Absence Of C4 Research Articles

Design and Synthesis of New Benzophenone Derivatives with In Vivo Anti-Inflammatory Activity through Dual Inhibition of Edema and Neutrophil Recruitment.

A series of novel benzophenone derivatives containing a thiazole heterocyclic nucleus were designed by molecular hybridization. Molecular docking studies have demonstrated the inhibitory potential of the designed compounds against cyclooxygenase (COX) isoenzymes. These compounds were synthesized, characterized, and evaluated for their anti-inflammatory properties by the croton oil-induced ear edema assay to examine their effect on both prostaglandin (PG) production and neutrophils recruitment. The thiazole derivatives displayed a potent effect in terms of reducing ear edema. The analysis suggested that the presence of 4-phenyl-2-hydrazinothiazole and the absence of C4′-OCH3 on the benzophenone derivative structure are strongly related to the inhibition of PG production. In addition, the derivatives 2e, 3a and 3c concomitantly inhibit PG production and neutrophil recruitment, which may be a mechanism of action better than of common NSAIDs due to their inability to inhibit the neutrophil recruitment. Thus, these compounds can be considered as potential lead compounds toward the development of new anti-inflammatory drugs with an innovating mechanism of action.

The lectin pathway in renal disease: old concept and new insights.

The complement system is composed of a network of at least 40 proteins, which significantly contributes to health and disease. The lectin pathway (LP) is one of three pathways that can activate the complement system. Next to protection of the host against pathogens, the LP has been shown to play a crucial role in multiple renal diseases as well as during renal replacement therapy. Therefore, several complement-targeted drugs are currently being explored in clinical trials. Among these complement inhibitors, specific LP inhibitors are also being tested in renal abnormalities such as in immunoglobulin A nephropathy and lupus nephritis. Using various in vitro models, Yaseen et al. (Lectin pathway effector enzyme mannan-binding lectin-associated serine protease-2 can activate native complement component 3 (C3) in absence of C4 and/or C2. FASEB J 2017; 31: 2210-2219) showed that Mannan-associated serine protease2 can directly activate C3 thereby bypassing C2 and C4 in the activation of the LP. These new findings broaden our understanding of the mechanisms of complement activation and could potentially impact our strategies to inhibit the LP in renal diseases. In support of these findings, we present data of human renal biopsies, demonstrating the occurrence of the LP bypass mechanism in vivo. In conclusion, this review provides a detailed overview of the LP and clarifies the recently described bypass mechanism and its relevance. Finally, we speculate on the role of the C4 bypass mechanism in other renal diseases.

Association between C4, C4A, and C4B copy number variations and susceptibility to autoimmune diseases: a meta-analysis

Although several studies have investigated the association between C4, C4A, and C4B gene copy number variations (CNVs) and susceptibility to autoimmune diseases, the results remain inconsistency for those diseases. Thus, in this study, a comprehensive meta-analysis was conducted to assess the role of C4, C4A, and C4B CNVs in autoimmune diseases in different ethnic groups. A total of 16 case-control studies described in 12 articles (8663 cases and 11099 controls) were included in this study. The pooled analyses showed that a low C4 gene copy number (GCN) (<4) was treated as a significant risk factor (odds ratio [OR] = 1.46, 95% confidence interval [CI] = 1.19–1.78) for autoimmune diseases compared with a higher GCN (>4). The pooled statistical results revealed that low C4 (<4) and low C4A (<2) GCNs could be risk factors for systemic lupus erythematosus (SLE) in Caucasian populations. Additionally, the correlation between C4B CNVs and all type of autoimmune diseases could not be confirmed by the current meta-analysis (OR = 1.07, 95% CI = 0.93–1.24). These data suggest that deficiency or absence of C4 and C4A CNVs may cause susceptibility to SLE.

Lectin pathway effector enzyme mannan-binding lectin-associated serine protease-2 can activate native complement C3 in absence of C4 and/or C2.

All 3 activation pathways of complement-the classic pathway (CP), the alternative pathway, and the lectin pathway (LP)- converge into a common central event: the cleavage and activation of the abundant third complement component, C3, via formation of C3-activating enzymes (C3 convertases). The fourth complement component, C4, and the second component, C2, are indispensable constituents of the C3 convertase complex, C4bC2a, which is formed by both the CP and the LP. Whereas in the absence of C4, CP can no longer activate C3, LP retains a residual but physiologically critical capacity to convert native C3 into its activation fragments, C3a and C3b. This residual C4 and/or C2 bypass route is dependent on LP-specific mannan-binding lectin-associated serine protease-2. By using various serum sources with defined complement deficiencies, we demonstrate that, under physiologic conditions LP-specific C4 and/or C2 bypass activation of C3 is mediated by direct cleavage of native C3 by mannan-binding lectin-associated serine protease-2 bound to LP-activation complexes captured on ligand-coated surfaces.-Yaseen, S., Demopulos, G., Dudler, T., Yabuki, M., Wood, C. L., Cummings, W. J., Tjoelker, L. W., Fujita, T., Sacks, S., Garred, P., Andrew, P., Sim, R. B., Lachmann, P. J., Wallis, R., Lynch, N., Schwaeble, W. J. Lectin pathway effector enzyme mannan-binding lectin-associated serine protease-2 can activate native complement C3 in absence of C4 and/or C2.

The Lectin Pathway of Complement Activation Is a Critical Component of the Innate Immune Response to Pneumococcal Infection

The complement system plays a key role in host defense against pneumococcal infection. Three different pathways, the classical, alternative and lectin pathways, mediate complement activation. While there is limited information available on the roles of the classical and the alternative activation pathways of complement in fighting streptococcal infection, little is known about the role of the lectin pathway, mainly due to the lack of appropriate experimental models of lectin pathway deficiency. We have recently established a mouse strain deficient of the lectin pathway effector enzyme mannan-binding lectin associated serine protease-2 (MASP-2) and shown that this mouse strain is unable to form the lectin pathway specific C3 and C5 convertases. Here we report that MASP-2 deficient mice (which can still activate complement via the classical pathway and the alternative pathway) are highly susceptible to pneumococcal infection and fail to opsonize Streptococcus pneumoniae in the none-immune host. This defect in complement opsonisation severely compromises pathogen clearance in the lectin pathway deficient host. Using sera from mice and humans with defined complement deficiencies, we demonstrate that mouse ficolin A, human L-ficolin, and collectin 11 in both species, but not mannan-binding lectin (MBL), are the pattern recognition molecules that drive lectin pathway activation on the surface of S. pneumoniae. We further show that pneumococcal opsonisation via the lectin pathway can proceed in the absence of C4. This study corroborates the essential function of MASP-2 in the lectin pathway and highlights the importance of MBL-independent lectin pathway activation in the host defense against pneumococci.

Targeting of mannan-binding lectin-associated serine protease-2 confers protection from myocardial and gastrointestinal ischemia/reperfusion injury.

Complement research experienced a renaissance with the discovery of a third activation route, the lectin pathway. We developed a unique model of total lectin pathway deficiency, a mouse strain lacking mannan-binding lectin-associated serine protease-2 (MASP-2), and analyzed the role of MASP-2 in two models of postischemic reperfusion injury (IRI). In a model of transient myocardial IRI, MASP-2-deficient mice had significantly smaller infarct volumes than their wild-type littermates. Mice deficient in the downstream complement component C4 were not protected, suggesting the existence of a previously undescribed lectin pathway-dependent C4-bypass. Lectin pathway-mediated activation of C3 in the absence of C4 was demonstrated in vitro and shown to require MASP-2, C2, and MASP-1/3. MASP-2 deficiency also protects mice from gastrointestinal IRI, as do mAb-based inhibitors of MASP-2. The therapeutic effects of MASP-2 inhibition in this experimental model suggest the utility of anti-MASP-2 antibody therapy in reperfusion injury and other lectin pathway-mediated disorders.

An Alternative Role of C1q in Cell Migration and Tissue Remodeling: Contribution to Trophoblast Invasion and Placental Development

Fetal trophoblast cells invading the decidua in the early phase of pregnancy establish complex interaction with the maternal extracellular matrix. We discovered that C1q was widely distributed in human decidual stroma in the absence of C4 and C3 and was actively synthesized by migrating extravillous trophoblasts. The cells expressed the messages for the three chains of C1q and secreted this complement component that interacted with the proteins of the decidual extracellular matrix. Solid phase-bound C1q promoted trophoblast adhesion and migration, and cell binding to C1q resulted in activation of ERK1/2 MAPKs. Ab inhibition experiments showed that the receptors for the globular head of C1q/p33 and α(4)β(1) integrin were both involved in this process and were colocalized on the cell surface following binding of C1q to trophoblasts. We also found that C1q(-/-) mice manifested increased frequency of fetal resorption, reduced fetal weight, and smaller litter sizes compared with wild-type mice. C1q deficiency was associated with impaired labyrinth development and decidual vessel remodeling. Collectively, these data suggest that C1q plays an important role in promoting trophoblast invasion of decidua and that defective local production of C1q may be involved in pregnancy disorders, such as pre-eclampsia, characterized by poor trophoblast invasion.

Antigen localization within the splenic marginal zone restores humoral immune response and IgG class switch in complement C4-deficient mice.

Defects of early complement components (C1, C4 and C2) are associated with the development of systemic lupus erythematosus (SLE). The availability of complement knockout mice has increased our knowledge on the role of complement in the regulation of adaptive immunity. An impaired removal of apoptotic bodies, a disturbed clearance of IgG immune complexes (ICs) and an insufficient B-cell regulation via complement receptors CD21/CD35 have been discussed as explanations for the induction of autoimmunity; however, a unifying hypothesis for the loss of B-cell tolerance in the absence of C1 or C4 is still lacking. Using IgM-containing ICs, we observed a significant accumulation of antigen within the splenic marginal zone (MZ) of C4-deficient mice but not in C3-deficient or complement receptors CD21/CD35-deficient mice. The targeting of antigen toward the MZ restored adaptive immunity (antibody response and class switch) in C4-deficient animals. A new explanation for the association of SLE and complement C4 deficiency would be that in the absence of C4, natural antibodies (IgM type) localize more self-antigen toward the MZ so that the auto-antibody response is increased and autoimmune disease ensues. As such, an inadequate localization of self-antigens might be responsible for the annulment of peripheral B-cell tolerance in the absence of C4.

Regulation of B-cell tolerance by complement C4

Systemic lupus erythematosus (SLE) is a B-cell-dependent autoimmune disease characterized by autoantibodies specific for nuclear antigens such as dsDNA and histones. Apoptotic blebs released from dying cells are thought to represent a major source of autoantigen. The cellular and molecular basis for SLE is not known but a major risk factor is deficiency in products of natural immunity. For example, individuals deficient in serum complement proteins C1q or C4 almost always develop lupus. This presents a paradox as the complement system is known to mediate pathogenesis in SLE. In addition to its known role in inflammation, the complement system participates in humoral antibody responses via enhancement of B-lymphocyte activation, germinal center survival and maintenance of long-term effector responses. Two nonmutually exclusive hypotheses for a protective role of complement are termed the clearance hypothesis and the B-cell tolerance hypothesis. The former proposes that complement (and natural immunity) protects by clearance of apoptotic blebs and sequesters them from the adaptive immune system. The tolerance hypothesis proposes that complement and natural immunity act in concert to localize lupus-antigens to sites within the primary and secondary lymphoid compartments where developing B cells undergo negative selection. To examine directly a role for complement C4 in protection from self-reactive B cells, we have bred the deficient mice with mice bearing rearranged immunoglobulin heavy chain and light chain anti-chromatin transgenes (Tg) (termed 564) specific for chromatin. On a normal C4-sufficient background, the self-reactive Tg B cells are blocked at the immature or transitional stage of development. By contrast, in the absence of C4, the self-reactive B cells continue to develop and enter the mature compartment. Moreover, they appear to secrete self-reactive antibody of both IgM and IgG isotypes. These results identify a functional role for C4 in regulation of self-reactive B cells and support the B-cell tolerance hypothesis.

Complement C4 is protective for lupus disease independent of C3.

The role of complement C3 in mediating systemic lupus erythematosus (SLE) was examined using a double-knockout C3(null)C4(null) Fas (CD95)-deficient mouse model. Results from this study reveal significant lymphadenopathy, splenomegaly, elevated titers of anti-nuclear Abs and anti-dsDNA Abs, an increased number of anti-dsDNA-producing cells in ELISPOT assay, as well as severe glomerulonephritis in the double-deficient mice. Based on these clinical, serological, and histological parameters, we find that autoimmune disease in the double-knockout group is similar in severity to that in C4(null) lpr mice, but not to that in C3(null) lpr mice. The development of severe SLE in the absence of both classical and alternative complement pathways suggests that it is the absence of C4, and not the presence of C3, that is critical in SLE pathogenesis. Thus, complement C4 provides an important protective role against the development of SLE.

IgG and Complement-Mediated Tissue Damage in the Absence of C2: Evidence of a Functionally Active C2-Bypass Pathway in a Guinea Pig Model

In vitro complement-mediated lysis of heavily sensitized sheep erythrocytes by C4-deficient (C4D) guinea pig and C2-deficient (C2D) human sera was demonstrated some years ago. It was postulated that these "complement-bypass" pathways resulted from activation of C1 and components of the alternative pathway. We used normal, C2D, and C4D guinea pigs in a Forssman shock model to test the in vivo relevance of the C2- and C4-bypass pathways of complement activation. High concentrations of both anti-Forssman Ab and C2D or C4D guinea pig serum induced efficient lysis of sheep erythrocytes in vitro. The most efficient lysis was observed when IgG Ab and C2D guinea pig serum were used. Blocking either the classical pathway (treatments with EGTA-Mg2+ or soluble recombinant complement receptor type 1 (sCR1)) or the alternative pathway (treatment with heating at 50 degrees C, sCR1, or soluble recombinant CR1 lacking the first of the four long homologous repeat sequences (sCR1[desLHR-A])) inhibited lysis; both pathways were required for lysis of sheep erythrocytes by C2D and C4D guinea pig sera. i.v. injection of anti-Forssman Ab in normal guinea pigs resulted in rapid death from pulmonary shock, whereas C4D guinea pigs had no adverse effect. Surprisingly, C2D guinea pigs either died in a delayed fashion or had a sublethal reaction. sCR1 treatment prevented Forssman shock in both normal and C2D guinea pigs, whereas sCR1[desLHR-A] prevented Forssman shock only in C2D animals. Our results suggest that the C2-bypass pathway occurs in vivo to produce tissue damage. Activation of complement in the absence of C2 appears to be far more efficient than in the absence of C4.

Characterization of neutralizing monoclonal antibodies to linear and conformation-dependent epitopes within the first and second variable domains of human immunodeficiency virus type 1 gp120.

A number of linear and conformation-dependent neutralizing monoclonal antibodies (MAbs) have been mapped to the first and second variable (V1 and V2) domains of human immunodeficiency virus type 1 (HIV-1) gp120. The majority of these MAbs are as effective at neutralizing HIV-1 infectivity as MAbs to the V3 domain and the CD4 binding site. The linear MAbs bind to amino acid residues 162 to 171, and changes at residues 183/184 (PI/SG) and 191/192/193 (YSL/GSS) within the V2 domain abrogate the binding of the two conformation-dependent MAbs, 11/68b and CRA-4, respectively. Surprisingly, a change at residue 435 (Y/H or Y/S), in a region of gp120 near the CD4 binding site (M. Kowalski, J. Potz, L. Basiripour, T. Dorfman, W. C. Goh, E. Terwilliger, A. Dayton, C. Rosen, W. Haseltine, and J. Sodroski, Science 237:1351-1355, 1987; L. A. Lasky, G. M. Nakamura, D. H. Smith, C. Fennie, C. Shimasaki, E. Patzer, P. Berman, T. Gregory, and D. Capon, Cell 50:975-985, 1987; and U. Olshevsky, E. Helseth, C. Furman, J. Li, W. Haseltine, and J. Sodroski, J. Virol. 64:5701-5707, 1990), abrogated gp120 recognition by both of the conformation-dependent MAbs. However, both MAbs 11/68b and CRA-4 were able to bind to HIV-1 V1V2 chimeric fusion proteins expressing the V1V2 domains in the absence of C4, suggesting that residues in C4 are not components of the epitopes but that amino acid changes in C4 may affect the structure of the V1V2 domains. This is consistent with the ability of soluble CD4 to block 11/68b and CRA-4 binding to both native cell surface-expressed gp120 and recombinant gp120 and suggests that the binding of the neutralizing MAbs to the virus occurs prior to receptor interaction. Since the reciprocal inhibition, i.e., antibody inhibition of CD4-gp120 binding, was not observed, the mechanism of neutralization is probably not a blockade of virus-receptor interaction. Finally, we demonstrate that linear sequences from the V2 region are immunogenic in HIV-1-infected individuals, suggesting that the primary neutralizing response may be directed to both V2 and V3 epitopes.

Organic chemistry of dinuclear metal centres. Part 12. Synthesis, X-ray crystal structure, and reactivity of the di-µ-alkylidene complex [Ru2(CO)2(µ-CHMe)(µ-CMe2)(η-C5H5)2]: alkylidene linking

Upon treatment with methyl-lithium followed by HBF4·OEt2 a carbon monoxide ligand of the µ-alkylidene complex [Ru2(CO)2(µ-CO)(µ-CMe2)(η-C5H5)2](1) is converted into µ-ethylidyne, giving [Ru2(CO)2(µ-CMe)(µ-CMe2)(η-C5H5)2]+(2). This is deprotonated readily by water to form the µ-vinylidene complex [Ru2(CO)2(µ-CCH2)(µ-CMe2)(η-C5H5)2](3), which quantitatively regenerates (2) with HBF4·OEt2. Addition of NaBH4 to (2) results in hydride attack on µ-CMe to yield the di-µ-alkylidene complex [Ru2(CO)2(µ-CHMe)(µ-CMe2)(η-C5H5)2](4) as cis and trans isomers. The structure of the trans isomer has been established by X-ray diffraction. Crystals are triclinic, space group P, with Z= 2 in a unit cell for which a= 8.474(2), b= 7.802(3), c= 12.989(5)A, α= 99.42(3), β= 96.96(3), and γ= 107.73(3)°. The structure was solved by heavy-atom methods and refined to R 0.026 (R′ 0.031) for 4 092 independent intensities. A ruthenium–ruthenium single bond of 2.701(1)A is symmetrically bridged by ethylidene [mean Ru–C 2.079(3)] and isopropylidene [mean Ru–C 2.107(3)A] ligands to form an approximately planar Ru2C2 ring with a non-bonding Me2C··CHMe distance of 3.20 A. Upon thermolysis the alkylidenes link to evolve Me2CCHMe, Me2CHCHCH2, and Et(Me)CCH2. The absence of C4 and C6 hydrocarbons indicates that the alkylidene coupling occurs intramolecularly, and the electronic and stereochemical requirements of this process are discussed. Unlike mono-µ-alkylidene complexes, [Ru2(CO)2(µ-CO)(µ-CR2)(η-C5H5)2], the cis and trans forms. of (4) do not interconvert thermally below 145 °C, but u.v. irradiation effects a slow trans to cis isomerisation. U.v. irradiation of (4) in the presence of dimethyl acetylenedicarboxylate promotes ethylidene–alkyne linking to form [Ru2(CO)(µ-CMe2){µ-C(CO2Me)C(CO2Me)CHMe}(η-C5H5)2], but with ethyne both of the alkylidenes are lost and the ruthenium–ruthenium double-bonded complex [Ru2(µ-CO)(µ-C2H2)(η-C5H5)2] is produced.

Metabolism of C4 and linkage analysis in a kindred with hereditary incomplete C4 deficiency.

We studied a kindred in which C4 deficiency had been discovered. Unlike families with total absence of C4, in this kindred C4 deficiency was found to be incomplete, autosomal dominant, not caused by null alleles, and not associated with a high incidence of systemic lupus erythematosus. The deficient state was caused by hyposynthesis of C4, not by hypercatabolism. The locus for incomplete C4 deficiency was not closely linked to the major histocompatibility complex. The abnormal autosomal dominant allele is, apparently, rare, and how it causes decreased synthesis of C4 is unknown.

Activation by Some T-Independent Antigens and B Cell Mitogens of the Alternative Pathway of the Complement System

A number of T-independent antigens and B cell mitogens were examined for their ability to activate C3 via the alternative pathway of the complement system. Loss of hemolytically active C3, generation of anaphylatoxin activity, and immunoelectrophoretic conversion of C3 and factor B, were checked in normal and C4-deficient guinea pig serum, and, in some cases, in normal human serum. As judged by their activity in these assays, 10 lipopolysaccharides of different origin and constitution, pneumococcus type III polysaccharide, levan, dinitrophenylated aminoethyl-dextran, dinitrophenylated (D-glutamic acid, D-lysin) copolymer, polymerized flagellin, and pokeweed mitogen were all capable of initiating the alternative pathway, but differed with respect to their potency, their relative activity in the presence or absence of C4, and their ability to inhibit C3-turnover at high concentrations. Polyvinylpyrrolidone of intermediate molecular weight (4 x 10(4) daltons) was only active if the most sensitive assay was used (anaphylatoxin generation). Other species of polyvinylpyrrolidone, depolymerized pneumococcal polysaccharide, aminoethyl-dextran, [D-glutamic acid, D-lysin] copolymer, phytohemagglutinin and concanavalin A failed to activate C3. C3-consumption by concanavalin A was due to nonspecific binding.

Experimental Autoimmune Renal Cortical Tubulointerstitial Disease in Guinea Pigs Lacking the Fourth Component of Complement (C4)

Abstract C4d guinea pigs develop experimental autoimmune renal tubulointerstitial disease indistinguishable from the disease observed in guinea pigs with circulating C4. These data demonstrate that an antibody-mediated autoimmune renal disease can occur in the absence of C4. We conclude: 1) complement may, at least in part, play a role in the pathogenesis, 2) complement activation does not occur via the classical sequence in this model, and 3) that activation and tissue injury may be mediated by an alternate complement pathway.

Mechanisms of immune lysis of red blood cells in vitro. I. Paroxysmal nocturnal hemoglobinuria cells.

The effect of five different reactions which activate complement (antibody activation, reduction in ionic strength, acidification, cobra venom factor (CoF) activation, and inulin activation) upon normal and PNH cells was investigated, using normal serum and serum devoid of the fourth component of complement (C4) activity from patients with hereditary angioneurotic edema (HANE) as a source of complement. Both normal and HANE serum lysed paroxysmal nocturnal hemoglobinuria (PNH) cells when complement was activated by acidification, CoF and inulin, indicating that the terminal steps of complement were activated in the absence of C4, presumably by the alternate or properdin pathway. Normal but not HANE serum lysed cells coated with anti-I, indicating that complement was activated by the C1-dependent classic pathway. HANE serum partially supported lysis by serum at reduced ionic strength, indicating that the activation of terminal complement components had occurred through both of the pathways of activation. The amount of the third component of human complement (C3) which was bound to the membrane of lysed and unlysed cells by these procedures was determined by anti-C3 absorption and was found to differ for each method of complement activation. In general, more C3 was bound to lysed cells than to unlysed cells. For given conditions, more was bound to PNH cells than to normal cells. However, very much less bound C3 was required for lysis of the PNH cells than for lysis of normal cells. These two phenomena, especially the latter, account for the marked lysis of PNH cells when complement is activated. Normal cells treated with AET (aminoethylisothiouronium bromide) did not bind more C3 than untreated cells and the lysed cells had less bound C3 than lysed PNH cells.