Development and characterization of novel anti-C5 monoclonal antibodies capable of inhibiting complement in multiple species

Over the last decade there has been an explosion in complement therapies; one-third of the drugs in the clinic or in development target C5 protein. Eculizumab, a monoclonal antibody (mAb) that binds C5 and blocks its cleavage by the convertase, is the current reference standard treatment for atypical haemolytic uraemic syndrome (aHUS) and paroxysmal nocturnal haemoglobinuria (PNH) and in clinical trials for many other diseases. Here we describe a panel of novel anti-C5 mAb, including mAb that, like Eculizumab, are efficient inhibitors of complement but, unlike Eculizumab, inhibit across species, including human, rat, rabbit and guinea pig. Several inhibitory anti-C5 mAb were identified and characterized for C5 binding and lytic inhibitory capacity in comparison to current therapeutic anti-C5 mAb; three clones, 4G2, 7D4 and 10B6, were selected and further characterized for ligand specificity and affinity and cross-species inhibitory activity. The mAb 10B6 was human-specific whereas mAb 4G2 and 7D4 efficiently inhibited lysis by human, rabbit and rat serum, and weakly inhibited guinea pig complement; 7D4 also weakly inhibited mouse complement in vitro The rat C5-cross-reactive mAb 4G2, when administered intraperitoneally in a rat model of myasthenia gravis, effectively blocked the disease and protected muscle endplates from destruction. To our knowledge this is the first report of an anti-C5 function blocking mAb that permits preclinical studies in rats.     

Exposure to Organic Dust Causes Activation of Human Plasma Complement Factors C3 and B and the Synthesis of Factor C3 by Lung Epithelial Cells <Emphasis Type="Italic">In Vitro</Emphasis>

Exposure in swine confinement buildings induces an intense airway inflammation. Twenty-two volunteers, of whom eleven wore a half-mask, were exposed for 3 hr in a swine barn. Blood samples were drawn before and after exposure. The ratio C3b/totalC3 in plasma decreased from 6.8 to 5.0% (p = 0.02) without mask and from 6.6 to 5.9% (p = 0.01) with mask (p = 0.67 between groups). The ratio Bb/totalB decreased from 14.5 to 13.5% (p < 0.01) without and 14.6–13.3% (p = 0.09) with mask (p = 0.25 between groups). Epithelial cells (A549) incubated up to 24 hr with 0.1 mg/mL dust suspensions were analysed for C3, IL-6 and IL-8 secretion. Cumulative C3 synthesis of dust stimulated cell cultures was 43,000 pg/mL compared to 25,000 pg/mL in unstimulated cells. Cumulative dust-induced IL-6 and IL-8 secretion was 200 and 3000 pg/mL, respectively and below detection in unstimulated cells.The activation of complement in vivo and induced C3 synthesis by epithelial cells suggests a role of complement in the airway reaction to organic dust exposure.     

Functional and biochemical characterizations of avian T lymphocyte antigens identified by monoclonal antibodies

Seven monoclonal antibodies (mAb) were used to characterize antigens present on chicken T lymphocytes and on natural killer cells by flow cytometry, radioimmunoprecipitation and by effects on cell-mediated cytotoxicity and mitogen-induced proliferation. mAb CTLA8 and 5 stained 73% of thymus, 44% of spleen and 51% of peripheral blood lymphocytes (PBL), respectively, and immunoprecipitated 65- and 45-kDa proteins from detergent extracts of ¹²⁵I surface-labeled thymocytes. Pretreatment of splenic lymphocytes with mAb CTLA5 and 8 in the presence of rabbit complement (C) eliminated the concanavalin A (Con A)-induced T cell proliferative responses. mAb CTLA3, 4 and 9 stained 43% of thymus, 36% of spleen and 18% of PBL, and immunoprecipitated 33–35-kDa proteins. Pretreatment of spleen cells with mAb 4 or 9 plus C reduced, but did not eliminate, the Con A-induced proliferative response and significantly reduced both major histocompatibility complex (MHC)-restricted and non-MHC-restricted cellular cytotoxicity. mAb CTLA1 and 6 stained 58% of thymus, 13% of spleen and 19% of PBL. mAb CTLA 1 and 6 immunoprecipitated a 65-kDa protein. mAb CTLA l and 6 had no effect on the Con A-induced blastogenesis and CTLA 6 caused no decrease in virus-specific cytotoxic T lymphocyte and natural killer activity. These results indicate that (a) mAb CTLA 5 and 8 identify antigens on mature T lymphocytes that are similar in tissue distribution, molecular mass and function to the mammalian CD5 antigen; (b) mAb CTLA 3, 4 and 9 detect the avian homologue of CD8 antigen; and (c) mAb CTLA l and 6 identify the avian homologue of CD4 antigen.     

Gene polymorphisms of TNF-α−308 (G/A), IL-10−1082 (G/A), IL-6−174 (G/C) and IL-1Ra (VNTR) in Egyptian cases with type 1 diabetes mellitus

Background. Type 1 diabetes (T1D) is a genetically conditioned autoimmune disease in which cytokines play an important role.

Objectives. To check for the association of polymorphisms of cytokine genes with type 1 diabetes.

Subjects. This work included 50 cases with T1D and 98 healthy individuals from the Nile Delta region of Egypt. Cases included 20 males and 30 females with a median age of 25 and range of 15–50 years.

Methods. DNA was amplified using PCR with sequence-specific primers for detection of polymorphisms related to tumor necrosis factor (TNF)-α^{? 308} (G/A), interleukin (IL)-10^{? 1082} (G/A), IL-6^{? 174} (G/C), and IL-1Ra (VNTR).

Results. Cases with T1D showed significant higher frequency of genotypes of TNF-α^{? 308} AA (p < 0.001, odds ratio (OR) = 7.91), IL-6-17CC (p < 0.05, OR = 3.36) and IL-1Ra A1A1 (p < 0.05, OR = 3.68) with significant lower frequencies of TNF-α^{? 308} GA, and IL-1Ra A1A2 genotypes (p < 0.001 and < 0.05, respectively). They also showed significant higher frequency of TNF-α^{? 308} allele A (p < 0.05, OR = 2.0), IL-1Ra allele A1 (p < 0.05, OR = 2.98) with a significant lower frequency of TNF-α^{? 308} G allele and IL-1Ra A2 allele (p < 0.05). No significant difference was detected among cases in relation to IL-10^{? 1082} (G/A) genotypes or alleles nor in relation to age, sex, consanguinity or family history of the disease.

Conclusions. Polymorphisms related to TNF-α and IL-1Ra genes may be considered genetic markers for T1D among Egyptians with a potential impact on family counseling and management.     

Synthesis of Complement Components C5, C6, C7, C8 and C9 in Vitro by Human Monocytes and Assembly of the Terminal Complement Complex

Monocytes cultured under serum-free conditions secreted protein which bound covalently and non-covalently to agarose beads, an activator of the alternative pathway of complement. There was a significantly binding of monoclonal anti-C3c antibodies, polyclonal anti-C5, anti-C6, anti-C7, anti-C8, and anti-C9 antibodies, and of a monoclonal antibody against a neoantigen of polymerized C9 to agarose beads incubated with the monocytes for 24, 48, 72 or 96 h. From these results, we conclude that monocytes produce C5, C6, C7, C8 and C9 that assemble as the terminal complement complex on the surface of the agarose beads. Activation by agarose of the alternative pathway with generation of particle bound C3 and C5 convertases is a prerequisite for the subsequent formation of the terminal complement complex. Whether SC5b-9 or the membrane attack of complement (C5b-9) is formed on the beads will be examined.     

The immunoregulatory role of IL-35 in patients with interstitial lung disease

Pulmonary fibrosis involves various types of immune cells and soluble mediators, including TGF-β and IL-35, a recently identified heterodimeric cytokine that belongs to the IL-12 cytokine family. However, the effect of regulatory IL-35 may play an important role in fibrotic diseases. The aim of this paper is to explore the immunoregulatory role of IL-35 in the development of fibrosis in interstitial lung disease (ILD). To gain a better understanding of this issue, the concentrations of IL-35 and different profibrotic cytokines in fibrotic (F-ILD) and non-fibrotic (NF-ILD) patients by ELISA were compared to that of intracellular IL-35 and IL-17 on CD4+ T cells stimulated in the presence of BAL or with different ratios of recombinant IL-35 (rIL-35) and TGF-β (rTGF-β), which were evaluated by flow cytometry. We observed that BAL concentration of IL-35 was lower in F patients (p < 0.001) and was negatively correlated with concentrations of TGF-β (p < 0.001) and IL-17 (p < 0.001). In supplemented cell cultures, BAL from NF but not F patients enhanced the percentage of IL-35 + CD4+ T (p < 0.001) cells and decreased the percentage of IL-17 + CD4+ T cells (p < 0.001). The percentage of IL-35 + CD4+ T cells correlated positively with BAL concentration of IL-35 (p = 0.02), but correlated negatively with BAL concentrations of IL-17 (p = 0.007) and TGF-β (p = 0.01). After adjusting the concentrations of recombinant cytokines to establish a TGF-β: IL-35 ratio of 1:4, an enhanced percentage of IL-35 + CD4+ T cells (p < 0.001) but a decreased percentage of IL-17 + CD4+ T cells (p < 0.001) was observed. After adding recombinant IL-35 to the BAL from F patients until a 1:4 ratio of TGF-β: IL-35 was reached, a significantly increased percentage of IL-35 + CD4+ T cells (p < 0.001) and a decreased percentage of IL-17 + CD4+ T cells (p = 0.003) was found. These results suggest that IL-35 may induce an anti-fibrotic response, regulating the effect of TGF-β and the inflammatory response on CD4+ T cells. In addition, the TGF-β: IL-35 ratio in BAL has been shown to be a potential biomarker to predict the outcome of F patients with ILD.     

Meta-Analysis of Associations Between Interleukin-10 Polymorphisms and Susceptibility to Vasculitis

Objective: This study determined whether interleukin-10 (IL-10) polymorphisms are associated with susceptibility to vasculitis.

Methods: A meta-analysis was conducted of the associations between the IL-10 -1082 G/A, -819 C/T, and -592 C/A polymorphisms and the haplotype of the IL-10-1082 G/A, -819 C/T, -592 C/A polymorphisms and vasculitis.

Results: A total of 21 comparative studies involving 4121 patients and 5504 controls were considered in the meta-analysis. Meta-analysis revealed no association between the IL-10-1082 G allele and vasculitis in all study subjects (OR?=?0.927, 95% CI?=?0.780–1.102, p?=?0.389). However, disease-specific meta-analysis showed an association between Wegener’s granulomatosis (WG) and the IL-10-1082 G allele (OR?=?0.729, 95% CI?=?0.547–0.971, p?=?0.031). Meta-analysis revealed an association between vasculitis and the IL-10-819 C allele (OR?=?0.804, 95% CI?=?0.706–0.916, p?=?0.001) in all study subjects and Behcet’s disease (BD) (OR?=?0.724, 95% CI?=?0.679–0.781, p?<?1.0?×?10^?9). Meta-analysis of the IL-10-592 C allele showed an association with vasculitis in all study subjects (OR?=?0.805, 95% CI?=?0.619–0.938, p?=?0.005) and BD (OR?=?0.718, 95% CI?=?0.661–0.781, p?<?1.0?×?10^?9). Meta-analysis of the IL-10 haplotype revealed an association between the GCC haplotype and vasculitis in Europeans (OR?=?1.239, 95% CI?=?1.105–1.513, p?=?0.035).

Conclusions: This meta-analysis showed that IL-10 polymorphisms are associated with vasculitis susceptibility, especially in WG and BD.     

A T cell activation antigen,Ly6C,induced on CD4+ Th1 cells mediates an inhibitory signal for secretion of IL-2 and proliferation in peripheral immune responses

A T cell activation antigen, Ly6C, is considered to be involved in the autoimmunity of some autoimmune-prone mice; however, the function of Ly6C remains largely unknown. We prepared a rat anti-mouse Ly6C monoclonal antibody (mAb) (S14) that inhibits the proliferation of peripheral T cells stimulated with anti-CD3 mAb in vitro. S14 mAb, the specificity of which is confirmed by a cDNA transfectant, recognizes Ly6C antigen preferentially expressed on a part of CD8⁺ T cells in peripheral lymphoid organs. The immunohistochemical analysis demonstrates that Ly6C appears on CD8⁺ T cells in the conventional T cell-associated area of BALB/c but not of nonobese diabetic (NOD) mice, confirming the absence of Ly6C⁺ T cells in NOD mice. Addition of soluble S14 mAb to the culture does not influence the proliferation of T cells in vitro; however, the S14 mAb coated on the plate clearly inhibits the proliferation and IL-2 production of anti-CD3-stimulated peripheral T cells. The T cells are arrested at the transitional stage from G₀/G₁ to S+G₂/M phases, but they are not induced to undergo apoptotic changes in vitro. This inhibitory signal provided through the Ly6C molecule inhibited IL-2 secretion in a subpopulation of the activated CD4⁺ T cells. Ly6C is expressed on T cell clones of both Th1 and Th2 cells, but the cytokine secretion from Th1 clones is preferentially inhibited. These results suggest that Ly6C mediates an inhibitory signal for secretion of cytokines from Th1 CD4⁺ T cells, potentially causing the inhibition of immune response in peripheral lymphoid tissues.     

Fas (CD95)-transduced signal preferentially stimulates lupus peripheral T lymphocytes

Fas (CD95) is a cell surface receptor whose biological function in circulating peripheral T cells is not well understood. To address the question of abnormal T cell sensitivity to Fas stimulation in systemic lupus erythematosus (SLE), we studied Fas-transduced stimulation and apoptosis in peripheral blood T cells from patients with SLE and normal control. Immobilized anti-Fas monoclonal antibodies (mAb) (imCH-11; IgM type) significantly stimulated SLE T cell proliferation compared to T cells from normal donors and patients with rheumatoid arthritis ( p < 0.003 and p < 0.005, respectively). The soluble form of CH-11 and other immobilized anti-Fas mAb (UB-2, ZB-4; IgG type) failed to stimulate lupus T cells while immobilized human Fas ligand did. Furthermore, imCH-11 induced IL-2 and IL-6 mRNA expression. However, imCH-11 activation failed to induce expression of the T cell activation surface molecules CD25 and CD69. Addition of exogenous ceramide, a second messenger for Fas-mediated apoptosis signaling, also induced T cell proliferation in SLE and normal controls. Moreover, fumonisin B1, a specific ceramide synthase inhibitor, and caspase inhibitors markedly suppressed imCH-11 induced T cell proliferation, suggesting that the ceramide pathway may be involved in Fas-transduced stimulation signals in SLE T cells. These results show that SLE T cells have an alteration in the Fas signal transduction pathway leading to cell proliferation. This defect may be important in Fas-mediated peripheral immune homeostasis.     

Anti-CD5 extends the proliferative response of human CD5+ B cells activated with anti-IgM and interleukin-2

The CD5 T cell glycoprotein which is expressed by a subset of B cells has been shown to be involved in T cell activation and proliferation. No similar studies, to date, have addressed the role of CD5 on the B cell subset. CD5⁺ and CD5^? B cells were sorted and stimulated with anti-CD5 monoclonal antibody (mAb) in vitro. The activation and proliferative responses of these two populations, as measured by analysis of proliferation marker, did not differ following anti-μ and interleukin (IL)-2 stimulation. The addition of anti-CD5 did not change the responsiveness of such activated CD5⁺ B cells but resulted in a decrease in CD25 expression. Pre-activation of B cells with phorbol 12-myristate 13-acetate, which increased CD5 expression, failed to alter the proliferative response of CD5⁺ B cells to anti-μ and IL-2 with or without addition of anti-CD5 mAb. Anti-μ and IL-2 treatment of CD5⁺ cells resulted in optimal proliferation measured at day 3 which decreased by day 6. However, addition of anti-CD5 mAb at day 3 prevented this decline in proliferative response. This dose-dependent effect was observed only when the anti-CD5 mAb was presented to the B cells in cross-linked form. Co-stimulation of CD5 did not lower the threshold of antigen to which the B cells responded. Taken together, these data support a functional role for CD5 on B cells acting as an accessory signal, following their primary activation through the B cell receptor complex and highlight differences in the role of CD5 associated with the T cell receptor complex.     

Complement plays a central role in Candida albicans-induced cytokine production by human PBMCs

In experimental studies, the role of complement in antifungal host defense has been attributed to its opsonizing capability. In this study, we report that in humans an activated complement system mainly augments Candida albicans-induced host proinflammatory cytokine production via C5a-C5aR signaling, while phagocytosis and intracellular killing of Candida are not influenced. By blocking the C5a-C5aR signaling pathway, either with anti-C5a antagonist antibodies or with the C5aR antagonist W-54001, C. albicans-induced IL-6 and IL-1β levels were significantly reduced. Recombinant C5a augmented cytokine production. In addition, using serum from patients with various complement deficiencies, we demonstrated a crucial role of C5, but not C6 or the membrane attack complex, in C. albicans-induced IL-6 and IL-1β production in monocytes. These findings reveal a central role of anaphylatoxin C5a in augmenting host proinflammatory cytokine production upon contact with C. albicans, and define the role of the complement system in anti-Candida host defense in humans.     

Combined inhibition of complement and CD14 abolish E. coli-induced cytokine-, chemokine- and growth factor-synthesis in human whole blood

The relative role of complement and CD14 in E. coli-induced cytokine synthesis in an in vitro human whole blood model of sepsis was examined. Fresh lepirudin-anticoagulated whole blood was incubated with E. coli for 2h. Monoclonal antibodies or a C5a receptor antagonist were used to block complement. Inflammatory mediators (n=27) were measured by multiplex technology, selected cytokine mRNA by real time PCR, and CD11b, oxidative burst and phagocytosis by flow cytometry. E. coli significantly increased 18 of the 27 inflammatory mediators, including proinflammatory cytokines (TNF-alpha, IL-6, INF-gamma and IL-1beta), chemokines (IL-8, MCP-1, MIP-1alpha, MIP-1beta, eotaxin and IP-10), growth factors (VEGF, FGF-basic, G-CSF and GM-CSF) and other interleukins (IL-9, IL-15 and IL-17). Notably, the increases in all mediators were abolished by a combined inhibition of CD14 and complement using anti-C2 and anti-factor D in combination, whereas the relative effect of the inhibition of complement and CD14 varied. In comparison, a C5a receptor antagonist and anti-CD14 in combination reduced cytokine synthesis less efficiently. Real time PCR analysis confirmed that the cytokine synthesis was blocked at the mRNA level. Similarly, E. coli-induced CD11b up-regulation, oxidative burst and phagocytosis was totally inhibited by CD14, anti-C2 and anti-factor D in combination after 2h incubation. In conclusion, the combined inhibition of complement using anti-C2, anti-factor D and CD14 almost completely inhibits the E. coli-induced inflammatory response. The combined approach may therefore be a new treatment regimen in Gram-negative sepsis.     

CD38 and Interleukin 6 Gene Polymorphism in Egyptians with Diffuse Large B-Cell Lymphoma (DLBCL)

Given the importance of understanding the genetic variations involved in the pathogenesis of non-Hodgkin’s lymphoma (NHL), this pilot study was designed to investigate the impact of CD38 (184C/G; rs6449182) and IL-6 (?174 G/C; rs1800795) gene polymorphism on susceptibility of Egyptians to diffuse large B cell lymphoma (DLBCL); major types of NHL. To the best of our knowledge, this study is the first one that examines CD38 polymorphism in the NHL. Genotyping polymorphism is performed using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) for CD38 and Mutagenically separated PCR (MS-PCR) for IL-6 in 100 Egyptian NHL patients with DLBCL subtype and 119 normal controls. The serum level of IL-6 was measured using Enzyme-linked immunosorbent assay (ELISA). CD38 (184C/G) genotype is significantly increased in NHL patients (p?<?0.01), while the GG genotype is significantly increased in controls (p?<?0.05). Only two genotypes were found (GG and GC) in IL-6 (?174), no CC in our NHL patients and only one case in the controls. Insignificant change in IL-6 (?174 G/C) genotypes was recorded. Significantly increased serum IL-6 (p?<?0.05) was positively correlated (r?=?0.17; p?<?0.05) with the disease. Taken together, our data stressed the importance of CD38 gene polymorphism in developing DLBCL. Our pilot study indicates that CD38 (184) CG genotype might play a role in DLBCL susceptibility in Egyptians. Additional prospective studies on larger population are needed to confirm our findings.     

High pentraxin-3 plasma levels associate with thrombocytopenia in acute Puumala hantavirus-induced nephropathia epidemica

Our aim was to investigate whether plasma levels of the long pentraxin-3 (PTX3) associate with the severity of Puumala hantavirus-induced nephropathia epidemica (NE). Sixty-one prospectively identified consecutively hospitalized NE patients were examined. Plasma PTX3, interleukin (IL)-6, terminal complement complex SC5b-9, complement component C3, C-reactive protein (CRP), creatinine, sodium, kynurenine, and tryptophan levels, as well as the blood cell count, were determined for up to five consecutive days after hospitalization. Receiver operating characteristic (ROC) analysis revealed that the maximum PTX3 level >101.6 ng/ml (high PTX3) showed a sensitivity of 71% and a specificity of 89% for detecting platelet level <50 × 10⁹/l, with an area under the curve (AUC) value of 0.78 (95% confidence interval [CI] 0.63–0.94). High PTX3 level was also associated with several other variables reflecting the severity of the disease: patients with high PTX3 level had higher maximum blood leukocyte (16.1 vs. 9.7 × 10⁹/l, p < 0.001), plasma IL-6 (16.9 vs. 9.0 pg/ml, p = 0.007), and creatinine (282 vs. 124 μmol/l, p = 0.007) levels than patients with low maximum PTX3 level. They also had longer hospital stays (8 vs. 5 days, p = 0.015) compared to patients with low PTX3 level. High plasma PTX3 levels are associated with thrombocytopenia and the overall severity of NE.     

NF-κB signaling pathway-enhanced complement activation mediates renal injury in trichloroethylene-sensitized mice

Both NF-κB pathway and complement activation appear to be involved in kidney damage induced by trichloroethylene (TCE). However, any relationship between these two systems has not yet been established. The present study aimed to clarify the role of NF-κB in complement activation and renal injury in TCE-sensitized BALB/c mice. Mice were sensitized by an initial subcutaneous injection and repeated focal applications of TCE to dorsal skin at specified timepoints. NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) was injected (intraperitoneal) before the final two focal TCE challenges. In the experiments, mice had their blood and kidneys collected. Kidney function was evaluated via blood urea nitrogen (BUN) and creatinine (Cr) content; renal histology was examined using transmission electron microscopy (TEM). Kidney levels of phospho-p65 were assessed by Western blot and kidney mRNA levels of interleukin (IL)-1β, IL-6, IL-17, tumor necrosis factor (TNF)-α, and p65 by real-time quantitative PCR. Presence of C3 and C5b-9 membrane attack complexes in the kidneys was evaluated via immunohistochemistry. The results showed there was significant swelling, vacuolar degeneration in mitochondria, shrinkage of microvilli, disappearance of brush borders, segmental foot process fusion, and glomerular basement membrane thickening (or disrobing) in kidneys from TCE-sensitized mice. In conjunction with these changes, serum BUN and Cr levels were increased and IL-1β, IL-6, IL-17, and TNFα mRNA levels were elevated. Levels of p65 and phospho-p65 protein were also up-regulated, and there was significant C3 and C5b-9 deposition. PDTC pretreatment attenuated TCE-induced up-regulation of p65 and its phosphorylation, complement deposition, cytokine release, and renal damage. These results provide the first evidence that NF-κB pathway has an important role in TCE-induced renal damage mediated by enhanced complement activation in situ.     

GM1, a ganglioside that specifically enhances immunoglobulin production and proliferation in human plasma cells

The effects of gangliosides on human plasma cell responses were studied. Among the various gangliosides tested, only GM1 enhanced immunoglobulin (Ig) production and proliferation in the human plasma cell lines, IM-9 and AF-10, while other gangliosides (GM2, GM3, GD1a, GD1b, GD3, GT1b, and GQ1b) had no effect. Among the various cytokines tested, including interleukin (IL)-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, interferon (IFN)-α and IFN-γ, only IL-6 enhanced Ig production and proliferation in IM-9 and AF-10 cells. However, the enhancement of plasma cell responses by GM1 was specific and was not mediated by IL-6, since GM1 activity was blocked by anti-GM1 monoclonal antibody (mAb), but not by control IgM, anti-IL-6 Ab or the anti-IL-6 receptor mAb, PM1. Conversely, the enhancement by IL-6 was blocked by anti-IL-6 Ab and PM1, but not by anti-GM1 mAb. GM1, but not other gangliosides, also enhanced Ig production and proliferation in freshly separated plasma cells from patients with plasma cell leukemia and in plasma cells generated in vitro. These actions of GM1 were specifically blocked by anti-GM1 mAb, but not by anti-IL-6 Ab or PM1. These results indicate that GM1 may be an important regulator of plasma cell responses.     

基于补体C5a在炎症性肠病中的作用研究

目的探索2,4,6-三硝基苯磺酸(TNBS)诱导炎症性肠病(IBD)发病机制中补体C5a的作用。方法选择SPF级雄性Sprague-Dawley(SD)大鼠60只,鼠龄6~8周,体质量180~220 g。随机分为3组,即空白对照组、TNBS IBD组、TNBS+抗C5a单克隆抗体干预组(简称治疗组)。IBD大鼠建模方法采用TNBS联合乙醇诱导灌肠法。治疗组在建模后立即每只腹腔注射抗C5a单克隆抗体1.5 mg干预。利用酶联免疫吸附分析(ELISA)法检测不同时间段大鼠血清中炎性介质C5a和白细胞介素(IL)-6、肿瘤坏死因子α(TNF-α)的表达水平;另给予C5a拮抗剂后,再次检测大鼠血清中IL-6、TNF-α的表达水平。结果C5a、IL-6、TNF-α伴随着炎症的进展而增高,伴随炎症的转归出现下降趋势,即血清中C5a、IL-6、TNF-α的变化趋势与炎症反应程度呈正向相关性;给予C5a拮抗剂后,TNBS IBD组与治疗组相比,IL-6、TNF-α的表达水平有明显下调趋势,差异有统计学意义(P<0.05)。结论初步判断抗C5a单克隆抗体可能通过抑制IL-6、TNF-α的表达,有效地降低了IBD炎症反应,提示C5a在IBD发病机制中发挥着重要作用。     

Significant Impact of IL-6 -174G/C but Inverse Relation with -634 C/G Polymorphism in Patients with Non-Small Cell Lung Cancer in Kashmiri Population

To study the possible role of proinflammatory interleukin 6 -174 G>C (rs 1800795) and -634 C>G (rs 1800796) polymorphism in the pathogenesis of non-small cell lung cancer (NSCLC). A total of 190 NSCLC patients and 200 healthy controls were evaluated for polymorphic analysis of -174 G/C and -634 C/G by PCR-RFLP followed by DNA sequencing. A significant association was observed in the genotypic and allelic distribution of IL-6 -174 G/C in the NSCLC group as compared to control group [OR?=?2.7 (1.77–4.11), p?<?0.0001]. Smokers with the -174C allele were found to be significantly associated with NSCLC (p?=?0.01), while 634C/G SNP showed an inverse relation [OR-0.4, p?<?0.0001]. The present investigation revealed a significant association of the IL6 -174 G/C gene promoter polymorphism with NSCLC, and thus, the IL-6 -174G/C genotype can be considered as one of the biological markers in the etiology of NSCLC.     

Covalent binding of C3b to monoclonal antibodies selectively up-regulates heavy chain epitope recognition by T cells

Protein C3 of the complement system is known for its role in the nonspecific immune response. Covalent binding of C3b to antigen upon complement activation also plays a significant role in specific T cell immune response. C3b-antigen complexes can bind to complement receptors on the antigenpresenting cell, and the C3b antigen link (most often an ester link) remains fairly stable inside the cells. In this study, IgG1,χ and IgG2a,χ murine monoclonal antibodies (mAb) were used as antigens; covalent complexes between mAb and C3b were produced and puritied in vitro from purified proteins; human B cell lines and T cell clones were raised from tumor patients who received mAb injections for cancer therapy or diagnosis. Recognition of epitopes of these mAb by T cell clones when the mAb were processed alone or bound to C3b was compared. IgG or IgG-C3b complexes presented by B cell lines were able to stimulate proliferation of χ light chain-specific T cell clones at similar concentrations. In contrast, IgG-C3b complex recognition by heavy chain-specific T cell clones required 100-fold less IgG-C3b than uncomplexed IgG. As C3b was shown to be covalently bound only to the IgG heavy chains in the complexes, C3b chaperoning is restricted to only the IgG heavy chain and selectively influences intracellular steps of IgG heavy chain processing. This differential modulation of C3b suggests an early dissociation of IgG heavy and light chains in antigenpresenting cells.