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PubMed1989

Distribution of complement in the sclera.

In the present study, we compared hemolytic activities of C1, C4, C2, C3, C5, C6 and C7 in the anterior and posterior sclera. Additionally, we used radial immunodiffusion to measure levels of Factor B, IgG, IgA and albumin in the anterior and posterior sclera. Except for C1, complement levels were significantly higher in the posterior than anterior sclera. Additionally, levels of immunoglobulins as well as albumin were significantly higher in the posterior than anterior sclera. These results suggest that the posterior sclera has a better adjacent vascular supply than the anterior sclera. On the other hand, the results of this study show that the anterior sclera has more C1, the recognition unit of the classical pathway, than the posterior sclera. Because there is nearly twice as much C1 in the anterior sclera, it may be easier for antigen-antibody complexes, whether formed in the sclera itself or derived from the neighboring vessels, to set off the complement cascade in the anterior sclera. This finding may help explain why scleritis associated with immune complex disease is more common in the anterior than posterior sclera.

Adult↗
PubMed1989

Complement activity and selected hematologic variables in newborn foals fed bovine colostrum.

Serum complement activity and selected hematologic variables were evaluated in 5 newborn foals fed bovine colostrum (principal group) and 6 foals allowed to nurse their dam (control group). Also, bovine colostrum was evaluated for anti-equine antibodies. Precolostral serum hemolytic and conglutinating complement activities were low and increased similarly in foals of both groups to reach adult values between 1 and 3 weeks after birth. Bovine colostrum strongly agglutinated, but did not hemolyse principal foals' RBC and blood containing all known equine blood group alloantigens. Hemolysis was not detected after administration of bovine colostrum. Physiologic anemia developed in foals of principal and control groups during the first week of life. Erythrocyte osmotic fragility in foals of the principal group prior to and after the ingestion of colostrum remained unchanged. However, at 36 hours after birth, there was a significant decrease in erythrocyte osmotic fragility in foals fed homologous colostrum.

Animals↗
PubMed1987

The effects of capsaicin denervation on leucocyte and complement components of the inflammatory response.

Primary afferent unmyelinated nerves containing substance P modulate the inflammatory response to injury through a process known as neurogenic inflammation. Rats treated as neonates with capsaicin, to destroy these nerves permanently, have a reduced capacity to mount an inflammatory response as measured by swelling or plasma extravasation. In this study we examined some aspects of the immunological response to injury in capsaicin-treated rats. One question addressed was whether changes in the inflammatory response are due to abnormal innervation or to a direct toxic effect of capsaicin. It was observed that the histological appearance of thymus, splenic white pulp and popliteal lymph nodes was unaltered in capsaicin-treated rats. However, there was an increase in leucocyte number in splenic red pulp. There was also an increase in the white cell numbers in the peripheral circulation (7 +/- 1 X 10(6)/mL for normal rats and 14 +/- 2 X 10(6)/mL in capsaicin-treated rats, p less than 0.001, Student's t-test). This affected all white cell subgroups. Complement levels in capsaicin-treated rats were not different from normals and both groups showed a similar depletion 6 days following antigenic stimulation with sheep red cells. These data support the view that the reduced cellular response to injury in the capsaicin-treated rat is secondary to the effect of the chemical on afferent nerves and not to a primary toxic effect on cells of the inflammatory response.

Animals↗
PubMed1985

Hemolytic plate assay for quantification of active human complement component C3 using methylamine-treated plasma as complement source.

A hemolytic plate assay specific for active human complement component C3 is described. The method is well suited for tracing active C3 during preparative purification or for screening of plasma samples. The assay is based on activation of the alternative pathway of complement by unmodified rabbit erythrocytes. Plasma treated with methylamine supplies the essential complement components other than C3. The lytic reaction is complete in 5 h at 37 degrees C and is unchanged by incubation overnight. The dose-response curve, i.e., lysis diameter versus logarithm of C3 concentration, is linear within 0.1-10 times normal plasma concentrations of C3. The standard deviation is below 10%. The hemolytic agarose plates are easy and inexpensive to prepare, and they can be stored at 4 degrees C for 2 weeks before use. This paper describes the optimal conditions of the assay and proves its specificity. Its use in C3 preparation and plasma screening for C3 is discussed.

Animals↗
PubMed1989

A hemolytic assay for the measurement of equine complement.

A hemolytic assay was developed for the measurement of functional equine complement activity. The assay utilizes antibody sensitized chicken erythrocytes as the target cell and was specific for classical pathway (antibody dependent) complement activity. The assay was found to be reproducible and more sensitive than previous reports using other species of target cells. Total serum complement (CH50) values were determined for five mares and their foals and followed over a period of 3 months.

Animals↗
PubMed1992

The effects of complement depletion on corneal inflammation in rats.

There are indications that complement activation may be involved in inflammatory processes of the cornea. To investigate the role of the complement system in experimental keratitis, rats were depleted of their plasma complement by treatment with cobra venom factor (CVF). intraperitoneal injection of CVF resulted in undetectable complement serum activity for 6 days when measured by a hemolytic assay. The corneal inflammatory response, induced by a single intracorneal injection of heterologous serum into nonsensitized CVF-treated rats, was suppressed significantly. The onset of the clinical symptoms of keratitis was delayed, and the severity was reduced. In addition, analysis of the serum antibody titers showed impaired antibody synthesis in the CVF-treated group. When keratitis was induced by an intracorneal antigen challenge in sensitized rats, no difference was observed when comparing the clinical signs of keratitis of CVF-treated animals with sham-treated animals. In addition, CVF treatment did not alter the course of lipopolysaccharide (LPS)-induced corneal inflammation. These findings suggest that the role of plasma complement activation in antigen- and LPS-induced keratitis appears to be limited. The implications for the immunopathologic mechanisms underlying keratitis are discussed.

Animals↗
PubMed1991

The role of surface charge in the activation of the classical and alternative pathways of complement by liposomes.

We have studied the complement-activating properties of liposomes. We show that surface charge is a key determinant of complement-activating liposomes. The nature of the charge, whether negative or positive, appears to dictate which pathway of the complement system is activated. Phosphatidylcholine:cholesterol (PC:CHOL, 55:45 mol/mol) liposomes were made to exhibit a positive or negative surface charge by the addition of cationic or anionic lipids, respectively. Normal human or guinea pig serum was incubated with liposomes, followed by determining the residual hemolytic activity of the serum as a measure of complement activation. Negatively charged liposomes containing phosphatidyl-glycerol, phosphatidic acid, cardiolipin, phosphatidylinositol, or phosphatidylserine activated complement in a Ca(2+)-dependent manner suggesting activation occurred via the classical pathway. Positively charged liposomes containing stearylamine or 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane activated complement via the alternative pathway. Neutral liposomes, PC:CHOL (55:45) and PC:CHOL:dipalmitoylphosphatidylethanolamine (35:45:20), failed to activate complement as measured by the hemolytic assays. We show that unsaturated liposomes are more potent complement activators than saturated liposomes and that 45 mol% cholesterol promotes complement protein-liposome interactions. Immunoblot analysis of phosphatidylglycerol-containing liposomes showed that C3b and C9 were associated with these liposomes. Thus, the complement consumption measured in the hemolytic assays represents active cleavage of the complement components and not passive adsorption to the liposome surface. These studies suggest that membranes composed of net charged phospholipids can activate the complement system. This observation underlines the importance in biologic membranes of complement regulatory proteins that protect normal cells from complement attack.

Animals↗
PubMed1987

Identical complement concentrations in blood obtained from central venous catheters, arterial lines, and antecubital phlebotomy.

In vitro complement activation has been detected during passage of blood through tubing used in hemodialysis and cardiopulmonary bypass surgery, in addition to tubing being investigated for vascular grafts. Because tubing from arterial lines and central venous catheters are composed of similar materials and are used when blood is withdrawn from patients for complement assays, a study was undertaken to assess the degree of complement activation in blood obtained from these sites. Complement activation was determined by a terminal complement complex enzyme-linked immunosorbent assay (ELISA), C1rC1s-C1 inhibitor complex ELISA, and 50% complement hemolytic activity (CH50) assay. Blood simultaneously obtained from the arterial lines and central venous catheters had identical terminal complement complex, C1rC1s-C1 inhibitor complex, and CH50 levels when compared with blood withdrawn by antecubital phlebotomy. Because blood may be obtained from the arterial lines and central venous catheters for complement assays, the pain and possible bruising from antecubital phlebotomy could be eliminated. Furthermore, blood may now be withdrawn from indwelling catheters without fear of in vitro complement activation causing a misinterpretation of results.

Arteries↗
PubMed1990

Specific, sensitive, precise, and rapid functional chromogenic assay of activated first complement component (C1) in plasma.

We present a new functional assay for the first complement component (C1) in plasma, based on its activation by inhibition of the C1-esterase inhibitor (C1-inh) when monospecific antiserum to C1-inh is added to the plasma. After maximal activation, we can determine the concentration of activated C1 by using an amidolytic rate assay with a chromogenic substrate. We have optimized the assay conditions with respect to incubation time, concentration of antiserum to C1-inh, ionic strength, and pH. Our method determines specifically the concentration in plasma of free activated C1, not complexes of activated C1 with C1-inh, and is not influenced by the concentration of C1-inh in the test sample. Concentrations of C1 correlated significantly with activities determined by a hemolytic assay (r = 0.55, t = 4.09, P less than 0.001). The estimated interassay CV was 5% and the intra-assay CV was 1%. The sensitivity, imprecision, and practical test performance of our assay are superior to those of conventionally used hemolytic assays.

Adult↗
PubMed1992

Assessment of complement activation in clinical samples. Comparison of immunochemical and functional measurements of complement components with quantitation of activation fragments.

We have compared functional and immunochemical measurements of complement components with assays measuring the generation of activation fragments, for the assessment of classical pathway activation in vitro and in vivo. The generation of the C3a, C3b/C3bi cleavage fragments of C3, and of the C4d cleavage fragment of C4 measured by ELISA and RIA, was correlated with the decrease in total complement hemolytic activity (CH50) and in functional activity of C3 and C4 in normal human serum in which the classical pathway had been activated with aggregated IgG. The decrease in CH50 in in vitro activated serum was also correlated with the generation of C5a and soluble SC5b-9 complexes. In contrast, no or little increase in the concentration of C3a, C3b/C3bi and C4d was observed in plasma samples from patients with low CH50 and with low levels of immunochemical C3 and C4, indicating that fragment quantitation assays provide no information on the presence and extent of complement activation in vivo. Analysis of samples from patients expressing the four C4 genes and patients having one or two C4 null alleles indicated that a ratio of hemolytic C4 to C2 > or = 1 was indicative of complement activation without C4 deficiency, whereas a ratio below 1 was indicative of C4 deficiency with or without classical pathway consumption. Classical pathway activation and C4 deficiency in clinical samples are best predicted by the concomitant assessment of immunochemical levels of C3 and C4 and hemolytic levels of C4, C2 and C3.

Complement Activation↗
PubMed1989

Expression of hemolytically active human complement component C1r proenzyme in insect cells using a baculovirus vector.

The gene of human C1r has been expressed in a baculovirus-insect-cell system via the pAc373 transplacement vector. The full-length cDNA copy was inserted into the pAc373 vector downstream from the strong polyhedrin promoter of the baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV). Spodoptera frugiperda cells were cotransfected with the resultant plasmid, pAcC1r, and the wild-type AcNPV DNA. Recombinant viruses, which drove the expression of C1r protein, were selected by plaque morphology and ELISA. Insect cells infected with the recombinant virus produced and secreted human C1r protein, at a level of 1-2 mg/l of medium. The expressed C1r was isolated from the medium by chromatofocusing. On reducing gels only a single Coomassie-staining band was observed, and this band migrated at 80-83 kD characteristic of the unactivated C1r proenzyme. Its identification as C1r was immunologically confirmed on Western blots. C1 reconstituted from purified C1r expressed in insect cells together with human C1q and C1s proved biologically active in a hemolytic assay. Thus, the baculovirus-insect-cell system is capable of expressing and secreting a sophisticated, multifunctional human complement subcomponent in its biologically activatable form.

Animals↗
PubMed1976

Serum and blister fluid anticomplementary activity in pemphigus and bullous pemphigoid. Sucrose density gradient studies.

By sucrose density gradient ultracentrifugation and standard hemolytic complement assays, complement fixing activity of about 19 S and greater was found in blister fluids of one patient with pemphigus and three of five patients with bullous pemphigoid. Control blister fluids, including experimentally induced blisters, lacked such activity. Complement fixing activity was apparent, however, in 6 of 10 pemphigus sera and in two of five bullous pemphigoid sera tested. The material present in both pemphigus and bullous pemphigoid appears to activate the classical complement pathway.

Antigen-Antibody Complex↗
PubMedDate not supplied

Protamine-coated Cuprophan. A potential nonthrombogenic hemodialysis membrane with improved blood compatibility.

A protamine-coated Cuprophan hemodialysis membrane possessing improved blood compatibility was developed. Protamine was covalently immobilized onto the inner walls of the Cuprophan hollow fibers, using the cyanogen bromide (CNBr) activation method. Studies conducted with aqueous solutions indicated that the immobilization process did not introduce deterioration of the membrane mechanical and mass transfer properties; the permeability of several compounds through the protamine-coated membrane was unchanged. The modified membrane was rendered nonthrombogenic by the adsorption of heparin to the protamine-bound surfaces. Heparin adsorbed on the immobilized protamine retained about 20% of its initial anticoagulant activity. Preliminary studies using the hemolytic complement assay indicated that complement activation by the protamine-coated membrane was statistically far less than that by the untreated membrane.

Cellulose↗
PubMed1991

C1q-bearing immune complexes detected by a monoclonal antibody to human C1q in rheumatoid arthritis sera and synovial fluids.

Using a monoclonal antibody directed against the C-chain of human C1q, we detected C1q-bearing immune complexes (IC) in sera and synovial fluids of rheumatoid arthritis (RA) patients. In a sandwich-ELISA, C1q-bearing IC were captured by the solid-phase monoclonal antibody and then detected with peroxidase-labeled F(ab')2-antibodies to either human IgG or IgM. The results of this assay were compared to an ELISA-modification of the C1q-solid-phase binding assay (C1q-SPBA). C1q-bearing IC were detected in 81.1% of RA-sera and the 65.2% of RA-synovial fluids. IgG as well as IgM was present in 72.6% of the sera and 70% of the synovial fluids which were positive in both assays. Most RA sera that were only positive for C1q-bearing IC, contained IgG alone (81.5%). The corresponding synovial fluids showed IgG alone (53%) or both IgG and IgM (41.1%). IgM alone (25%) could be detected in sera, e.g. in juvenile forms of RA. The levels of IC were higher in synovial fluid than in paired serum. In comparison to normal human serum (NHS) and patients with osteoarthritis, complement activity (CH50 titers) and C1q-values in patients with RA were frequently elevated. Since the formation of C1q-bearing IC is an indicator for the classical complement pathway activation, an assay with monoclonal anti-C1q antibody may be a useful tool in the diagnosis of rheumatoid diseases.

Antibodies, Monoclonal↗
PubMed1990

Protective and inactivating effects of neutrophil myeloperoxidase on C1q activity.

This study investigated the interaction between neutrophil myeloperoxidase (MPO) and the C1q component of the complement system. Using a dot-spot assay, MPO was found to bind to C1q in a dose-dependent manner. The specificity of this reaction was proved by the inhibitory effect of F(ab')2 antibodies to C1q and by the inability of MPO to bind to C1r, C1s and IgG. The interaction between MPO and C1q did not influence the enzymatic activity of the peroxidase but resulted in a more stable C1q as assessed by hemolytic assay for C1q. The protective effect of MPO on C1q did not require the presence of H2O2 in the reaction mixture nor was it inhibited by sodium azide, whereas it was abolished by heating the peroxidase. Lactoferrin and lysozyme, unlike MPO, were ineffective in protecting C1q from functional decay. Addition of H2O2 and chloride to MPO and C1q led to a complete inactivation of C1q, which could not be induced by H2O2 alone. The hypochlorite, which is known to be generated during the reaction of MPO with H2O2 and chloride, exhibited a similar inactivating effect on C1q, which was prevented by an external source of methionine.

Chlorides↗
PubMed1990

Leukocyte depletion ameliorates free radical-mediated lung injury after cardiopulmonary bypass.

Activated leukocytes and oxygen free radicals have been implicated in the pathogenesis of lung injury associated with cardiopulmonary bypass. To determine whether leukocyte depletion could prevent this injury, we used a dog model simulating routine cardiac operations. Mongrel dogs (11 to 17 kg) were subjected to cardiopulmonary bypass with a bubble oxygenator and cooled to 27 degrees C. After aortic crossclamping and cardioplegic arrest for 90 minutes, control animals (n = 5) were rewarmed and weaned from bypass, and their condition was then stabilized for 90 minutes. Leukocyte-depleted animals (n = 5) had a leukocyte filter incorporated in the bypass circuit. During bypass, circulating leukocyte counts decreased by 60% in control dogs, and by 97% in leukocyte-depleted animals. Free radical generation (estimated by spectrophotometric assay of plasma conjugated dienes) was significantly reduced by leukocyte depletion during and after bypass. Total hemolytic complement activity and the titer of C5 decreased markedly immediately after the onset of bypass in both the control and leukocyte-depleted animals. Pulmonary function after bypass was better preserved in leukocyte-depleted animals. These data suggest that depletion of circulating leukocytes contributes to lung injury during cardiopulmonary bypass and is associated with increased oxygen radical activity, pulmonary edema, and vasoconstriction. Leukocyte depletion substantially reduced the pulmonary injury seen after cardiopulmonary bypass.

Animals↗
PubMed1992

Heparin and modified heparin inhibit complement activation in vivo.

Heparin regulates C activity in vitro, but has not been examined for this activity in vivo. The present study investigated the ability of commercial heparin and derivatized (N-desulfated, N-acetylated) heparin (Hep-NAc) with greatly diminished anticoagulant activity to inhibit C activation in guinea pigs. Catheters were placed in the right atrium of guinea pigs and kept patent with frequent saline flushes. The next day, heparin, Hep-NAc, or saline was given and 2.5 min later cobra venom factor or saline was given. Blood was drawn at intervals and assayed for total hemolytic C, C3 hemolytic activity, free hemoglobin, and activated partial thromboplastin time. Total hemolytic C and C3 activity decreased less rapidly in heparin- and Hep-NAc-pretreated animals than in non-pretreated animals, indicating that both heparins inhibited C activation. Heparin and Hep-NAc also inhibited cobra venom factor-induced hemolysis. This study demonstrates that commercial heparin and modified heparin inhibit C activation in vivo. This represents an important step in the development of an oligosaccharide drug to regulate C activation.

Animals↗