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PubMed1990

Neutralization of human immunodeficiency virus type 1 by complement occurs by viral lysis.

The ability of complement to inactivate human immunodeficiency virus (HIV) in the presence of specific antibody was evaluated. HIV was treated with complement and/or antibody, and then its titer was determined on the CD4+ H9 cell line. While complement alone had no effect on the HIV titer, complement plus subneutralizing levels of antibody resulted in titer reductions. Complement sources deficient in membrane attack component C5 or C8 did not inactivate antibody-treated HIV, suggesting that neutralization occurred via lysis. This possibility was investigated by assessing release of reverse transcriptase (RT) from the virion. Antibody plus complement, but neither reagent alone, released RT from HIV in a dose-dependent manner. Release of RT did not occur with C5- or C8-deficient sera, also indicating a requirement for membrane attack components. These studies show that complement can neutralize HIV via the classical complement pathway and that this neutralization occurs via C5b-9-mediated viral lysis. Thus, complement may play a major role in resistance to disease by lysing HIV and preventing infection of Fc- and complement receptor-positive cells, as well as CD4+ cells.

Antibody Formation↗
PubMed2005

Activation of the extracellular signal-regulated kinase by complement C5b-9.

Extracellular signals may be transmitted to nuclear or cytoplasmic effectors via the mitogen-activated protein kinases. In the passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury, proteinuria, and activation of phospholipases and protein kinases. This study addresses the complement-mediated activation of the extracellular signal-regulated kinase (ERK). C5b-9 induced ERK threonine202/tyrosine204 phosphorylation (which correlates with activation) in GEC in culture and PHN in vivo. Expression of a dominant-inhibitory mutant of Ras reduced complement-mediated activation of ERK, but activation was not affected significantly by downregulation of protein kinase C. Complement-induced ERK activation resulted in phosphorylation of cytosolic phospholipase A2 and was, in part, responsible for phosphorylation of mitogen-activated protein kinase-associated protein kinase-2, but did not induce phosphorylation of the transcription factor, Elk-1. Activation of ERK was attenuated by drugs that disassemble the actin cytoskeleton (cytochalasin D, latrunculin B), and these compounds interfered with the activation of ERK by mitogen-activated protein kinase kinase (MEK). Overexpression of a constitutively active RhoA as well as inhibition of Rho-associated kinase blocked complement-mediated ERK activation. Complement cytotoxicity was enhanced after disassembly of the actin cytoskeleton but was unaffected after inhibition of complement-induced ERK activation. However, complement cytotoxicity was enhanced in GEC that stably express constitutively active MEK. Thus complement-induced ERK activation depends on cytoskeletal remodelling and affects the regulation of distinct downstream substrates, while chronic, constitutive ERK activation exacerbates complement-mediated GEC injury.

Actins↗
PubMed2004

Membrane complement regulatory proteins in autoimmune and inflammatory tissue injury.

The complement system plays a complex role in the pathogenesis of autoimmune diseases. It inhibits autoimmunity development by helping to maintain self-tolerance and/or by facilitating the disposal of immune complexes and apoptotic cell antigens. On the other hand, complement activation is thought to contribute significantly to end organ damage in antibody-mediated autoimmune and inflammatory conditions, although the relevant importance of complement and Fe receptor pathways in these processes has recently been debated. To avoid autologous complement-mediated tissue injury, host cells normally express a number of soluble and membrane-bound complement regulatory proteins. Recent studies with gene knockout mice have suggested that membrane-bound complement regulatory proteins may critically determine the sensitivity of host tissues to complement injury in autoimmune and inflammatory disorders. Evidence is also accumulating to support the hypothesis that membrane complement regulatory proteins may not only inhibit complement-mediated injury during the effector phase of autoimmunity but also influence the adaptive immune response through complement-dependent or -independent mechanisms. The latter mechanism is likely related to their potential as cell surface signaling molecules.

Animals↗
PubMedDate not supplied

Binding of complement C3 to Klebsiella pneumoniae treated with sub-MIC cefodizime and chemiluminescence response of human polymorphonuclear leukocytes.

The binding of complement C3 to the cell surface of Klebsiella pneumoniae exposed to human serum complement after treatment with or without sub-MIC of antibiotics was examined by double diffusion immunoprecipitation against anti-human complement C3, and the production of oxygen-derived radicals by human polymorphonuclear leukocytes stimulated by complement-opsonized K. pneumoniae after treatment with or without sub-MIC of antibiotics was measured using the chemiluminescence (CL) assay. Complement C3 bound to the cell surface of K. pneumoniae treated with cefodizime was detected after exposure to human serum complement. The CL response induced by complement-opsonized bacteria after treatment with cefodizime was much higher than the response induced by nontreated bacteria or complement-opsonized bacteria after treatment with other antibiotics. These findings indicate that treatment with sub-MIC cefodizime make K. pneumoniae more susceptible to opsonization by complement and promotes the specific phagocytosis mediated by complement receptors.

Cefotaxime↗
PubMed2001

Complement activation in patients with congestive heart failure: effect of high-dose intravenous immunoglobulin treatment.

BACKGROUND: Increasing evidence implicates innate immunity in the pathogenesis of congestive heart failure (CHF). In the present study, we examined the possible role of complement, an important part of innate immunity, in CHF. METHODS AND RESULTS: Complement activation was analyzed in systemic and coronary circulation in 39 patients with CHF and 20 healthy control subjects. In a double-blind, placebo-controlled study, we have recently reported that high-dose intravenous immunoglobulin (IVIG) improves left ventricular ejection fraction (LVEF) in these patients. To examine if this improvement was related to IVIG-induced effects on complement, we also examined complement activation during induction (first week) and maintenance therapy (6 months) with IVIG or placebo. Our main findings were: (1) We found enhanced systemic complement activation involving classic, alternative, as well as terminal pathway in patients with CHF compared with healthy control subjects. (2) Particularly enhanced complement activation was found in coronary sinus, representing venous drainage from the heart. (3) The systemic complement activation was further enhanced during IVIG but not during placebo therapy, particularly during induction therapy. (4) Although IVIG improved LVEF in patients with CHF, the degree of IVIG-mediated complement activation was negatively correlated with this improvement of LVEF. CONCLUSIONS: This study further supports the involvement of innate immunity in the pathogenesis of CHF. Our findings suggest that complement may be added to the list of possible therapeutic targets in CHF and that future studies with specific complement inhibitors may be of interest in this disorder.

C-Reactive Protein↗
PubMed1975

Complement fixation by rheumatoid factor.

The capacity for fixation and activation of hemolytic complement by polyclonal IgM rheumatoid factors (RF) isolated from sera of patients with rheumatoid arthritis and monoclonal IgM-RF isolated from the cryoprecipitates of patients with IgM-IgG mixed cryoglobulinemia was examined. RF mixed with aggregated, reduced, and alkylated human IgG (Agg-R/A-IgG) in the fluid phase failed to significantly reduce the level of total hemolytic complement, CH50, or of individual complement components, C1, C2, C3, and C5. However, sheep erythrocytes (SRC) coated with Agg-R/A-IgG or with reduced and alkylated rabbit IgG anti-SRC antibody were hemolyzed by complement in the presence of polyclonal IgM-RF. Human and guinea pig complement worked equally well. The degree of hemolysis was in direct proportion to the hemagglutination titer of the RF against the same coated cells. Monoclonal IgM-RF, normal human IgM, and purified Waldenström macroglobulins without antiglobulin activity were all inert. Hemolysis of coated SRC by RF and complement was inhibited by prior treatment of the complement source with chelating agents, hydrazine, cobra venom factor, specific antisera to C1q, CR, C5, C6, or C8, or by heating at 56 degrees C for 30 min. Purified radiolabeled C4, C3, and C8 included in the complement source were bound to hemolysed SRC in direct proportion to the degree of hemolysis. These data indicate that polyclonal IgM-RF fix and activate complement via the classic pathway. The system described for assessing complement fixation by isolated RF is readily adaptable to use with whole human serum.

Animals↗
PubMed1982

A quantitative analysis of the interactions of antipneumococcal antibody and complement in experimental pneumococcal bacteremia.

The mechanism of protection of type-specific antipneumococcal antibody and complement in bacteremia was investigated with purified rabbit antibody and a guinea pig model of pneumococcal bacteremia. IgG and IgM were isolated from the sera of rabbits immunized with type 7 pneumococci (Pn), and their binding to Pn was quantitated. The number of antibody-binding sites on the pnuemococcal capsule was also determined. Pn were incubated with various amounts of the immunoglobulin preparations before intravenous injection into nonimmune guinea pigs. Whereas 120 molecules of IgM per Pn were sufficient to enhance bloodstream clearance of Pn, 1,400 molecules of IgG per bacterium were required to produce this effect. As the amount of either IgG or IgM added to the Pn was increased, the rate of bloodstream clearance accelerated. In striking contrast, greater than 1,000 molecules of IgM had no effect on the rate of clearance in C4-deficient guinea pigs, which cannot activate complement via the classic pathway. Similarly, 5,000 molecules of IgG had only minimal effect in C4-deficient guinea pigs, and 24,000 molecules of IgG had no effect in guinea pigs depleted of complement by cobra venom factor. Thus, the in vivo opsonic effects of both IgG and IgM anticapsular antibody are mediated via their ability to activate complement. IgG anti-pneumococcal cell wall antibody, raised by intravenous injection of rabbits with unencapsulated Pn, had no effect on the rate of bloodstream clearance of Pn or on the polymorphonuclear leukocyte killing of type 7 Pn in an in vitro bacterial assay. Because the opsonic effects of anticapsular antibody required complement activation, the ability of anticell wall IgG to activate complement was compared with the two classes of anticapsular antibody. As judged by depletion of C3 and C4 from guinea pig serum, as well as by the fixation of radiolabeled C3 to Pn, IgM anticapsular antibody was the best complement activator. However, anticell wall IgG was somewhat more active than anticapsular IgG in each of these tests of complement activation and fixation. When equivalent amounts of C3 were fixed to Pn by each of the three antibodies, Pn sensitized with IgG and IgM anticapsular antibodies caused immune adherence, whereas Pn sensitized with anticell wall IgG did not. This may explain the failure of anticell wall antibody of mediate complement-dependent phagocytosis of Pn in vivo or in vitro. Although anticell wall IgG is capable of activating complement and fixing C3 to Pn, it is not opsonic; the most likely reason is that the nonopsonic antibody mediates C3 deposition in sites on the Pn that cannot interact efficiently with phagocytic cell C3 receptors.

Animals↗
PubMedDate not supplied

Immunohistochemistry of complement response on human renal cell carcinoma biopsies.

The goal of the study was to characterize the complement humoral and cellular antitumor responses on primary renal cell carcinoma biopsies. As an original observation, complement activation was found on 11/22 cases. Classical complement pathway activation was characterized by tumor C1q complement protein and IgG deposition (5/22 cases). Alternative or nonimmune complement pathway activation was seen as tissue deposition of C3 (6/22 cases). The membrane attack complex was present in cases with alternative complement pathway activation at the sites of tumor necrosis. Renal cell carcinomas with complement activation overexpressed at least one of the complement regulatory factors (membrane cofactor protein, decay accelerating factor, membrane attack complex inhibitor) and major histocompatibility complex class II molecules. Tumor infiltrating lymphocytes were present in most of the renal cell carcinomas with complement activation (8/11). However, the number of tumor-infiltrating lymphocytes was correlated with the intensity of major histo-compatibility complex-II expression in 18/22 cases. Detection of complement activation and immune cell infiltrates on renal cell carcinoma primary biopsies may serve as a new predictive factor for immunotherapy.

Adult↗
PubMed1998

Characterization of bovine serum factor triggering the lysis of liposomes via complement activation.

Our previous studies have shown that the degree of damage to a liposome corresponds to the variability of the animal species from which the serum comes, and that a complement activating factor (CAF) plays an important role in inducing the activation of the complement system, ultimately leading to the lysis of the liposomes. In this study, our attention focused on the characterization of the bovine serum factor (bCAF) that is involved in complement-mediated immune lysis of the liposome. The active fraction containing CAF partially purified with PEG and ammonium sulfate results in marked activation of the complement system via the alternative pathway when interacted with CAF-depleted serum, whereas the active fraction or CAF-depleted serum alone does not activate the complement. The interaction between lipopolysaccharide (LPS), heparin, zymosan or their mixture in place of CAF and CAF-depleted serum does not result in any significant activation of the complement system. Results from pretreatment with rabbit anti-bovine IgM IgG and rabbit anti-bovine IgG IgG indicate that activation of the complement system is not attributable to the antibody which is generally involved in activation of complement via the classical pathway. The results have further been proven by pretreatment with Concanavalin A (Con A) sepharose and protein G sepharose ruling out the possibility of antibody-mediated activation of complement. Our studies on collagenase and trypsin digestion demonstrate that the relative activity of CAF does not diminish with increase in collagenase concentration, and decreases with increase in trypsin concentration, strongly indicating that CAF does not have a collagen-like domain in its structure. The relative activity of CAF is dramatically inhibited after reduction with 2-mercaptoethanol (2-ME), clearly demonstrating that CAF is sensitive to reduction with 2-ME and confirming a sulhydryl-dependent protein. The optimal activity of CAF is observed in the range of 35-45 degrees C and its half-life at 37 degrees C is about 105 h. Furthermore, the relative activity of CAF increases and gradually approaches a plateau level with the increase of Mg2+ concentration. Obviously, complement activation induced by CAF depends on adequate Mg2+ concentration, confirming that this dependence is characteristic of the alternative pathway.

Ammonium Sulfate↗
PubMed1995

Contradictory roles for antibody and complement in the interaction of Brucella abortus with its host.

The ability of serum complement to kill bacteria has been linked to host resistance to Gram-negative bacteria. A mechanism for killing extracellular organisms during early invasion, following release from infected phagocytic cells, or during bacteremia would contribute to a host's ability to resist disease. In fact, the ability of serum complement to kill bacteria has been linked to disease resistance. Brucella abortus are Gram-negative intracellular pathogens. Resistance to these bacteria involves the coordinated activities of the cellular and humoral immune systems. The existence of serum-resistant forms of B. abortus has been established, and it has been shown that these bacteria can resist the killing action of complement even in the presence of specific antibody. Antibody is usually necessary for complement-mediated killing of smooth (virulent) forms of Gram-negative bacteria. An anomolous situation exists with some isolates of smooth B. abortus. Sera containing high titers of specific antibody do not support killing unless they are diluted. In the bovine, this phenomenon is associated with IgG1 and IgG2 antibodies. This finding may account for the lack of positive correlation between antibody levels and resistance to disease, which has led, perhaps wrongly, to the idea that antibody and complement are not important in resistance to brucellosis. Available evidence suggests that antibody may have contradictory roles in the interactions between a host and bacteria. Avirulent (rough) forms of the organism would be rapidly killed by complement shortly after invasion, but serum-resistant smooth forms of the organism would survive and invade resident phagocytic cells. During the process of invasion and phagocytosis, the bacteria would initiate an immune response. With time, some B. abortus organisms would be released from infected phagocytic cells. In the early stages of this process, the bacteria would encounter IgM antibody and low concentrations of IgG antibody. These would cause complement-mediated killing, and infection would be restricted to resident phagocytic cells. However, the immune response to B. abortus antigens would be intensified, and IgG antibody levels would increase. High concentrations of antibody do no support complement-mediated killing of extracellular B. abortus, but the bacteria would be opsonized by antibody and complement component fragments. This would lead to increased phagocytosis of extracellular B. abortus as they appear, and concomitant extension of disease. Because of high levels of antibody would block complement-mediated killing of B. abortus, resistance to disease at this point would be dependent on cell-mediated immunity.

Animals↗
PubMed1999

The local production of complement in the pathogenesis of renal inflammation.

The deposition of complement components is a feature of many immune mediated human glomerular diseases. Experimental models provide evidence that complement activation within the glomerulus has a pathogenic role in immune complex and antibody mediated glomerulonephritis. It was thought that the complement components deposited within the kidney were derived from the systemic circulating pool. However, recent work has shown that the kidney is able to produce many of the components of the complement cascade. In vitro work has shown that cells of glomerular and tubular origin can produce complement, as can inflammatory cells present within the kidney during inflammation. Analysis of human biopsy material has shown that expression of complement genes is increased during inflammatory renal disease implicating local complement synthesis as a potential source of complement components. At present no direct evidence for a pathogenic role of local complement synthesis exists. In this review we consider the current experimental evidence which suggests that local production of complement may be contributing to renal injury in a variety of diseases.

Complement System Proteins↗
PubMed1976

The indiction by complement of a change in KSCN-dissociable red cell membrane lipids.

During complement lysis of antibody-sensitized sheep erythrocytes (EA) there was a larger loss of membrane phospholipids than during lysis elicited by hypotonic buffer. In addition, membranes prepared from complement-lysed EA had a marked reduction in KSCN (2.4 M)-dissociable membrane cholesterol and phospholipids, as compared to membranes from EA lysed hypotonically. Complement lysis caused a mild reduction in the amount of KSCN-dissociable membrane hexose but no change in the amount of dissociable protein. The impairment in dissociation of membrane lipids was related to the action of C8; it did not occur with membranes from EA that were treated with heat-inactivated (56 degrees C for 30 min) human serum, C4-deficient guinea pig serum, C6-deficient rabbit serum, or the first seven human complement components. EA lysed with limited amounts of complement exhibited a partial impairment in KSCN-dissociable lipids. Membranes from erythrocytes lysed with melittin showed a large increase in dissociation by KSCN of lipids, proteins,and hexoses. Membranes from erythocytes lysed with lysolecithin or phospholipase C showed, in addition to a reduction in dissociable lipid, a much larger reduction in dissociable hexose than a membranes from complement-lysed cells. These profiles of reactivity with 2.4 M KSCN inidcate that the membrane pertubations caused caused by complement may be specific. We conclude that complement-lysis is accompanied by a major rearrangement of membrane cholesterol and phospholipid which could be demonstrated in membranes from cells lysed by only one or very few complement lesions. Therefore, it appears that the lesions induce a propragated change in the lipid organization which extends throughout large areas of the membrane. This change might be responsible for the impairment of membrane permeability that follows the action of complement and results in cell destruction.

Animals↗
PubMed1991

[Role of complement in experimental glomerulonephritis].

The complement system is composed by 26 plasmatic proteins. The activation of either the classical or alternative complement pathway leads to the formation of the membrane attack complex C5b-C9 (MAC) which is capable of producing damage of the cellular membrane. MAC has been identified in renal biopsies from human and experimental, immune and nonimmune renal diseases, but it has not been possible to demonstrate any enzymatic activity on the glomerular basement membrane components (GBM). MAC can produce a lytic or a nonlytic effect on renal cells depending upon the dose used. The lytic effect in vitro has been demonstrated in epithelial, mesangial and endothelial cells, whereas the lytic effect in vivo has been described in a model of acute glomerulonephritis produced by the administration of monoclonal antibody anti-Thy 1.1. which reacts with mesangial cells. The nonlytic effect of MAC on renal cells is characterized by alterations in cell metabolism which can lead the production of prostaglandins, type IV collagen, reactive oxygen species, and a growth factor resembling interleukin I which can contribute to glomerular damage, to the modification of the filtration barrier permeability and hemodynamic changes in experimental glomerulonephritis. The effector mechanisms by which the complement system participates in the pathogenesis of glomerulonephritis are different in the various experimental models of nephritis. In nephrotoxic nephritis the complement pathway participates at least in 3 different ways: a) complement-neutrophil mediated injury, b) MAC dependent mechanism and c) producing hemodynamic alterations. In acute serum sickness the complement system beyond C2 is not necessary for the development of proteinuria and glomerular inflammation, but the MAC assembly seems to be important for the formation of large deposits. In the chronic serum sickness model, the complement system participates in the early proteinuria as well as in the histological expression of the disease. In the heterologous phase of Heymann's nephritis, the proteinuria is complement dependent whereas in the autologous phase the damage depends upon cellular mediated immunity. In passive Heymann's nephritis the complement system and particularly MAC, has a central role for the histological lesion of the epithelial cell as well as in the proteinuria. In conclusion, the complement system can mediate renal damage by the following mechanism: a) Releasing chemotactic factors which result in neutrophil recruitment and neutrophil mediated glomerular damage.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Neutralization of porcine transmissible gastroenteritis virus by complement-dependent monoclonal antibodies.

Monoclonal antibodies (MAB) to each of the 3 major structural proteins of transmissible gastroenteritis virus (TGEV) of swine were compared, using their virus-neutralizing (VN) activity in the presence or absence of guinea pig, rabbit, or swine complement. The MAB against the peplomer protein had similar VN titers for TGEV in the presence or absence of complement from any source. The MAB against the matrix protein had VN activity for TGEV only in the presence of complement, whereas MAB specific for the nucleocapsid protein did not possess VN activity in the presence or absence of complement. Serum from sows containing antibodies against TGEV peplomer and matrix proteins had slightly higher VN titers in the presence of complement than in the absence of complement. High concentrations of complement from swine serum (128 U) had little effect on the infectivity of TGEV for swine testes cells, whereas 32 U of complement from rabbit serum and 64 U of complement from guinea pig serum were able to neutralize virulent and attenuated TGEV in the absence of known antibodies for TGEV. Complement (less than or equal to 8 U) from any source did not decrease the infectivity of TGEV by greater than 0.5 log10 units.

Animals↗
PubMed1985

Anti-glomerular basement membrane antibody-induced experimental glomerulonephritis: evidence for dose-dependent, direct antibody and complement-induced, cell-independent injury.

The immunopathogenesis of anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM-GN) involves the triad of antibody fixation to the glomerular basement membrane, local complement activation and polymorphonuclear leucocyte (PMN) infiltration. We sought to investigate the potential for contributions to renal injury from each component of this triad (i.e., antibody, complement, and PMN). This study compares the ability of antibody to induce injury in normal, PMN-depleted and dual (i.e., PMN + complement)-depleted rabbits by assessing the quantity of kidney-fixed antibody (KFA) necessary to induce proteinuria. (The KFA levels are expressed in micrograms of antibody bound per gram renal cortex.) Normal animals developed significant proteinuria at KFA 60 +/- 6 micrograms/g. PMN-depleted animals were injury free at KFA 60 +/- 6 micrograms/g, but developed heavy proteinuria at KFA 140 +/- 5 micrograms/g. The protection from injury by PMN depletion at lower KFA levels confirms the important contribution of PMN to injury in this model. Furthermore, the demonstration of a clearcut threshold for injury despite PMN-depletion indicates that glomerular antibody deposition and complement activation can cause direct PMN-independent injury. Dual depletion studies showed a significantly higher KFA threshold for injury in PMN + complement-depleted animals than in PMN-depleted, complement intact animals (195 +/- 10 micrograms/g, cf. 140 +/- 5 micrograms/g; p less than 0.01). The occurrence of injury despite dual mediator depletion demonstrates that antibody itself can produce direct, complement and PMN independent renal injury. Together the PMN depletion and dual depletion studies indicate that antibody-induced glomerular complement activation produces direct, PMN-independent renal injury that can be prevented by complement depletion. Thus each of the potential mediators studied is individually capable of producing renal injury in anti-GBM-GN. In addition to the well established injurious role of PMN, this study demonstrates that glomerular complement activation and anti-GBM antibody deposition may both produce neutrophil-independent components of renal injury. The relative contributions of the individual mediators to injury is dependent on the quantity of antibody deposited within the glomerulus.

Animals↗
PubMed1983

Pneumococcal sonicate-induced biphasic granulocytopenia and its dissociation from pulmonary leukostasis. The contribution of pneumococcal products and complement to early and late granulocytopenic phases.

The pathogenesis of pneumococcus (PNC)-induced granulocytopenia is unclear. We studied its relationship to pulmonary leukostasis and the possible roles of PNC constituents and complement. Nonviable PNC was infused into normal and 99% C3-depleted rabbits. PNC challenge induced both granulocytopenia and pulmonary leukostasis in normal animals; complement-depleted animals displayed granulocytopenia without pulmonary leukostasis. Therefore an intact complement system was not essential to the granulocytopenia, whereas pulmonary leukostasis was complement-dependent. Rabbits infused with serum, plasma, or nonpyrogenic normal saline, each after in vitro incubation with PNC, developed significant granulocytopenia (p less than 0.001, p less than 0.01, and p less than 0.001, respectively) with maximal mean percent decreases of -98%, -97%, and -91%, respectively. When the animals were sacrificed at 3 hr, no pulmonary leukostasis was found. The granulocytopenia persisted for 3 hr after infusion of either PNC-exposed serum or plasma, whereas the granulocytopenia induced by PNC-exposed saline was of less than or equal to 1 hr duration. If serum or plasma complement was inactivated prior to PNC incubation, subsequent infusion also induced significant granulocytopenia of less than 1 hr duration (p less than 0.05 and p less than 0.01). PNC-exposed saline that was subsequently heat-treated induced this same early (less than or equal to 1 hr) granulocytopenia (p less than 0.05). Control animals infused with serum, plasma, or saline unexposed to PNC displayed neither granulocytopenia nor pulmonary leukostasis. Therefore neither prior in vitro complement inactivation nor heat treatment after PNC incubation prevented the early granulocytopenic phase; in vitro complement inactivation totally aborted the late phase. The complement-independent, heat-stable early granulocytopenic phase was further investigated. Its granulocytopenia-inducing activity did not require the presence of PNC capsular polysaccharide and was resistant to trypsin treatment. With ultrafiltration, its molecular weight was 100,000 to 300,000. Thus PNC-induced granulocytopenia is a multifactorial phenomenon involving both complement-dependent and complement-independent mechanisms as well as contribution by PNC constituents or by-products.

Agranulocytosis↗
PubMed1982

[Chinese hamster cells with mutation in the hypoxanthine-guanine phosphoribosyltransferase locus. III. The biochemical characteristics of the hypoxanthine-guanine phosphoribosyltransferase of complementing mutants].

The paper presents a biochemical study of hypoxanthine-guanine phosphoribozyltransferase (HRPT) in mutant clones of Chinese hamster cells showing an ability for complementation. In order to characterize HPRT, its kinetic properties, temperature sensitivity and electrophoretic mobility in polyacrylamide gel were assayed. According to the complementation map, the nine mutant clones studied can be divided into four complementation groups. All these clones have been shown to be mutants with respect to the HPRT structural gene, as they synthesize the structurally and functionally altered enzyme. A comparative biochemical analysis of HPRT in the four complementation groups revealed substantial differences in mutant enzymes from different groups; hence, the possibility of complementation on the molecular level. All biochemical characteristics of HPRT tested are similar in clones belonging to one and the same complementation group, which could indicate that they have the same structural variant of the enzyme, regardless of the manner in which the mutants were obtained. Having revealed the similarity and the distinctive features of mutant enzymes within complementation groups, the biochemical analysis confirmed the results of complementation analysis and added the structural information concerning mutant variants of the enzyme. Thus, the complementation map of the HPRT gene yielded by hybridological analysis has been tested and confirmed by an independent biochemical study. Complementation analysis applied to the HPRT mutants made it possible to identify qualitatively distinct groups. Each of these groups may be regarded as an allele of the gene, and the sum of the groups may be regarded as a series of multiple alleles.

Animals↗
PubMed1977

Cholesterol-dependent human complement activation resulting in damage to liposomal model membranes.

Human (but not guinea pig) complement-mediated damage. It was concluded that human complement was activated spontaneously by liposomes containing a high concentration (71 mol %) of cholesterol. This occurred in the absence of any recognizable antigen or antibody, and did not occur at a low concentration (43 mol %) of cholesterol. Activation of complement resulted in membrane damage and release of trapped liposomal glucose. The complement activity was inhibited by preheating (56 degrees C, 30 min), 10 mM Mg2EDTA3 or EGTA, and by prior adsorption with insoluble immune complexes. Almost all human sera had some reactivity, but it ranged from very low levels (less than 7% liposomal glucose release) to very high levels (greater than 50% glucose release). Complement activation appeared to be mediated by a serum factor which could be removed by adsorption and which was partially heat labile. The factor was transferred by adding heated high reacting human serum to unheated low reacting human serum, or to guinea pig serum. The serum factor, although quantitatively diminished in potency due to heat lability, caused equal activation of each of these two latter complement sources in the presence of high cholesterol liposomes. It did not cause activation of C4-deficient guinea pig complement. These data suggested that the classical complement pathway was activated. The liposomal membrane composition had an influence on this phenomenon. Activities of about half of the human sera were enhanced when galactosyl ceramide, or ceramide alone, was present in the liposomes. Activity was enhanced by longer fatty acyl chain lengths of lecithin when dimyristoyl-, dipalmitoyl-, or distearoyllecithin was employed in the liposomes. Liposomes containing sphingomyelin as the only phospholipid were not sensitive to cholesterol-dependent complement-mediated damage. It was concluded that human complement was activated in the presence of high concentrations of membrane cholesterol and that this was caused by an uncharacterized serum factor and was influenced by the lipid composition of the membrane.

Ceramides↗