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Characterization of immune complexes detected by the 125I-C1q binding assay in breast cancer.

The aim of the present study was to isolate and characterize immune complexes measured by the 125I-C1q binding assay in breast cancer sera. The C1q binding assay detects immune complexes by their binding to 125I-C1q and subsequent precipitation with PEG. We purified the C1q binding material from these precipitates by superose 6 gel filtration and cation exchange chromatography. The isolation steps were monitored by the C1q binding assay and the purified material was analyzed by SDS--PAGE and immunodiffusion. The C1q binding material isolated from two breast cancer sera contained complexes of IgM, C4b binding protein (C4-bp), and C1q. The C4-bp fraction showed significant C1q binding activity which increased after the addition of IgM. This C4-bp was not different from C4-bp isolated from normal serum. The IgM fraction, however, differed from normal IgM by its binding to C4-bp and C1q and its stronger affinity to the cation exchange column. These unique properties were not due to rheumatoid factor activity. They might be caused by a different glycosylation pattern or by complex formation with an as yet unknown polysaccharide antigen.

Antibodies, Neoplasm↗

Characterization of a non-functional form of C1q found in patients with a genetically linked deficiency of C1q activity.

A genetically defective form of C1q was purified from the sera of patients suffering from an immune complex related disease and who were homozygous for the defect. The defective C1q was haemolytically inactive and did not bind to immune aggregates or IgG-Sepharose. It showed the following similarities to the normal C1q molecule: a high glycine content and the presence of hydroxyproline and hydroxylysine; subunits with apparent mol. wts of 70,000 and 56,000, when examined by SDS-polyacrylamide gel electrophoresis under non-reducing conditions; preferential incorporation of 125I-label into only one of the types of chain present in the molecule, in a manner similar to that found for the C-chain of normal C1q. However, the defective molecule had an apparent mol. wt of approximately 155,000 in non-dissociating conditions, which is approximately one-third of the mol. wt of the normal molecule. Also, the material in the defective molecule preparation which corresponded, on the basis of mol. wt, to the disulphide-linked A-chain-B-chain dimer of normal C1q differed from that found in the normal molecule in that it did not appear to be sensitive to reducing agents. Collagenase and pepsin treatment of specific immunoprecipitates containing the radiolabelled defective molecule indicated that it is, like the normal molecule, composed of collagenous and non-collagenous domains.

Amino Acids↗

Patients with inflammatory arthritic diseases harbor elevated serum and synovial fluid levels of free and immune-complexed glucose-6-phosphate isomerase (G6PI).

In K/BxN mice, anti-glucose-6-phosphate isomerase (G6PI) IgG antibodies (Abs) cause joint-specific inflammation and destruction. Anti-G6PI Abs are also present in humans with inflammatory arthritis, especially among patients with rheumatoid arthritis (RA). A contributing factor to the induction of such autoantibodies may be upregulated expression of the corresponding antigen G6PI in affected tissues and/or increased levels of G6PI in the circulation. To determine G6PI levels and the presence of free G6PI and/or G6PI-containing immune complexes in sera and synovial fluids (SF) of patients with different arthritides, serum and SF obtained concomitantly from 91 clinically well-defined arthritis patients were assessed in a blinded manner for G6PI enzymatic assay and for G6PI protein concentration by ELISA. Sera and SF from patients with immune-based inflammatory arthritis contained significantly higher levels of G6PI enzymatic activity compared to sera or SF from patients with non-immune-based inflammatory arthritis or healthy controls. In addition, significantly higher levels of total G6PI protein concentration (including both enzymatically active and inactive forms) were present in sera of RA patients vs. those with other immune-based or non-immune-based inflammatory arthritis.G6PI in sera and SF were present both as G6PI-containing immune complexes and as free G6PI, with the majority of free G6PI existing as tetramers with lesser amounts of dimers and monomers. Levels of G6PI enzymatic activity in the sera of most immune-based inflammatory arthritis patients are elevated and may reflect ongoing inflammation and cell destruction. The high serum levels of enzymatically inactive forms of G6PI in RA relative to those in other arthritic diseases are partially due to G6PI-containing immune complexes, a portion of which also contains C1q. Overall, our study supports the notion that elevated G6PI levels present in patients with immune-based inflammatory arthritis may contribute to elevated levels of anti-G6PI Abs and G6PI/anti-G6PI immune complexes. This, in turn, may trigger production of proinflammatory cytokines and perpetuate the inflammatory process.

Arthritis↗

Circulating immune complexes in sickle cell-beta zero thalassemia.

A serum fraction from patients with sickle cell-beta zero thalassemia prepared by treatment with polyethyleneglycol showed increased amounts of C1q-precipitable immune complexes, i.e., 216 micrograms/dl (range, 141-266 micrograms/dl) vs 181 micrograms/dl (range, 152-228 micrograms/dl) for controls (P less than 0.05), as well as increased amounts of protein. Levels of IgG, IgA, IgM, C3, C4 and factor B in the same fraction were within the normal range.

Adolescent↗

[Adult papular acrodermatitis (Gianotti's disease). Report of 3 cases].

The authors report three cases of adult papular acrodermatitis with circulating HBs antigen. The eruption was followed by a benign icteric hepatitis which lasted from 30 to 45 days. In two cases, HBs antigen disappeared in a one month period, in one case the antigen has been present for more than three months. Direct immunofluorescence staining exhibits c3 deposits in the vessels of the dermal lesions, without any deposition of immunoglobulins or fibrinogen. We were unable to demonstrate the presence of HBs and " e " antigens in the skin lesions (using FITC conjugated specific antiserums). Serum protein concentrations of complement components C1q, C4,C3, C3PA were normal as measured by radial immunodiffusion. The percentage of circulating B and T cells was normal, as essayed by E-RFC, EAC-RFC and sIg. Thus, adult papular acrodermatitis, as well as the infantile form, does represent a sign of invasion of a benign viral hepatitis.

Acrodermatitis↗

Interleukin-2, soluble interleukin-2 receptor and tumor necrosis factor in sera from patients with rheumatoid arthritis.

Interleukin-2 (IL-2), soluble interleukin-2 receptor (IL-2R) and tumor necrosis factor (TNF) have been measured in sera from 47 patients affected by classic rheumatoid arthritis (RA) using an enzyme-linked immunosorbent assay. The patients were divided into 4 groups as follows: group A, 18 patients with inactive disease; group B, 19 patients with active disease under treatment with non-steroidal antiinflammatory drugs (NSAID) and second-line drugs; group C, 5 patients with active disease under treatment with NSAID and cyclosporine A (CSA) for at least 4 months; group D, 5 patients in the same condition as patients of group C, but treated with azathioprine (AZA) instead of CSA. IL-2 was undetectable in all patients except two, both characterized by active disease. Soluble IL-2R levels were above the upper limit of the normal range in most of the patients studied, but the mean value ( +/- 1 SD) was significantly higher in patients of group B (1,288 +/- 421 U/ml) than in patients of group A (686 +/- 205 U/ml) and group C (842 +/- 414 U/ml). In two patients affected by active RA treated with pulse methylprednisolone therapy (1 g/day for 3 alternate days) the values of soluble IL-2R dropped from 948 to 662 U/ml and from 660 to 518 U/ml, respectively. No statistically significant correlation was observed between the serum level of IL-2R and the RF titre or percentage of C1q-binding activity, respectively. TNF was found within the normal range in all patients except one, who was characterized by active arthritis, high number of rheumatoid skin nodules and extremely high RF titre.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-Inflammatory Agents, Non-Steroidal↗

IgG subclass antibody production in human serum sickness.

The role of IgG-subclass antibodies in the spectrum of immunologic disorders has not yet been fully defined. In an attempt to understand its role in an immune complex-mediated disease, we studied patients who developed serum sickness (SSX) after treatment with an equine-derived immunoglobulin, antithymocyte globulin (ATG), for bone marrow failure. The predominant IgG subclass produced was IgG1, representing nearly 80% of all IgG anti-ATG activity present. The appearance of IgG anti-ATG antibodies and C1q-containing immune complexes was closely correlated with symptoms of SSX. Although other antibody isotypes were present and may have contributed to the patients' symptoms, it is evident that IgG1 is the predominant IgG subclass produced in human SSX caused by a heterologous protein.

Adolescent↗

Immune responses in patients with obstructive jaundice of infancy. Anti-LSP and Clq binding in sera.

To investigate possible involvement of immune responses in the pathogenesis of obstructive jaundice in infancy we measured antibody to liver specific lipoprotein (LSP) by radio immunoassay and immune complexes by their ability to bind C1q in sera from 16 patients with extrahepatic biliary atresia and 16 with neonatal hepatitis and 13 age matched controls. Anti-LSP was present in 6 of 16 with preoperative biliary atresia and 6 of 16 with hepatitis. Mean percentage C1q bound was higher in hepatitis (22 SD 15%) than preoperative biliary atresia (11.1 SD 2.3%). Nine of 16 hepatitis patients had elevated C1q binding as compared with 1 of 16 with biliary atresia. The highest value for anti-LSP and C1q binding were found in sera from patients with histologically severe hepatitis and hepatitis associated with specific viral or bacterial causes. Anti-LSP was significantly raised 5 months post-operatively in all of 6 patients with biliary atresia and poor biliary drainage but only 2 of 5 survivors. Elevated C1q binding was detected in 6 of 7 with poor drainage and 1 of 7 survivors at the same stage. Anti-LSP and C1q binding fell in 4 patients with neonatal hepatitis on recovery. These findings suggest that immunological mechanisms, possibly involving antibody to hepatocyte membrane components and immune complexes, may be involved in the pathogenesis of progressive liver disease in biliary atresia.

Antigen-Antibody Complex↗

Aspects of humoral immunity in a prospective study of Type I (insulin-dependent) diabetic subjects treated with insulins of different purity.

In 41 Type 1 (insulin-dependent) diabetic patients, islet cell antibodies, anti-insulin antibodies, and immune complexes measured by two different methods (the C1q solid phase assay and the conglutinin binding test) were studied at diagnosis, and the influence of treatment with insulins of different purity was investigated during the first year of treatment. Twenty subjects were treated with conventional insulins (group 1) while 21 were treated with monocomponent porcine insulins (group 2). The prevalence of islet cell antibodies significantly decreased during the 12-month study period in the 41 patients. From the first month anti-insulin antibodies were always significantly higher in group 1 than in group 2. At diagnosis the prevalence of both types of immune complexes in the 41 patients was higher than in normal subjects. The immune complexes measured by the C1q solid phase method showed a significant and progressive reduction during the follow-up period, whereas the immune complexes assayed by conglutinin showed no significant variation in the same period. The presence of C1q immune complexes was found to correlate with the occurrence of islet cell antibodies both at diagnosis and during the follow-up period. The presence of conglutinin immune complexes, on the other hand, tended to parallel the increase of anti-insulin antibody levels.

Adolescent↗

Loss of suppressor T cell function and circulating immune complexes in alcoholic cirrhosis.

In the present work Concanavalin-A-induced T suppressor function has been studied in 28 patients with active alcoholic cirrhosis and 22 patients with inactive cirrhosis. Control subjects were 14 alcoholics without liver disease and 70 healthy individuals with normal liver function tests. Suppressor T cell function was decreased in active alcoholic cirrhosis whereas this change was not observed in inactive cirrhosis nor in alcoholics without liver disease. The suppressor T cell activity of normal individuals was inhibited by heat-inactivated serum pretreatment of patients with alcoholic cirrhosis. The data suggest that there is some kind of serum factor in alcoholic cirrhosis able to affect lymphocyte suppressor activity. No linear correlation could be established between serum level of circulating immune complexes (CIC), determined by solid-phase C1q-binding assay, and suppressor T cell function. There were no differences between sera of alcoholic cirrhosis patients with or without CIC regarding their inhibition of suppressor T cell activity of normal individuals. These results suggest that these serum factors are not related to the presence of CIC.

Adult↗

Detection of circulating immune complexes in patients with squamous cell carcinoma of the oral cavity.

Circulating immune complexes (CICs) have been quantitated from the sera of untreated oral cancer patients and those treated with radiation or surgery using C1q binding assay in terms of percent binding activity (C1q BA) and microgram/ml equivalent of aggregated human gamma globulin (AHG). Sera from a total of 108 oral cancer patients and 47 normal healthy donors were evaluated for CICs. Out of these, 48 patients were tested before treatment and 60 were tested 6 months to 1 yr after treatment. Levels of CICs were elevated in 70.8% patients before treatment (mean % C1q BA 29.6 +/- 2.2) when compared to healthy controls (4.2% positive, mean % C1q BA 9.0 +/- 0.8). Treated patients showing no evidence of the disease had reduced CIC levels, only 11.7% patients showing C1q BA at the level of 13.2 +/- 1.4. On the other hand, treated patients showing recurrence of the disease had much higher CIC levels (mean % C1q BA 42.8 +/- 3.5, 92.3% positive) even higher than the untreated patients. Status of CIC levels as well as recurrence rate in patients treated with either radiation or surgery were comparable. One out of 4 individuals with premalignant changes such as oral leukoplakia showed elevated levels of CICs.

Adult↗

Binding of immunoglobulins and immune complexes to cartilage derived extracts.

Cartilage extracts with affinity for heat aggregated immunoglobulins were prepared from human articular and bovine nasal cartilage. These extracts, containing predominantly collagen, also bound both to immune complexes (IC) prepared in vitro and to immunoglobulins from sera of many patients with rheumatoid arthritis (RA). Cryoprecipitation of rheumatoid sera removed material reacting with the extract and density gradient fractionation of a positive serum showed correlation between binding to the extract and to C1q. These results indicate that the binding materials in rheumatoid sera were likely to be IC. We suggest that some assays which apparently demonstrate anti-collagen autoantibodies in fact measure IC. These findings also have implications for models of the pathogenesis of RA.

Animals↗

Circulating immune complexes detected by C1q solid phase assay in leprosy.

Sixty-three sera of patients with leprosy were tested for the presence of circulating immune complexes (CIC) by C1q solid phase assay (C1qSPA). The mean values in CIC levels in leprosy patients were very high in comparison to native and European controls. No difference was found in the tuberculoid and lepromatous forms, but there was a good correlation between the presence of CIC and autoantibodies.

Adolescent↗

[Circulating immune complexes in a population of subjects seropositive for rheumatoid factor].

The level of immune complexes has been detected with the C1q binding test in 179 subjects positive by Latex agglutination test (titre greater than 1/20). The average of C1q binding was 5.36%, significantly higher than in 30 normal blood donors. A positive correlation was observed between the percent of C1q binding and, respectively, the titre of the latex test and the erythrocyte sedimentation rate. Of the 36 patients with rheumatoid arthritis, 16 (45.7%) showed an high level of C1q binding activity (greater than 5%), as well as 4 of the 8 patients with chronic hepatitis (50%) and 11 of the 31 patients with infectious diseases. No immune complexes were detected among the 11 healthy latex positive subjects.

Antigen-Antibody Complex↗

Cloning of the mouse homolog of the 126-kDa human C1q/MBL/SP-A receptor, C1qR(p).

Binding of C1q to cell surfaces has been shown to mediate a number of biological activities including enhancement of phagocytosis and stimulation of superoxide production. Several C1q binding proteins have been proposed as candidate receptors for these functions. The 126-kDa human C1q membrane receptor, termed C1qR(p), has recently been cloned. This molecule is believed to play a role in the enhancement of phagocytosis in monocytes and macrophages, and its expression has been shown to be restricted to cells of the myeloid lineage, endothelial cells, and platelets. Here we report the isolation and genomic characterization of the murine homolog of C1qR(p). Degenerate oligonucleotide primers based on the published human sequence were used to amplify a region of the murine homolog spanning from the carbohydrate recognition domain to the fourth epidermal growth factor (EGF) domain. This fragment was used as a probe to isolate the murine gene from a 129/Sv genomic lambda library. The predicted primary protein sequence displayed 68.1% identity with the human homolog. All the major structural domains were conserved between the two molecules. The coding sequence of the murine gene was contained within two exons separated by a small intron of approximately 250 bp. The structure of the human gene was found to be similar, with the position of the intron conserved. Cloning of the murine C1qR(p) will facilitate further investigation of the physiological function of this molecule.

Amino Acid Sequence↗

Evidence for enhanced rates of complement activation in serum from patients with newly diagnosed insulin-dependent diabetes mellitus exposed to rat islet cells and complement-dependent induction of islet cell apoptosis.

In this paper we report the concentration of terminal complement complexes (TCCs, SC5b-9, an index of complement activation) in newly diagnosed insulin-dependent diabetes mellitus (IDDM) patient serum and normal human serum. In the nine patients studied, levels of serum soluble TCCs were approximately 1.6-fold higher than in sera obtained from normal control individuals. On incubation of rat islet cells with diluted serum (10%, v/v, concentration), complement activation was increased at a significantly faster rate and the total TCC concentration was significantly higher in culture medium containing IDDM patient serum than in medium containing control serum. The concentration of anti-(glutamic acid decarboxylase) autoantibodies in newly diagnosed IDDM patient serum was on average 60-fold higher than in normal human control serum. IDDM patient serum (10%, v/v) induced apoptosis in islet cells, as determined by islet cell density changes and DNA fragmentation patterns. However, serum from IDDM patients was not able to induce apoptosis of the cells when complement components (C1q and C3) or antibodies were depleted. In addition, glutamine and the potent antioxidant 1-pyrrolidinecarbodithioic acid partially reversed cell death induced by IDDM patient serum in a concentration-dependent manner. The ATP concentration in islet cells incubated for 24 h in the presence of diluted IDDM patient serum was reduced to 4.4% of that observed in islet cells incubated in fetal calf serum or 7.3% of that observed in islet cells incubated in normal human serum. On the basis of these observations, we suggest that the pathway of IDDM patient serum-induced islet cell apoptosis may involve antibody-dependent complement activation, free radical generation and a precipitous fall in ATP levels.

Adolescent↗

Circulating immune complexes in patients with multiple sclerosis. A longitudinal study of serum and CSF by C1q and platelet binding tests.

Two hundred and twenty-eight paired serum and CSF samples collected from 31 patients with MS during a 2-3-year follow-up were analyzed for the presence of immune complexes (IC) by C1q RIA and PIPA (platelet [125I]protein A) techniques. One hundred and forty-four sera from 11 healthy individuals were analyzed as controls. In almost all patients (29/31) IC were detectable during some period of the disease, as tested by either of the techniques. The results obtained by C1q RIA and PIPA correlated positively with each other in MS when mean serum values of each patient were compared. The mean CSF IC levels detected by C1q RIA appeared to correlate to the mean IgG indexes, an indicator of the intrathecal rate of IgG synthesis. The amount of IC in serum and CSF fluctuated independently in MS. The results of the PIPA test for MS serum IC correlated significantly to the duration of the disease. The PIPA test results also showed that patients in stable or chronic phases of MS displayed IC in serum and CSF more often than patients with a relapsing/remitting course of disease but there was no clear correlation between fluctuations in IC levels in individual patients measured by C1q RIA or PIPA techniques and the disease course. Because of the lack of a clear correlation between the presence, quantity and fluctuation of IC and the clinical picture we suggest that those IC detected in the present study are probably not a precipitating factor in the pathogenesis of multiple sclerosis.

Antigen-Antibody Complex↗

Dermatitis herpetiformis: immune complex detection with C1q and monoclonal rheumatoid factor.

To determine the significance of circulating immune complexes in dermatitis herpetiformis, serum samples from thirty patients with active disease were tested by a C1q binding radioassay, while serum samples from twenty-one of these patients were tested by a monoclonal rheumatoid factor (mRF) inhibition radioassay. By direct immunofluorescence, all patients demonstrated typical IgA deposition in dermal papillae. Using the C1q binding assay, only seven of forty-two serum samples had elevated C1q binding activity, while by the mRF inhibition assay, thirteen of twenty-five samples had elevated immune complex levels. Nine of these latter thirteen positive serum samples, however, were minimally elevated. Thus, IgG and/or IgM containing immune complexes are infrequently present, or at very low levels, in sera of patients with active dermatitis herpetiformis.

Antigen-Antibody Complex↗