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PubMed2006

Phenotypic expression of factor H mutations in patients with atypical hemolytic uremic syndrome.

We investigated the phenotypic expression of factor H mutations in two patients with atypical hemolytic uremic syndrome (HUS). Factor H in serum was assayed by rocket immunoelectrophoresis, immunoblotting, and double immunodiffusion and in tissue by immunohistochemistry. Functional activity was analyzed by hemolysis of sheep erythrocytes and binding to endothelial cells. A homozygous mutation in complement control protein (CCP) domain 10 of factor H was identified in an adult man who first developed membranoproliferative glomerulonephritis and later HUS. C3 levels were very low. The patient had undetectable factor H levels in serum and a weak factor H 150 kDa band. Double immunodiffusion showed partial antigenic identity with factor H in normal serum owing to the presence of factor H-like protein 1. Strong specific labeling for factor H was detected in glomerular endothelium, mesangium and in glomerular and tubular epithelium as well as in bone marrow cells. A heterozygous mutation in CCP 20 of factor H was found in a girl with HUS. C3 levels were moderately decreased at onset. Factor H levels were normal and a normal 150 kDa band was present. Double immunodiffusion showed antigenic identity with normal factor H. Factor H labeling was minimal in the renal cortex. Factor H dysfunction was demonstrated by increased sheep erythrocyte hemolysis and decreased binding to endothelial cells. In summary, two different factor H mutations associated with HUS were examined: in one, factor H accumulated in cells, and in the other, membrane binding was reduced.

Animals↗
PubMed1980

Inherited deficiency of the ninth component of complement in man.

A 76-year-old man was found to have low (33% normal) serum complement (C) hemolytic activity, although C3 and C4 protein levels were normal. Further evaluation of his serum and plasma indicated that all C components were present in normal or elevated amounts except for C9, which was undetectable by both antigenic and functional assays. Addition of purified human C9 led to full restoration of the hemolytic activity. Family studies demonstrated that the deficiency was inherited as an autosomal codominant trait and was not linked with alleles at the HLA-A or HLA-B loci. The patient had no history of recurrent or unusual infections and no evidence of autoimmune disease. The availability of serum totally lacking in C9 permitted an investigation of the lytic capacity of the C5b-8 segment of the C attack mechanism, which was pursued in kinetic studies on the hemolysis of erythrocyte intermediates. These studies indicated that hemolysis occurred approximately 100 times slower in patient than in normal serum, using either EA or EAC1-7 intermediates as target cells. Serum bactericidal activity also was slower in patient serum, occurring at a rate about 1/35 that observed in normal serum. These studies provide direct independent evidence that cytolysis of erythrocytes and bacteria can be mediated by C5b-8, and allow a quantitative estimation of the increment in the rates of these reactions provided by normal serum levels of C9. The presence of readily detectable though slow hemolytic activity of C9-deficient serum may account for the difficulty in identifying individuals with this defect.

Aged↗
PubMed1996

Mouse complement component C4 is devoid of classical pathway C5 convertase subunit activity.

It has long been known that mouse C4 has unusually low hemolytic activity relative to the C4 of other mammalian species (e.g. human and guinea pig), the measurements being done in most cases using a C4-deficient guinea pig serum reagent in a one-step assay with EA. This low activity for mouse C4 previously had been attributed to "technical" difficulties such as lability of the protein during blood collection and partial species incompatibilities with guinea pig components. Recently, we presented evidence for the involvement of human C4 beta-chain residues 455-469, a putatively exposed hydrophilic segment, in contributing to a C5 binding site in the C4b subunit of the classical pathway C5 convertase, C4b3b2a. Given that there were five sequence differences between the human and mouse protein within this segment, we hypothesized that these substitutions may have compromised the C5 convertase subunit activity of mouse C4, thereby resulting in its low hemolytic activity. Using a multi-step hemolytic assay which was totally dependent upon C5 cleavage by the classical pathway, we found that mouse C4 was completely devoid of classical pathway C5 convertase subunit activity. We have been able to rule out the most obvious potential species incompatibilities (e.g. between C4mo and C5gp) as being responsible for this lack of activity. Moreover, we found that the low level of hemolytic activity of mouse C4 measured in the one-step assay can be ascribed totally to C5 cleavage, and subsequent terminal component assembly, by the alternative pathway C5 convertase, (C3b)2Bb. However, the assembly of the latter enzyme complex is dependent upon the presence of C3b molecules deposited initially via the classical pathway C3 convertase in which mouse C4b is a subunit. Finally, whereas conversion of human residues 458RP to the mouse-like sequence PL was sufficient to abrogate classical pathway C5 convertase subunit activity in human C4, the five substitutions which "humanized" the 452-466 segment of mouse C4 (corresponding to human residues 455-469) were on their own insufficient to impart this activity to mouse C4. This implies that, in addition to the 455-469 beta-chain segment of human C4, there are other regions of the molecule contributing to C5 binding which are also non-conserved between human and mouse C4.

Amino Acid Sequence↗
PubMed1983

Mechanisms of immune red cell destruction, and red cell compatibility testing.

The immune destruction of red cells can occur as a complement-mediated intravascular process, or extravascularly, where the red cells are destroyed by macrophages following interaction with cell-bound IgG1, IgG3, and/or C3b. Many of the factors that affect this in vivo destruction are not taken into account during in vitro pretransfusion compatibility testing. At present, even by use of more elaborate tests, it is difficult to accurately predict the fate of a transfused unit of blood. By using some simple information, such as antibody specificity and thermal range, it is sometimes possible to predict the outcome of transfusing a unit of blood that is incompatible in vitro. At other times it may be necessary to utilize 51Cr-labeled red cells to determine the risk of transfusing such units. Because of the paucity of reported clinical correlations, macrophage/monocyte monolayer assays are of little practical value at present.

Anemia, Hemolytic, Autoimmune↗
PubMed2002

Bacteriolytic activity of rainbow trout (Oncorhynchus mykiss) complement.

The total bacteriolytic activity comprising of the classical, alternative and possible lectine pathways as well as the bacteriolytic activity of the alternative pathway (AP) of rainbow trout (Oncorhynchus mykiss) complement was assessed in temperatures ranging from 0 to 35 degrees C against a recombinant strain Escherichia coli containing two reporter genes gfp and lucFF. At 35 degrees C there was no difference between the total (TC) activity and the activity of the AP, but at 10 degrees C the TC was notably higher than the AP. Total activity peaked at 30 degrees C and gradually grew smaller towards 0 degrees C. The activity of the AP was similarly temperature-dependent, but CB50 value was found to be beyond measurable range at temperatures below 10 degrees C. When compared to human serum complement, the peak human TC activity at 37 degrees C was four times higher than the TC of rainbow trout at 30 degrees C. Human TC activity was 10.1-fold lower at 25 degrees C when compared to the activity at 37 degrees C. At 37 degrees C the human AP bacteriolytic activity was 4.5-fold less effective than human TC, but at 25 degrees C there was no difference between human TC and AP. In contrast to previous reports where AP activity of fish was assayed as hemolytic activity our study showed that the bacteriolytic activity of AP was lower than that of TC and very low at temperatures below 10 degrees C suggesting that the earlier proposed particular importance of AP in fish should be reconsidered.

Animals↗
PubMed1995

Regulation of the function of the first component of complement by human C1q receptor.

A membrane-associated receptor for the C1q subcomponent of complement is widely distributed among different cell types. While a number of possible physiological functions of the C1q receptor (C1qR) on different cell types have been described, the way in which C1qR regulates complement activity remains unclear. This report describes the mechanism by which C1qR regulates activation of the first component of complement, C1. Using purified components of complement, we were able to show that membrane-associated C1qR as well as detergent-solubilized C1qR, purified from polymorphonuclear leukocytes, human umbilical vein endothelial cells or an endothelial cell line, EA.hy 926, are able to inhibit complement-mediated lysis of C1q-sensitized erythrocytes. Using hemolytic assays, we were able to demonstrate that C1qR prevents the association of C1q with C1r and C1s to form macromolecular C1. In addition, incubation of C1qR with the collagen-like stalks, but not with the globular heads of C1q, inhibits the effect of C1qR. This demonstrates that C1qR exerts its complement inhibitory effect by binding to the collagen-like stalk of C1q. No complement regulatory effect of C1qR was observed on preformed macromolecular C1. These data suggest that besides such-well-known complement regulatory molecules as CD55 (DAF), CD46 (MCP), CD35 (CR1) and CD59 (HRF), C1qR too is able to regulate complement activity.

Carrier Proteins↗
PubMed1997

The modified nucleoside 2-methylthio-N6-isopentenyladenosine in tRNA of Shigella flexneri is required for expression of virulence genes.

The virulence of the human pathogen Shigella flexneri is dependent on both chromosome- and large-virulence-plasmid-encoded genes. A kanamycin resistance cassette mutation in the miaA gene (miaA::Km Sma), which encodes the tRNA N6-isopentyladenosine (i6A37) synthetase and is involved in the first step of the synthesis of the modified nucleoside 2-methylthio-N6-isopentenyladenosine (ms2i6A), was transferred to the chromosome of S. flexneri 2a by phage P1 transduction. In the wild-type bacterium, ms2i6A37 is present in position 37 (next to and 3' of the anticodon) in a subset of tRNA species-reading codons starting with U (except tRNA(Ser) species SerI and SerV). The miaA::Km Sma mutant of S. flexneri accordingly lacked ms2i6A37 in its tRNA. In addition, the mutant strains showed reduced expression of the virulence-related genes ipaB, ipaC, ipaD, virG, and virF, accounting for sixfold-reduced contact hemolytic activity and a delayed response in the focus plaque assay. A cloned sequence resulting from PCR amplification of the wild-type Shigella chromosome and exhibiting 99% homology with the nucleotide sequence of the Escherichia coli miaA gene complemented the virulence-associated phenotypes as well as the level of the modified nucleoside ms2i6A in the tRNA of the miaA mutants. In the miaA mutant, the level of the virulence-associated protein VirF was reduced 10-fold compared with the wild type. However, the levels of virF mRNA were identical in the mutant and in the wild type. These findings suggest that a posttranscriptional mechanism influenced by the presence of the modified nucleoside ms2i6A in the tRNA is involved in the expression of the virF gene. The role of the miaA gene in the virulence of other Shigella species and in enteroinvasive E. coli was further generalized.

Alkyl and Aryl Transferases↗
PubMed1982

Rheumatoid factor inhibition of in vitro binding of IgG complexes in the human glomerulus.

We studied the effects of rheumatoid factor (RF) on binding of immune complexes to activated C3 (C3b) receptors in vitro. IgM fraction of serum containing RF activity (IgM-RF), IgM isolated from pooled normal human serum and have no RF activity (IgM-control), bovine serum albumin, and Veronal buffered saline solutions were used in a C3b assay system consisting of aggregated human IgG (AggHuIgG) coupled to sheep erythrocytes (SRBC) with guinea pig and normal human serum complement. The number of glomerular bound AggHuIgG-SRBC with IgM-control and bovine serum albumin or Veronal buffered saline was similar, while the number of bound cells with IgM-RF was reduced significantly, This effect was seen with both guinea pig and normal human serum complements. Supernatant hemolytic complement activity was maintained with IgM-RF, but reduced with control solutions. The blocking factor was shown to be RF by serial dilutions of IgM-RF resulting in inverse correlations with latex flocculation and inhibition of SRBC binding, absorption of blocking from IgM-RF with insolubilized AggHuIgG, and failure of IgM-control to block binding. IgM-RF did not directly interfere with activation of complement, but blocked attachment of C3 to AggHuIgG and formation of C3b capable of reacting with glomerular receptors. These results showed that IgM-RF can inhibit binding of AggHuIgG complexes to human glomeruli. This in vitro phenomenon may represent a possible protective mechanism of RF in vivo in diseases with immuno complexes.

Antigen-Antibody Complex↗
PubMed1986

Complement activation by 19S IgM rheumatoid factor: relationship to disease activity in rheumatoid arthritis.

19S IgM rheumatoid factor (RF) in rheumatoid arthritis (RA) are polyclonal autoantibodies directed against the Fc piece of IgG. Rheumatoid patients with RF tend to have aggressive synovitis, nodules, and extraarticular manifestations. Although RF titer does not correlate with disease activity, RF activates complement (C) by the classical pathway. Thus, we postulated that selective stimulation of cell clones producing efficient C activating RF molecules might be associated with disease flares, independent of changes in serum RF concentration. To address the question, 42 patients with RA were evaluated prospectively. Serum RF concentration was measured by radioimmunoassay (RIA) and C activating activity by hemolytic assay. We then calculated the mean hemolysis (MH) of sensitized sheep erythrocytes (SRC) produced/ml of RF serum (MH/ml) and MH/microgram of RF as an expression of RF C activating properties (CAP). The following observations were made: RF CAP varied among the patients studied; RF CAP varied over time in individual patients; RF CAP differences varied in both groups independently from RF concentration; RF CAP correlated with both systemic and articular disease activity; and total RF concentration correlated with articular findings and nodules but less well with systemic disease activity.

Adult↗
PubMed1984

Inhibition of complement-mediated hemolysis in gel by rheumatoid factors.

When subjected to a hemolysis in gel (HIG) assay for the detection of complement deficiency, 9 of 37 sera from patients with classical rheumatoid arthritis produced impaired lysis of sensitized sheep erythrocytes. All sera were normal in a test for the alternative pathway and no major abnormalities were found within the complement system. Using a two-step HIG technique, with guinea-pig serum as the complement source, all sera were shown to inhibit lysis of sheep erythrocytes sensitized with rabbit IgG. Lysis of IgM-coated erythrocytes was not inhibited. The agglutination titers in a Waaler-Rose test, and the areas of inhibition in the two-step HIG assay with IgG-sensitized erythrocytes, were correlated (r = 0.80, p less than 0.001). Absorption of serum with rabbit IgG coupled to Sepharose 4B, reduced the capacity to inhibit immune hemolysis. The eluate from IgG-Sepharose contained rheumatoid factors and also inhibited immune hemolysis. The findings suggested that rheumatoid factors in serum were responsible for inhibition in the HIG assays used.

Animals↗
PubMed1989

Functional heterogeneity of sialic acid binding agglutinins of rat uteri towards in vitro lymphocyte transformation.

The sialic acid binding agglutinins (SAS) are purified from three different stages (proestrus [P], estrus [E], diestrus [D]) of rat uterine homogenate. All three proteins are capable of inhibiting the in vitro phytohemagglutinin (PHA)-induced blastogenic response of lymphocytes and thymocytes as manifested by inhibition of 3H-thymidine incorporation into cells. The lymphocytes isolated from pregnant female rats were found to be more susceptible to inhibition. In resting lymphocyte cultures (RLC), however, the P- and E-SAS proteins acted differently from the D-SAS by stimulating lymphocyte proliferation in the cultures initiated from normal rat lymphocytes. At the same dose these proteins were inhibitory to the RLC, where cultured lymphocytes were isolated from the pregnant female. Reverse hemolytic plaque formation assay results also indicated functional heterogeneity between these proteins. P-SAS induced a potent polyclonal activation of the B cell, whereas E- and D-SAS did not show much activation. SAS proteins do not have any complement-like activity. These results, corroborated by cell viability count and 51Cr uptake tests, suggest that D-SAS has genuine lymphoblastogenesis-suppressive properties whereas P- and E-SAS-induced suppression in the presence of PHA is probably due to an increased cell-density-dependent cytotoxicity.

Agglutinins↗
PubMed1987

A natural auto-inhibitory factor of the terminal complement pathway in serum of Ctenodactylus gondi.

The serum of Ctenodactylus gondi, a Tunisian rodent, contains a unique inhibitor of the terminal complement pathway. The auto-inhibitor has been partially characterized as a heat-stable euglobulin that is slightly retarded on a DEAE-ion exchange column at pH 7 and elutes as a symmetrical peak on Sephacryl S-300 in the mol. wt region of approximately 200,000. The inhibitor acts by preventing attachment of cytolytic C5b-9 complexes to natural target cells. It does not appear to affect formation and function of C3-convertase, does not exert inhibitory effects at stages later than C5b-7 formation, and also does not prevent formation of SC5b-9 in serum. That the factor prevents attachment of C5b-7/C5b-9 to cells has been demonstrated in hemolysis model systems using sheep EA + human serum, and in the C3-independent reactive lysis system with the use of ELISA methods and quantitative assays with radioiodinated C8. Addition of partially purified inhibitory factor to human sera or to sera of other animal species abolishes the hemolytic activities of these sera. The inhibitory factor of Gondi serum is the first inhibitor of the terminal pathway which has been shown to be capable of preventing cytolysis of cells undergoing complement attack under physiological conditions. The presence of this factor is probably partially responsible for the remarkable susceptibility of C. gondi towards bacterial and parasitic infections.

Animals↗
PubMed1984

An immunochemical method for assessing the function of the alternative complement pathway.

Activation of the alternative pathway of human complement (C) by soluble C activators resulted in a decrease of C9 antigen, measured by single radial immunodiffusion, concomitant with a decrease of C9 hemolytic activity. In the presence of ethylene glycol bis-(beta-aminoethylether)-tetraacetic acid (EGTA) and Mg2+, this decrease of C9 antigen in the presence of soluble activators such as dinitrophenylated bovine serum albumin depends mainly on the activation of the alternative C pathway. Therefore, an assay system to express the total activity of the alternative C pathway in human serum - the C9 depletion test (C9DT) -, was devised. C9DT showed significantly lower values for patients with systemic lupus erythematosus and rheumatoid arthritis than for control patients.

Antigens↗
PubMed1997

Activation of the alternative pathway of complement by calcium-loaded erythrocytes resulting from loss of membrane phospholipid asymmetry.

The aminophospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) are exposed on the outer membrane leaflet of deoxygenated and irreversibly sickled erythrocytes and senescent normal cells. PS exposure on erythrocytes results in the expression of procoagulant activity for the conversion of prothrombin to thrombin. Because liposomes or vesicles composed of aminophospholipids can activate the alternative pathway of complement, the possibility that increased exposure of PS and PE on intact erythrocytes would also make them capable of activating the alternative pathway was examined. Loss of normal membrane phospholipid asymmetry was induced by incubation of erythrocytes with calcium (Ca2+) and the calcium ionophore A23187. PS exposure on 60% of erythrocytes was confirmed by binding of fluorescein isothiocyanate-conjugated annexin V. Expression of procoagulant activity, measured with the Russell's viper venom clotting assay, was significantly increased on the Ca2+/A23187-treated erythrocytes. In addition, the erythrocytes became capable of activating the alternative pathway of complement, as judged by an increase in cell-bound C3b after incubation with serum and a decrease in alternative pathway hemolytic activity of the serum. The effect could be reversed by incubation of the Ca2+/A23187-treated erythrocytes under conditions that induced recovery of normal membrane phospholipid asymmetry. In contrast, tetrathionate-treated erythrocytes showed no increase in binding of annexin V and no procoagulant activity and failed to activate the alternative pathway of complement. These findings demonstrate that loss of phospholipid asymmetry in erythrocytes not only results in expression of procoagulant activity but also renders the cells capable of activating the alternative pathway of complement.

Annexin A5↗
PubMed1985

An improved method for preparation of C1-depleted serum and its application to hemolytic assay of C1.

Serum depleted of the first component of complement (C1D) was prepared by treating fresh human serum with Sepharose-IgG in the presence of triethylenetetramine-N,N,N',N'',N''',N'''-hexa-acetic acid and di-isopropyl fluorophosphate at acidic pH (5.2). The total hemolytic activity of this C1D could be increased to approximately 75% of that of the original serum by the addition of excess purified C1. A linear relation was obtained on determination of C1 hemolytic activity using C1D, and the C1 titers of sera from patients measured by this simple method showed a good correlation with those measured using intermediate cells.

Animals↗
PubMed1996

Hemolytic anemia in chronic large granular lymphocytic leukemia of natural killer cells: cytotoxicity of natural killer cells against autologous red cells is associated with hemolysis.

BACKGROUND: It has ben suggested that natural killer cells are effectors in some hemolytic transfusion reactions, but a direct mechanism of the destruction of red cells has not been demonstrated. CASE REPORT: Lytic activity of natural killer cells against autologous red cells was found in a patient with large granular lymphocytic leukemia of natural killer cells and hemolytic anemia, with no evidence of immune antibody or complement-mediated hemolysis. Large granular lymphocytes in the blood showed a CD2+ CD3- CD16+ CD57+ phenotype, cytotoxic activity against natural killer cell-sensitive K562 target cells, and no lytic activity against natural killer cell-resistant JY target cells. Cytotoxicity directed against red cells was quantified by a 51Cr-specific release assay with autologous and ABO-identical red cells used as target cells and the patients purified natural killer cells used as effector cells in the presence and absence of autologous serum. The cytotoxic activity of natural killer cells was directed against autologous red cells but not against allogenic red cells, and it was not enhanced by the presence of autologous serum in the medium. Clinical remission was induced by the administration of cyclophosphamide, after which no evidence of cytotoxic activity against the red cells could be found. CONCLUSION: Hemolytic anemia in the presence of a negative direct antiglobulin test can be attributed to a direct cytotoxic mechanism mediated by natural killer cells.

Aged↗
PubMedDate not supplied

Complement-fixing hidden rheumatoid factor in children with benign rheumatoid nodules.

Rheumatoid nodules have been described in many children without evidence of clinical disease. These have been referred to as benign rheumatoid nodules (BRN). Except for one report, no serological abnormalities have been demonstrated. Four children with BRN have been studied. By means of a hemolytic assay, high titers of hidden rheumatoid factor (RF) have been found in the blood, i.e., 19S IgM RF detected after acid separation of IgM-containing fraction from the serum. The median hidden RF titer of the children with BRN was 1:362 and in healthy and disease controls was 1:7. The difference was significant at P less than 0.001. The results indicate that patients with BRN have active complement-fixing 19S IgM RF in their serum. Thus the association of hidden RF with BRN raises the possibility that the deposition of immune complexes (19S IgM-7S IgG) leads to small vessel vasculitis and nodular formation.

Antigen-Antibody Complex↗