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PubMed1998

[Threshold mechanism of the control of cascade proteolysis].

The immune lysis rate of erythrocytes vs. the concentration of particular complement components was studied. It was found that in the presence of component C1 (but not C3) and factors B and D, the cells undergo lysis after the concentration of these compounds exceeds a "threshold" value. The threshold molar concentrations of factors D and B exceeded 5- and 20-fold, respectively, that of component C1. The multiplicity and different capacities of the thresholds allow the fragmentary activation of cascade proteolysis at stretches between neighboring thresholds without the total realization of the final effect of the system (cell lysis, clot formation, etc.). The resulting peptideby-products (bypass peptides) may possess their own biological activity. It is the generation of various bioregulators that appears to be the main function of the cascade proteolytic systems functioning in the subthreshold regime.

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↗
PubMed2004

The role of complement in the early phase of Leishmania (Leishmania) amazonensis infection in BALB/c mice.

Complement-depleted and -non-depleted BALB/c mice were inoculated with Leishmania (Leishmania) amazonensis promastigotes into the hind footpad to study the role of the complement system in cutaneous leishmaniasis. Total serum complement activity was measured by hemolytic assay and C3 fragment deposit at the inoculation site was determined by direct immunofluorescence in the early period of infection, i.e., at 3, 24, 48 h and 7 days post-infection. The inflammatory reaction and the parasite burden were evaluated in the skin lesion at 7 and 30 days post-infection. Total serum complement activity decreased in the early phase of infection, from 3 to 24 h, in non-depleted mice compared to non-infected and non-depleted mice. C3 fragment deposit at the site of parasite inoculation was present throughout the period of infection in non-depleted mice. In contrast, no C3 fragment deposit was observed at the inoculation site in complement-depleted mice. Complement-depleted mice showed a significant decrease in the inflammatory response and a significant increase in the number of parasites (70.0 +/- 5.3 vs 5.3 +/- 1.5) at 7 days of infection (P<0.05). A higher number of parasites were also present at 30 days of infection at the inoculation site of complement-depleted mice (78.5 +/- 24.9 vs 6.3 +/- 5.7). These experiments indicate that complement has an important role at the beginning of experimental cutaneous leishmaniasis caused by L. (L.) amazonensis by controlling the number of parasites in the lesion.

Animals↗
PubMed2001

Cold activation of serum complement in patients with chronic hepatitis C: study on activating pathway and involvement of IgG.

It has been documented that the serum complement activities measured by hemolytic assay (CH50) are decreased after storage of sera at a low temperature in some patients with chronic hepatitis C. However, the mechanism of this phenomenon has not been identified yet. Here, we tried to elucidate factors involved in the cold activation of complement (CAC). To clarify what pathway is activated in CAC, we measured complement cleavage products after cold storage of sera. C4d increased significantly after 12 h-storage at cold temperatures in 5 CAC (+) sera compared with 5 CAC (-) (P < 0.01) and 3 control sera (P < 0.05), while Bb did not increase in any of the groups. In order to determine whether IgG or IgG complex is necessary for CAC, 8 CAC (+) sera were incubated with Protein G Sepharose gel beads, and all of them retained hemolytic activities to some extent after cold storage. Column chromatography through Superose 6HR of CAC-positive serum identified the fractions containing molecules that induced CAC in normal serum, which were depleted by treatment with protein G Sepharose. In conclusion, CAC in hepatitis C seems to occur via a classical or lectin pathway, and the IgG complex produced in hepatitis C virus infection may be an important factor in inducing CAC, a common extrahepatic manifestation of hepatitis C.

Adult↗
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↗
PubMed1994

Complement activation on solid surfaces as determined by C3 deposition and hemolytic consumption.

Complement activation was investigated on hydrophilic and hydrophobic glass beads incubated in serum. Very little complement activation was detected with these surfaces, as indicated by a hemolytic assay and by measurement of the amount of iC3b appearing in the solution. However, preadsorption with IgG at the hydrophobic and hydrophilic beads resulted in complement activation on both surfaces. We also investigated dependent deposition of C3 at hydrophobic and hydrophilic silicon surfaces when the complement was activated. The chemistry of those surfaces is similar to the hydrophobic and hydrophilic beads. Ellipsometry, an optical method, was used for determination of the amounts of organic material deposited at the surface. C3 deposition was observed at the IgG precoated hydrophobic surface but not at the IgG-coated hydrophilic surface. The absence of C3 deposition at the hydrophilic surface was probably due to reversible binding of IgG. However, precoating of the hydrophilic surface with a double layer of IgG and anti-IgG resulted in C3 deposition also at the hydrophilic surface. The results illustrate that methods based on measuring deposition of C3 at surfaces, such as immunofluorescence or ellipsometry, cannot exclude surface-associated complement activation that is probably due to reversible binding of the complement components of the activated molecule. On the other hand, it has previously been shown that determination of complement activation in solution with the use of a C3a assay cannot exclude surface-associated activation due to immobilization of C3a at the surface. This methodologic question is an important issue because factors such as C3a and C5a act as soluble anaphylatoxins, whereas deposited factors such as C3 act as cellular receptors.

Adsorption↗
PubMed2006

Immunocytogenetic effects of gonadotropin releasing hormone analogue: Triptorelin Pamoate (Decapeptyl) during in vitro fertilization treatment.

In this study, the immunocytogenetic effects of Decapeptyl (Triptorelin Pamoate) were assessed in the peripheral blood lymphocytes of females undergoing in vitro fertilization (IVF) treatment. Blood samples were taken from 34 females (23 treated and 11 controls), cultured and examined for sister chromatid exchanges (SCE) and cell replication index (CRI). The SCE frequency increased around ovulation time in the controls, and around the time of human chorionic gonadotropin administration in the IVF group. However, the SCE rate was significantly higher in the latter group. Furthermore, the white blood cells (WBC) count was significantly higher on the day of ovum pick up compared to the day preceding luteinizing hormone (LH) and follicle stimulating hormone (FSH) treatment. Similar observations were recorded with respect to phagocytic activity tested by nitroblue tetrazolium (NBT) assay. The nitric oxide production abilities of macrophages were not significantly changed in the LH, FSH-treated group relative to its control. Finally, the 50% complement hemolytic activity (CH50) assay results indicated that Decapeptyl lacks a significant potential to affect the complement system.

Adult↗
PubMed2005

Interaction of poly(styrene sulfonic acid) with the alternative pathway of the serum complement system.

Bioartificial pancreas, in which the islets of Langerhans are enclosed in artificial membrane to be protected from the host immune system, is expected to be a promising medical device to treat patients who suffer from insulin-dependent diabetes. Our strategy for preparation of a bioartificial pancreas involves utilizing a membrane including polymeric materials that can inhibit the complement reaction. In this study, we examined the effects of poly(styrene sulfonic acid) (PSSa) on the alternative pathway of the serum complement system to identify the mechanism(s) involved. PSSa was dissolved in pooled normal human serum (NHS), and the mixtures were incubated at 37 degrees C for 30 min. Complement activities in sera were determined by hemolytic assays. Amounts of complement activation products released were determined by ELISA. Interactions of PSSa with complement components and fragments were examined with electrophoresis and immunoblotting. From these examinations, it appeared that the manner of PSSa effects on the alternative pathway (AP) highly depends on its concentration. PSSa seemingly acted as an activator when its concentration was 0.005 g/dl to 0.05 g/dl, while it acted as an inhibitor when its concentration was more than 0.1 g/dl. In terms of activation or inhibition of the AP, forming complex of PSSa with factor H induced activation, and that with factor D induced inhibition.

Biocompatible Materials↗
PubMed2000

Mouse complement components C4 and Slp act synergistically in a homologous hemolytic C4 assay.

Goals of the present study were to compare the hemolytic activities of mouse C4 and Slp in a homologous system and to study a possible interaction between these proteins during complement activation. As reagents for mouse C4 and Slp, we used serum of C4(- / -) knockout C57BL / 6 (C4(-) / Slp(-)) mice and sensitized rabbit erythrocytes as target cells. Sera to be tested contained none, either of the two or both proteins. We found that C4(-) / Slp(+) serum has some hemolytic C4 activity, but less than C4(+) / Slp(-) serum. Comparing C4 activities of C4(+) / Slp(-) and C4(+) / Slp(+) sera, we found a threefold enhanced activity in double-positive serum. Hemolytic C4 levels of mixtures of solely C4- and Slp-sufficient sera did not overlap with expected C4 levels, but rather these sera showed synergy. This explains the enhanced activity of double-positive serum. Similar results were observed for total complement activation. In conclusion, Slp has measurable, but poor C4 activity as compared with mouse C4. Using our homologous system, we showed that the enhanced classical pathway activity of double-positive sera is most probably based on synergy between C4 and Slp. Our results answer an old question as to why C4(+) / Slp(+) mice have higher complement levels than C4(+) / Slp(-) mice.

Animals↗
PubMed1980

Hemolytic complement activity in normal human donor corneas.

Normal human donor corneas were minced into small fragments and eluted for 16 to 23 hours at 4 degrees C. The corneal eluates were then studied for hemolytic complement activity of C1, C4, C2, C3, C5, C6, and C7 with 50% hemolysis (CH50) of sensitized sheep RBCs. Sera from ten normal volunteers were also assayed for hemolytic complement activity in CH50 units per milliliter. For each complement component, the mean hemolytic activity in corneas was compared with the mean hemolytic activity in sera. These comparisons suggest that molecular weight may be a factor in determining the concentration of complement components in the cornea. The present study provides normal values of hemolytic complement activity for further studies of complement consumption in corneal diseases.

Adult↗
PubMed1997

Molecular characterization of complement components (C3, C4, and factor B) in human saliva.

A molecular analysis of complement components (C3, C4, and factor B) in human saliva was performed by SDS-PAGE and immunoblotting. Complement C3 was detected as a molecule composed of a 115-kDa alpha-chain linked to a 70-kDa beta chain by disulfide bonds, and C3 levels ranged from 0.52 to 15.0 micrograms/ml (n = 15). C4 was detected as a triple-chain molecule (98-kDa alpha chain, 73-kDa beta chain, and 33-kDa gamma chain) linked by disulfide bonds, and C4 levels ranged from 0.086 to 4.8 micrograms/ml. Factor B was detected as a 100-kDa single chain, and factor B levels ranged from 0.042 to 0.62/microgram/ml. The sizes and subunit structures of the complement components in human saliva were compatible with those reported in human serum. The results of a hemolytic assay indicated that the complement molecules in human saliva were functionally active. These complement components may participate in the local immune and inflammatory responses in the oral cavity.

Complement C3↗
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↗
PubMed1984

Simplified assays of hemolytic activity of the classical and alternative complement pathways.

Simplified hemolytic assays for the classical (CP) and alternative (AP) pathways of complement (C) were developed. The CP function was tested with sensitized sheep erythrocytes in a diluent containing Ca2+ and Mg2+, while AP was tested with unsensitized rabbit erythrocytes in a diluent containing Mg2+-EGTA. In contrast to the commonly used hemolytic titration (CH50) assays, the present techniques tested the activity in reaction mixtures containing C at final dilutions which would not affect its function. These ranges fell between 1/1 and approximately 1/20 for CP and between 1/1 and approximately 1/3 for AP. With the adopted assay techniques single aliquots of serum were tested at single final dilutions of 1/8 for CP and 1/2 for AP, in the presence of excess target cells. Hemolysis was allowed to take place at 37 degrees C for 20 min. The number of cells lysed by CP and AP under these conditions was directly proportional to the dose of serum and unaffected by the presence of a large excess of target cells. Each pathway was tested independently of the other. Serum C levels, measured as described, correlated strongly with those determined by standard hemolytic titration (CH50) assays. The modified assays should offer less laborious alternatives for the functional assay of C than current routine procedures.

Animals↗
PubMed2003

Simple method to distinguish between primary and secondary C3 deficiencies.

Due to the increasing numbers of reported clinical cases of complement deficiency in medical centers, clinicians are now more aware of the role of the complement system in the protection against infections caused by microorganisms. Therefore, clinical laboratories are now prepared to perform a number of diagnostic tests of the complement system other than the standard 50% hemolytic component assay. Deficiencies of alternative complement pathway proteins are related to severe and recurrent infections; and the application of easy, reliable, and low-cost methods for their detection and distinction are always welcome, notably in developing countries. When activation of the alternative complement pathway is evaluated in hemolytic agarose plates, some but not all human sera cross-react to form a late linear lysis. Since the formation of this linear lysis is dependent on C3 and factor B, it is possible to use late linear lysis to routinely screen for the presence of deficiencies of alternative human complement pathway proteins such as factor B. Furthermore, since linear lysis is observed between normal human serum and primary C3-deficient serum but not between normal human serum and secondary C3-deficient serum caused by the lack of factor H or factor I, this assay may also be used to discriminate between primary and secondary C3 deficiencies.

Animals↗