Haixiang Jiang

Surfactant Protein A Regulates Complement Activation

Complement proteins aid in the recognition and clearance of pathogens from the body. C1, the first protein of the classical pathway of complement activation, is a calcium-dependent complex of one molecule of C1q and two molecules each of C1r and C1s, the serine proteases that cleave complement proteins. Upon binding of C1q to Ag-bound IgG or IgM, C1r and C1s are sequentially activated and initiate the classical pathway of complement. Because of structural and functional similarities between C1q and members of the collectin family of proteins, including pulmonary surfactant protein A (SP-A), we hypothesized that SP-A may interact with and regulate proteins of the complement system. Previously, SP-A was shown to bind to C1q, but the functional significance of this interaction has not been investigated. Binding studies confirmed that SP-A binds directly to C1q, but only weakly to intact C1. Further investigation revealed that the binding of SP-A to C1q prevents the association of C1q with C1r and C1s, and therefore the formation of the active C1 complex required for classical pathway activation. This finding suggests that SP-A may share a common binding site for C1r and C1s or Clq. SP-A also prevented C1q and C1 from binding to immune complexes. Furthermore, SP-A blocked the ability of C1q to restore classical pathway activity to C1q-depleted serum. SP-A may down-regulate complement activity through its association with C1q. We hypothesize that SP-A may serve a protective role in the lung by preventing C1q-mediated complement activation and inflammation along the delicate alveolar epithelium.

New therapies for hereditary angioedema: Disease outlook changes dramatically

Hereditary angioedema (HAE) is an autosomal dominant disease associated with episodic attacks of nonpitting edema that may affect any external or mucosal body surface. Attacks most often affect the extremities, causing local swelling, the GI tract, leading to severe abdominal pain, and the mouth and throat, at times causing asphyxiation. Most patients with HAE have low levels of the plasma serine protease inhibitor C1 inhibitor. The edema in these patients is caused by unregulated generation of bradykinin. Effective chronic therapy of patients with impeded androgens or plasmin inhibitors has been available for decades, but in the United States, we do not have therapy for acute attacks. Five companies have completed or are in the process of conducting phase 3 clinical trials, double-blind, placebo-controlled studies of products designed to terminate acute attacks or to be used in prophylaxis. Two companies, Lev Pharmaceuticals and CSL Behring, have preparations of C1 inhibitor purified from plasma that have been used in Europe for decades (trade names Cinryze and Berinert P, respectively). One company, Pharming, has developed a recombinant C1 inhibitor preparation. One company, Dyax, is testing a kallikrein inhibitor (ecallantide), and one company, Jerini, is completing testing of a bradykinin type 2 receptor antagonist (Icatibant). Although little has been published thus far, all of these products may prove effective. It is likely that HAE treatment will change dramatically within the next few years.

Wild-type adenoviruses from groups A–F evoke unique innate immune responses, of which HAd3 and SAd23 are partially complement dependent

Alternative human and non-human Ad serotype vectors are currently studied for gene therapy and/or vaccine applications to capitalize upon their likely ability to avoid pre-existing immunity to HAd5. However, relatively little attention has been given to the nature and scope of innate immune responses generated by alternative Ad serotypes. In this study, we characterized several innate immune responses after intravenous administration of wild-type Ad serotypes HAd31, HAd3, HAd5, HAd37, SAd23 and HAd41, representing groups A–F, respectively. Notably, biodistribution studies revealed significant differences between the serotypes, with high levels of HAd3 genomes found in the liver and lung, and HAd37 genomes found in the spleen after systemic administration. Relative to similar treatments with other Ad serotypes, HAd3 and SAd23 induced altered innate immune responses, illustrated by induction of higher levels of cellular gene transcription in several tissues, and higher plasma levels of cytokines and chemokines. We also investigated whether complement interactions have a role in HAd3- and SAd23-induced responses. We confirmed complement dependent gene transcription, plasma cytokine/chemokine responses, and liver toxicities incurred after administration of HAd3 and SAd23. This study highlights the potential benefits and/or limitations to the proposed use of alternative Ad serotypes for gene therapy or vaccine applications.

Immunoglobulin in the control of complement action.

Complement is a critical element of innate immunity, protecting individuals from a wide variety of microbial infections. This group of proteins is responsible for many features of in flammation and tissue damage. Because of its ability to mediate autoimmune tissue damage and to destroy host tissues, it is under tight regulation with many circulating and cell-membrane-bound complement regulatory proteins. The function of much of the circulating immunoglobulin has never been defined. We have advanced the hypothesis that one function of circulating immunoglobulin is to down-regulate complernent attack on host tissues in the presence of anti-self antibody. The data to support this hypothesis are reviewed. The data are consistent with the suggestion that one mechanism of action of intravenous immunoglobulin, used to treat patients with a variety of autoimmune diseases, is prevention of complement-mediated attack on host tissues.

Heparan Sulfate, Including That in Bruch’s Membrane, Inhibits the Complement Alternative Pathway: Implications for Age-Related Macular Degeneration

An imbalance between activation and inhibition of the complement system has been implicated in the etiologies of numerous common diseases. Allotypic variants of a key complement fluid-phase regulatory protein, complement factor H (CFH), are strongly associated with age-related macular degeneration (AMD), a leading cause of worldwide visual dysfunction, although its specific role in AMD pathogenesis is still not clear. CFH was isolated from individuals carrying combinations of two of the nonsynonymous coding variants most strongly associated with AMD risk, V62/H402 (risk haplotype variants), I62/Y402 (nonrisk haplotype variants), and V62/Y402. These proteins were used in two functional assays (cell surface- and fluid-phase–based) measuring cofactor activity of CFH in the factor I-mediated cleavage of C3b. Although no variant-specific differences in the cofactor activity were detected, when heparan sulfate (HS) was added to these assays, it accelerated the rate of C3b cleavage, and this effect could be modulated by degree of HS sulfation. Bruch’s membrane/choroid, a site of tissue damage in AMD, contains high concentrations of glycosaminoglycans, including HS. Addition of human Bruch’s membrane/choroid to the fluid-phase assay accelerated the C3b cleavage, and this effect was lost posttreatment of the tissue with heparinase III. Binding of CFH variants to Bruch’s membrane/choroid isolated from elderly, non-AMD donor eyes, was similar, as was the functional activity of bound CFH. These findings refine our understanding of interactions of HS and complement and support the hypothesis that these interactions play a role in the transition between normal aging and AMD in Bruch’s membrane/choroid.

Synergism from combined immunologic and pharmacologic inhibition of HER2 in vivo

The monoclonal antibody trastuzumab and the EGFR/HER2 tyrosine kinase inhibitor lapatinib improve the clinical outcome of patients with HER2‐overexpressing breast cancer. However, the majority of metastatic cancers will eventually progress, suggesting the need for other therapies. Because HER2 overexpression persists, we hypothesized that the anti‐HER2 immune response induced by cancer vaccines would be an effective strategy for treating trastuzumab‐ and lapatinib‐refractory tumors. Furthermore, we hypothesized that the antibody response could synergize with lapatinib to enhance tumor inhibition. We developed a recombinant adenoviral vector expressing a kinase‐inactive HER2 (Ad‐HER2‐ki) to use as a cancer vaccine. Vaccine‐induced polyclonal HER2‐specific antiserum was analyzed for receptor internalization and signaling effects alone and in combination with lapatinib. Ad‐HER2‐ki vaccine‐induced potent T cell and antibody responses in mice and the vaccine‐induced polyclonal HER2‐specific antiserum mediated receptor internalization and degradation much more effectively than trastuzumab. Our in vitro studies demonstrated that HER2 vaccine‐induced antibodies effectively caused a decrease in HER2 expression, but when combined with lapatinib caused significant inhibition of HER2 signaling, decreased pERK and pAKT levels and reduced breast tumor cell proliferation. In addition, a known mechanism of resistance to lapatinib, induction of survivin, was inhibited. The combination of Ad‐HER2‐ki plus lapatinib also showed superior antitumor efficacy in vivo. Based on these results, we feel clinical studies using this approach to target HER2‐overexpressing breast cancer, including trastuzumab‐ and lapatinib‐resistant tumors is warranted.

Novel adenovirus vectors 'capsid-displaying' a human complement inhibitor.

Adenovirus (Ad) vectors are currently the most commonly utilized gene transfer vectors in humans worldwide. Unfortunately, upon contact with the circulatory system, Ads induce several, innate, complement-dependent toxicities that limit the full potential for Ad-based gene transfer applications. Therefore, we have constructed several novel Ad5-based vectors, ‘capsid-displaying’ as fiber or pIX fusion proteins, a complement-regulatory peptide (COMPinh). These novel Ads dramatically minimize Ad-dependent activation of the human and non-human primate complement systems, as determined by several assays. In summary, our work has shown that a novel COMPinh-displaying Ad5 has the potential for broadening the safe use of Ad vectors in future human applications.

Subcutaneous infusion of human C1 inhibitor in swine.

Hereditary angioedema afflicts patients with unpredictable episodes of swelling that can be life threatening. Treatments approved by the Food and Drug Administration for routine prophylaxis include danazol given orally and the nanofiltered human C1 esterase inhibitor, CINRYZE, which is approved for intravenous administration. Approved for the treatment of acute attacks are the C1 esterase inhibitor, Berinert, given intravenously, and the kallikrein inhibitor, KALBITOR, given subcutaneously. C1 inhibitor has generally been non-toxic and neither pro-inflammatory nor pro-fibrotic, suggesting that it may be suitable for subcutaneous infusion. The current study used a swine model to compare blood levels of human C1 inhibitor following intravenous and subcutaneous infusion, and the effect of infusion route on heart and skin pathology. Levels of C1 inhibitor achieved with SC infusion compared favorably with levels achieved after IV infusion and were relatively more stable than those after IV infusion. Neither cardiac nor skin toxicity was observed.

Complement and the control of HIV infection: an evolving story.

Purpose of reviewThirty years ago, investigators isolated and later determined the structure of HIV-1 and its envelope proteins. Using techniques that were effective with other viruses, they prepared vaccines designed to generate antibody or T-cell responses, but they were ineffective in clinical trials. In this article, we consider the role of complement in host defense against enveloped viruses, the role it might play in the antibody response and why complement has not controlled HIV-1 infection. Recent findingsComplement consists of a large group of cell-bound and plasma proteins that are an integral part of the innate immune system. They provide a first line of defense against microbes and also play a role in the immune response. Here we review the studies of complement-mediated HIV destruction and the role of complement in the HIV antibody response. SummaryHIV-1 has evolved a complex defense to prevent complement-mediated killing reviewed here. As part of these studies, we have discovered that HIV-1 envelope, on administration into animals, is rapidly broken down into small peptides that may prove to be very inefficient at provident the type of antigenic stimulation that leads to an effective immune response. Improving complement binding and stabilizing envelope may improve the vaccine response.

Mechanisms by which HIV envelope minimizes immunogenicity

With many viruses, vaccines containing the appropriate envelope antigens have provided strong and long lasting immunity. Not so with HIV-1 envelope, despite two decades of experience with various envelope and core constituent vaccines, protection provided has been weak or absent. Our laboratory has been systematically investigating the characteristics of HIV-1 envelope gp140, the principle HIV-1 envelope protein heterodimer responsible for HIV infectivity. We have identified two properties of HIV-1 envelope gp140 that may be important factors in reducing immunogenicity. HIV envelope protein gp140 rejects complement binding. Such binding can be of vital importance, since an extensive literature suggests that complement binding markedly increases immunogenicity, and, more importantly, complement binding influences the type of immune response. For many antigens, C3 binding is required for normal transport of antigens into follicles to initiate a normal germinal center response, and in the absence of appropriate complement binding, the antibody response is reduced, short lived with short-lived memory cell formation, and for an unknown reason, the antibody response shows increased affinity maturation of antibody. These features are characteristic of the HIV-1 antibody response. Just as important is the finding that envelope gp140 is highly unstable on injection, is rapidly removed from the circulation, and is degraded into peptides. This short-lived antigen may be available on initial exposure to the immune system for too short a period of time, particularly in the absence of complement binding, to be an adequate immunogen.