The results indicate that, for the C5 convertases, decay accelerating activity is mediated primarily by site 1. A properly spaced site 2 has an important auxiliary role, which may involve its C3b binding capacity. Moreover, using homologous substitution mutagenesis, residues important in site 1 for dissociating activity were identified. Based on these results, we generated proteins onefourth the size of CR1 but with enhanced decay accelerating activity for the C3 convertases.

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The molecular mechanisms leading to the development of chronic lung allograft dysfunction following de novo development of antibodies to mismatched donor MHC remain undefined. We demonstrated that intrabronchial administration of antibodies to MHC class I resulted in induction of both innate and adaptive cellular immune responses characterized by a predominance of Th17 specific to lung associated self antigens Kα1-tubulin and Collagen-V leading to the development of obliterative airway lesions (OAD), correlate of chronic rejection following human lung transplantation. To determine the role of regulatory T cells (Treg) in the pathogenesis of OAD, we administered anti-MHC class I to mice, in which Treg were depleted by conditional ablation of FoxP3+cells. Under this condition, we observed a three-fold increase in pulmonary cellular infiltration, luminal occlusion and fibrous deposition when compared anti-MHC class I Ab administered mice maintaining FoxP3. OAD lesions were accompanied with enhanced accumulation of neutrophils along with self-antigen specific Th17 and humoral responses. However, IL-17-blockade or adoptive transfer of Treg abrogated OAD. We conclude that Treg exerts a suppressive effect on anti-MHC induced IL-8 mediated neutrophil infiltration and innate immune responses that leads to inhibition of Th17 immune responses to lung associated self antigens which is critical for development of OAD.