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Female SJL/J mice are more susceptible to development of experimental autoimmune myositis than most other mouse strains. Since complement has been implicated in the pathogenesis of inflammatory muscle disease in humans, quantitative and qualitative studies of complement C3 were undertaken in SJL/J and BALB/c mice to determine whether complement may influence disease susceptibility in SJL/J mice. In accordance with previous studies, mature male and female BALB/c mice were shown to have similar serum C3 concentrations. However, differences were found between mature male and female SJL/J mice. Male SJL/J mice have significantly higher serum C3 concentrations than SJL/J females and both sexes of BALB/c mice suggesting that serum C3 concentration may be variably influenced by sex in some mouse strains. Qualitatively, SJL/J mice were shown to have a different allotypic form of C3 (C3F) compared to the common electrophoretically slow form (C3S) found in BALB/c mice and most other mouse strains. Furthermore, studies on the decay rate of C3 revealed that C3b/iC3b fragments are converted to C3c/d at a faster rate in sera from female SJL/J mice compared to female BALB/c mice. Because removal and solubility of immune complexes is influenced by complement C3, it is possible that the more rapid decay of the phagocytic ligands C3b/iC3b may account for the increased susceptibility to development of autoimmune disease in female SJL/J mice.
Evidence on the role of the complement system in transplantation pathology has been accelerated by the discovery of C4d as an in situ marker of antibody-mediated rejection. However, a local or systemic source of complement expression during acute rejection is under discussion. Thus, we quantitatively analyzed local RNA expression of complement component C3 as a pivotal molecule in active humoral and cellular rejection of renal allografts. After laser microdissection, real-time RT-PCR was performed for C3 using RNA extracted from tubuli and glomeruli of 68 paraffin-embedded renal allograft biopsies. Protocol and indication biopsies with signs of humoral and/or cellular rejection were investigated. Quantitative expression analysis of cytokines (IFN gamma, MCP-1, IL2, IL8) potentially influencing local C3 expression was performed. We observed a significant increase in median expression level of C3 mRNA in tubuli of C4d-positive indication biopsies, and in tubuli from indication biopsies with signs of T-cell-mediated cellular rejection. Highest expression levels were found in C4d-positive indication biopsies with signs of cellular rejection. Biopsies with upregulated C3 showed increased IFN gamma expression, suggesting allograft-infiltrating T-cells as potential stimulus for local C3 expression. Therefore, locally synthesized complement component C3 contributes to both humoral and cellular rejection, with tubular epithelial cells being a major source.
Based on a method that uses the fourth component of complement (C4)-deficient guinea pig serum to quantify the hemolytic activity of C4, we developed an assay that allows the processing of a large number of individual samples with high reproducibility. In contrast to the conventional procedure using titration curves of each sample to be determined, we can show that a single appropriate dilution of the sample allows accurate quantitation of hemolytic activity. The reliability of the procedure is demonstrated using either C4A- or C4B- deficient and normal individual samples.
Susceptibility to hemolysis initiated by activated cobra venom factor (CoF) complexes is a characteristic that distinguishes the most complement-sensitive type III erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) from the intermediately sensitive type II and the normally sensitive type I cells. Recently we isolated a membrane constituent from normal erythrocytes that inhibits CoFBb-initiated hemolysis, and this protein was designated membrane inhibitor of reactive lysis (MIRL). To investigate the molecular basis of the variability in complement sensitivity among PNH erythrocytes, the surface expression of MIRL and decay accelerating factor (DAF) on the three phenotypes of PNH was quantified immunochemically. Both complement regulatory proteins were markedly deficient on the erythrocytes from a patient with predominately type III cells. The erythrocytes from patients with a majority of either type II or I cells were also significantly deficient in both MIRL and DAF. While cytofluorometric analysis confirmed the quantitative deficiencies, segregation of erythrocytes into discrete subpopulations that expressed either no MIRL or normal amounts of MIRL was not observed. The results of immunoprecipitation studies were consistent with quantitative, but not qualitative abnormalities of MIRL and DAF. Selective removal of the sensitive erythrocytes indicated that approximately 20% of the normal amount of MIRL is sufficient to protect cells from CoF-initiated lysis. These studies suggest that relatively subtle quantitative differences in membrane complement regulatory proteins underlie the variability in complement sensitivity of PNH erythrocytes.
Chromium is released during several industrial processes and has accumulated in some estuarine areas. Its effects on mammals have been widely studied, but relatively little information is available on its effects on fish. Gene expression changes are useful biomarkers that can provide information about toxicant exposure and effects, as well as the health of an organism and its ability to adapt to its surroundings. Therefore, we investigated the effects of Cr(VI) on gene expression in the sediment dwelling fish, winter flounder (Pseudopleuronectes americanus). Winter flounder ranging from 300 to 360 g were injected i.p. with Cr(VI) as chromium oxide at 25 microg/kg chromium in 0.15N KCl. Twenty-four hours following injections, winter flounder were euthanized with MS-222 and the livers were excised. Half of the livers were used to make cytosol and the other half were used to isolate mRNA for subtractive hybridization. Subtractive clones obtained were spotted onto nylon filters, which revealed several genes with potentially altered expression due to Cr(VI), including an alpha class GST, 1-Cys peroxiredoxin (a non-selenium glutathione peroxidase), a P-450 2X subfamily member, two elongation factors (EF-1 gamma and EF-2), and complement component C3. Semi-quantitative RT-PCR was performed and confirmed that Cr(VI) down-regulated complement component C3, an EST, and two potential glutathione peroxidases, GSTA3 and 1-Cys peroxiredoxin. In addition, cytosolic GSH peroxidase activity was reduced, and silver stained SDS-PAGE gels from glutathione-affinity purified cytosol demonstrated that a 27.1 kDa GSH-binding protein was down-regulated greater than 50%. Taken together, Cr(VI) significantly altered the expression of several genes including two potential glutathione peroxidases in winter flounder.
For complex diseases, underlying etiologic heterogeneity may reduce power to detect linkage. Thus, methods to identify more homogeneous subgroups within a given sample in a linkage study may improve detection of putative susceptibility loci. In this study we describe an ordered subsetting approach that utilizes disease-related quantitative trait data to complement traditional linkage analysis. This approach uses family-based lod scores derived from the initial genome screen and a family-based descriptor of the trait of interest. The goal of the approach is to identify more homogeneous subgroups of the data by ranking families based on their quantitative trait data. Permutation testing is used to assess statistical significance. This approach can be adapted to a variety of linkage methods and may provide a means to dissect some of the underlying heterogeneity in complex disease genetics.
We have developed a method for quantitating mannan-binding lectin (MBL)-induced activation of the complement system (MBL-C4-AC) in human plasma. This method and an assay for MBL concentration were applied to plasma samples from healthy individuals and patients with systemic lupus erythematosus (SLE), Crohn's disease (CD) and colorectal cancer (CRC). The MBL concentration was measured by an enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-MBL-antibodies and MBL-C4-AC by an ELISA using solid-phase mannan, incubating with plasma samples and quantitating the complement (C) activation by the use of antibodies against the C split-products C4b/C4c. The MBL concentration was nonsignificantly elevated in plasma from SLE-patients, whereas MBL-C4-AC was suppressed (P < 0.04). There was no correlation between MBL concentration and MBL-C4-AC in plasma from SLE-patients. In contrast, a significant correlation was found between the MBL concentration and MBL-C4-AC in plasma from healthy individuals. The C4 concentration was significantly reduced (P < 0.002) in plasma from the SLE patients and showed a significant correlation to MBL-C4-AC. The MBL-C4-AC assay was highly effective in discriminating the SLE patients from the other patient groups and healthy individuals.
Three adult patients, two with undifferentiated connective tissue disease and one with carcinoma, had a distinctive pathologic reaction pattern consisting of necrotizing myopathy, minimal cellular infiltration, and a microangiopathy with thick "pipestem" vessels and microvascular deposits of complement membrane attack complex. Quantitative analysis revealed focal capillary depletion. This pattern represents an immune-mediated microangiopathy and is distinct from that observed in other inflammatory myopathies.
The study of protein--protein interactions is central to understanding the chemical machinery that makes up the living cell. Until recently, facile methods to study these processes in intact, living cells have not existed. Furthermore, the assignment of function to novel proteins relies on demonstrating interactions of these proteins with proteins of known function. This review describes an experimental strategy, devised to study protein--protein interactions in any intact living cells based on protein-fragment complementation assays. Applications to quantitative analysis of interactions, allosteric processes and cDNA library screening are discussed. Recently, the feasibility of employing this strategy in genome-wide biochemical pathway mapping efforts has been demonstrated.
ATTAIN NAV (Access to Tailored Autism Integrated Care through Family Navigation) was delivered by family navigators to promote access to and engagement with mental health services for school-age autistic youth. This study used a mixed method, stepped wedge design to test the effects of family navigation on service and clinical outcomes while gathering information on implementation. Primary care providers from six clinics in California and 56 caregiver-child dyads enrolled in and completed the study. Clinics were randomized to either a technology-enhanced or standard family navigation condition. Caregivers completed assessments at baseline and post about child, family and services outcomes, and a subset participated in a post qualitative interview. Quantitative findings demonstrated improvements in child challenging behavior and parent activation across conditions although these improvements were more pronounced for families in the standard FN condition. At post-intervention, families in the standard FN condition reported higher levels of navigation satisfaction, a shorter time to attend their first mental health appointment, and higher engagement with their navigator. Qualitative findings complemented and expanded the quantitative survey findings. The ATTAIN NAV model of family navigation for autistic children with co-occurring mental health needs demonstrates promising implementation, service, and clinical benefits. Clinical Trials Registration. NCT05344378.
Two hundred and nine patients consecutively admitted to hospital with a tentative diagnosis of meningitis were screened for complement deficiency by measuring classical and alternative pathway serum haemolytic complement activity and the plasma concentration of C3d. Abnormal test results were followed up by quantitative immunochemical measurements of individual complement components. No patients with homozygous complement deficiency were found in our material. One patient with pneumococcal meningitis with probable heterozygous C2-deficiency was identified. Patients with purulent meningitis of various etiologies or meningococcal disease had significantly increased plasma C3d concentration at admission compared to patients with serous meningitis or without meningitis. Furthermore, increased plasma level of C3d at admission in patients with purulent meningitis or meningococcal disease was associated with an increased lethality. Our findings do not support the hypothesis that complement deficiency is commonly associated with sporadically occurring meningococcal disease or purulent meningitis.
Plasma protein alpha 1-antitrypsin (alpha 1-AT) and the serum complement component C3 have been quantitated by a laser nephelometric technique. The correlation coefficient between the laser nephelometric and the RID methods was r = 0.87 for alpha 1-AT, and r = 0.80 for C3 in normal serum. The alpha 1-AT concentration determined by the laser method ranged from 161 to 393 mg/dl in normal plasma, whereas the serum C3 concentration in normal serum was found to be 76.5 +/- 21.9 mg/dl. In pathological plasma, higher-than-normal alpha 1-AT levels were found (mean = 300 +/- 105.2 mg/dl), and these elevated levels are consistent with an acute-phase response. These studies indicate that laser nephelometric methods can be conveniently employed and automated for rapid immuno-quantitation of plasma and serum proteins.
In situ hybridization was used to localize and quantify gene expression in rheumatoid arthritis (RA) and osteoarthritis (OA) synovial tissue. Collagenase, tissue inhibitor of metalloproteinases (TIMP), HLA-DR, and complement (C2 and C3) gene expression was studied in synovial tissue from 23 patients with RA, OA, or other inflammatory arthropathies. Gene expression was highly compartmentalized: Collagenase, TIMP, and C2 messenger RNA (mRNA) were localized primarily to the synovial lining layer; HLA-DR mRNA was prominent in the lining and in some sublining lymphoid aggregates; the C3 probe hybridized only to sublining lymphoid aggregates. Relative mRNA levels were quantified using computer-assisted image analysis. There was significantly more collagenase, C2, C3, and HLA-DR mRNA in RA compared with OA patients. However, TIMP mRNA levels were similar in RA and OA. Expression of collagenase, TIMP, C2, C3, and HLA-DR genes correlated with the degree of synovial inflammation. The effect of intraarticular corticosteroid injection on synovial tissue gene expression was studied using serial percutaneous synovial biopsy samples from the knees of 3 RA patients. Joints were biopsied, injected with triamcinolone, and rebiopsied 1-2 weeks later. Histologic inflammation scores were lower in posttreatment synovia. Collagenase and TIMP mRNA, although abundant in presteroid samples, were nearly undetectable in post-steroid tissues. HLA-DR mRNA levels also were significantly decreased. C2 and C3 hybridization significantly decreased in 2 of 3 patients and 1 of 3 patients, respectively. Hence, clinical response to intraarticular steroid therapy was accompanied by histologic improvement and decreased expression of genes that play a role in articular destruction.(ABSTRACT TRUNCATED AT 250 WORDS)
Current evidence indicates that virtually all neuropsychiatric disorders, like many other common medical disorders, are genetically complex, with combined influences from multiple interacting genes, as well as from the environment. However, additive or epistatic gene interactions have proved quite difficult to detect and evaluate in human studies. Mouse phenotypes, including behaviors and drug responses, can provide relevant models for human disorders. Studies of gene-gene interactions in mice could thus help efforts to understand the molecular genetic bases of complex human disorders. The serotonin transporter (SERT, 5-HTT, SLC6A4) provides a relevant model for studying such interactions for several reasons: human variants in SERT have been associated with several neuropsychiatric and other medical disorders and quantitative traits; SERT blockers are effective treatments for a number of neuropsychiatric disorders; there is a good initial understanding of the phenotypic features of heterozygous and homozygous SERT knockout mice; and there is an expanding understanding of the interactions between variations in SERT expression and variations in the expression of a number of other genes of interest for neuropsychiatry and neuropharmacology. This paper provides examples of experimentally-obtained interactions between quantitative variations in SERT gene expression and variations in the expression of five other mouse genes: DAT, NET, MAOA, 5-HT(1B) and BDNF. In humans, all six of these genes possess polymorphisms that have been independently investigated as candidates for neuropsychiatric and other disorders in a total of > 500 reports. In the experimental studies in mice reviewed here, gene-gene interactions resulted in either synergistic, antagonistic (including 'rescue' or 'complementation') or more complex, quantitative alterations. These were identified in comparisons of the behavioral, physiological and neurochemical phenotypes of wildtype mice vs. mice with single allele or single gene targeted disruptions and mice with partial or complete disruptions of multiple genes. Several of the descriptive phenotypes could be best understood on the basis of intermediate, quantitative alterations such as brain serotonin differences. We discuss the ways in which these interactions could provide models for studies of gene-gene interactions in complex human neuropsychiatric and other disorders to which SERT may contribute, including developmental disorders, obesity, polysubstance abuse and others.
F(1) hybrids among New Zealand Black (NZB), New Zealand White (NZW), and BXSB lupus-prone strains develop accelerated autoimmunity in both sexes regardless of the specific combination. To identify BXSB susceptibility loci in the absence of the Y chromosome accelerator of autoimmunity (Yaa) and to study the genetics of this complementation, genome-wide quantitative trait locus (QTL) mapping was performed on female (BXSB x NZW)F(2) mice. Six QTL were identified on chromosomes 1, 4, 5, 6, 7, and 17. Survival mapped to chromosomes 5 and 17, anti-chromatin Ab to chromosomes 4 and 17, glomerulonephritis to chromosomes 6 and 17, and splenomegaly to chromosomes 1, 7, and 17. QTL on chromosomes 4 and 6 were new and designated as Lxw1 and -2, respectively. Two non-MHC QTL (chromosomes 1 and 4) were inherited from the BXSB and the rest were NZW-derived, including two similar to previously defined loci. Only two of 11 previously defined non-MHC BXSB QTL using male (Yaa(+)) crosses were implicated, suggesting that some male-defined BXSB QTL may require coexpression of the Yaa. Findings from this and other studies indicate that BXSB and NZB backgrounds contribute completely different sets of genes to complement NZW mice. Identification of susceptibility genes and complementing genes in several lupus-prone strain combinations will be important for defining the epistatic effects and background influences on the heterogeneous genetic factors responsible for lupus induction.
Mechanisms of immunologic injury causing glomerulonephritis are based on immune complex or autoantibody deposition with activation of complement. Circulating immune complex disease can be diagnosed and monitored with improved assays for immune complexes, DNA antibodies, complement levels, and complement activation products. Etiologic auto-antibodies for the Goodpasture antigen, neutrophil enzymes, and ones directly promoting complement activation are now quantitated for more accurate serologic diagnosis. The spectrum of postinfectious glomerulonephritis also presents several new measurements for assessing clinical status. Diagnosis and monitoring of glomerulonephritis now entails panel testing for these humoral abnormalities that are useful to establish the etiology and treatment regimens.
This study reports quantitative information on the concentration of complement receptor for C3b and C4b (CR1) on erythrocytes from normal individuals and patients with immune complex disease. The measurements were performed by an immunoradiometric assay using monoclonal antibodies against CR1. The antibody specificity was confirmed by immunoprecipitation of CR1 from extracts of surface-labeled cells, by inhibition of rosette formation between B lymphocytes and the erythrocytes intermediate EAC14oxy23b, and by the characteristic distribution of the antigen among cells of human peripheral blood. The number of CR1 molecules in erythrocytes from 52 normal individuals was estimated as 1,410 +/- 620. No significant differences in CR1 levels were observed when individuals were grouped by sex, age, or blood groups. In patients with SLE and rheumatoid arthritis, the number of CR1 molecules per RBC was significantly lower, i.e., 600 +/- 307 and 903 +/- 417, respectively. CR1 levels were normal in asthmatics undergoing long-term treatment with prednisone. In SLE patients, significant correlations were found between CR1 levels, C4 hemolytic titers, and levels of circulating immune complexes. In two out of four patients with SLE, CR1 levels increased significantly during remission, showing that the deficiency is, at least in part, reversible. The deficiency in CR1 could be genetically controlled or could represent an epiphenomenon caused by the interaction of the receptor with a ligand present in the circulation of patients.