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PubMed1977

Frequencies of mitogen-reactive B cells in the mouse. II. Frequencies of B cells producing antibodies which lyse sheep or horse erythrocytes, and trinitrophenylated or nitroiodophenylated sheep erythrocytes.

The B-cell mitogens LPS and lipoprotein stimulate 20-35 percent of all B cells in the spleen of 6- to 8-wk old C3H/Tif mice, as determined by limiting dilution analysis of precursors. Each reactive cell grows to a clone of IgM-secreting PFC, enumerated in a hemolytic plaque assay detecting all IgM secreting cells, regardless of v-region specificity. We have used these mitogens to reveal the total repertoire of Ig specificities produced by these mitogen-reactive B cells. We have determined in plaque assays with six different target erythrocytes the number of spleen cells limiting to one the number of mitogen-reactive B cells detected as specific IgM-secreting clones in each of these plaque assays. By this method, the absolute frequencies of precursor B cells with defined v-gene specificities could be calculated, for at least, one third of all B cells. The frequencies of specific IgM-plaque-forming B-cell clones within the total pool of mitogen-reactive B cells was 1 in 10 for NIP(12),-SRC, 1 in 50 for TNP(12)- SRC, 1 in 100 for NIP(1)-SRC, 1 in 160 for TNP(3)- SRC, 1 in 500 for HRC, and 1 in 1,000 for SRC. These frequencies were the same in the LPS- and in the lipoprotein-reactive B-cell population for TNP(30)- SRC and SRC.

Animals↗
PubMed1988

Maternal autoimmune disease influences self-tolerance in offspring: the role of suppressor cells and materno-foetal cell traffic.

Autoimmune haemolytic anaemia (AIHA) was induced in normal strain (CBA/Ca/T6) mice by repeated intraperitoneal injection of rat red blood cells (RRBC). Antibody production to cross-reactive antigens on mouse red blood cells (MRBC) and foreign antigens on RRBC was measured by the direct antiglobulin test (DGAT) and serum haemagglutination, respectively. RRBC primed female or male mice and sheep red blood cell (SRBC) primed controls were mated with naive partners and their progeny immunized with RRBC in adult life. The offspring of mothers but not fathers with active autoimmune disease showed a significant reduction in antibody response to self (MRBC) antigens, whereas the response to non-self (RRBC) was unaffected. Transfer of 30 X 10(6) spleen cells from the progeny of RRBC primed mothers into non-irradiated normal recipients resulted in selective suppression of the anti-self response following challenge with RRBC, provided that the cell donors had been boosted with RRBC 7-10 days before the transfer was performed. Thus the progeny of mothers with AIHA possessed self-reactive memory suppressor cells (Ts) shown previously to belong to the Thy-1+ I-J+ Ly-2+ T cell subset in this model. To test whether the Ts were of maternal or foetal origin the suppressor assay was repeated with spleen cells from the F1 offspring of RRBC primed B10.A(3R) (I-Jb) mothers and normal CBA(I-Jk) fathers. Pretreatment with anti-I-Jb serum plus complement completely abrogated suppression on adoptive transfer but anti-I-Jk serum failed to do so, indicating that the Ts were derived from the mothers. These findings emphasize the potential importance of Ts in induction of self tolerance during early ontogeny.

Anemia, Hemolytic, Autoimmune↗
PubMed1988

Reversible biotinylation of C1q with a cleavable biotinyl derivative. Application in C1q receptor (C1qR) purification.

Reversible biotinylation of human C1q without impairment of its physiologic functions has allowed us to develop a simple and rapid purification method for C1q receptor (C1qR). The biotinylating reagent, NHS-SS-biotin (Mr 606.7) contains an extended connector or cross-linker arm which limits steric hindrance and is bridged by a cleavable disulfide bond to the biotin component. Biotinylation was achieved by mixing C1q (in PBS, pH 7.4) with NHS-SS-biotin (dissolved in dimethyl formamide) in a 50:1 v/v and 1:25 mol/mol ratio and allowing the reaction to continue at room temperature for 4 h. The mixture was then dialyzed against PBS pH 7.4 (2 X 1 liter) and analyzed by SDS-PAGE and hemolytic assay using C1q depleted serum. Under these conditions neither denaturation of the protein nor loss of hemolytic activity was evident. Such biotinylated C1q (Bio-C1q) was used to pull out the C1qR from detergent-solubilized (1% NP-40 in PBS, pH 7.4 plus inhibitors) 125I-surface labeled membrane solution that had been first centrifuged (1 h, 45,000 X g, 4 degrees C) and then sequentially precleared with immobilized protein A, protein A-IgG and gelatin. The mixture of Bio-C1q and membrane solution was then incubated (20 h, 4 degrees C), applied to immobilized avidin (equilibrated with PBS, pH 7.4, 0.1% NP-40) and after washing, the bound C1qR was eluted with equilibrating buffer containing 1 M NaCl, and the C1q by same buffer containing 100 mM DTT. The eluted C1qR contained a major Mr 70,000 molecule which upon reduction electrophoresed with an apparent Mr of 85,000-90,000 as assessed by SDS-PAGE analysis. In addition, a faint single chain band of 30-40 kDa was eluted with the major band and may represent a non-covalently associated part of the C1qR molecule.

Avidin↗
PubMed1992

The proteoglycan decorin binds C1q and inhibits the activity of the C1 complex.

Decorin, a small collagen-binding dermatan sulfate proteoglycan, is widely distributed as a component of extracellular matrices. Using a solid phase binding assay, we showed that decorin bound C1q at physiologic pH and ionic strength. The interaction did not require divalent cations and was time and temperature dependent reaching equilibrium in 4 h at 37 degrees C. Binding was specific and saturable with an apparent dissociation constant of 7.6 x 10(-9) M. Decorin was shown to bind pepsin-derived fragments containing the collagenous domain of C1q and collagenase-derived fragments containing the globular domain of C1q. Because these fragments share a short sequence of amino acids, this finding suggests that decorin binds to a region of C1q located near the junction of the two domains. Competition studies using purified preparations of the decorin core protein and the glycosaminoglycan chains showed that only the former inhibited binding of decorin to C1q indicating that the interaction is mediated by the decorin core protein. Decorin was shown to inhibit the hemolytic activity of purified C1 as well as C1 in normal human serum. Approximately 50% inhibition was observed at a decorin concentration of 2 micrograms/ml. Inhibition was not observed if C1 was bound to Ag-complexed antibody. Furthermore, neither the core protein nor the glycosaminoglycan chain of decorin inhibited C1, indicating that the intact proteoglycan is necessary for functional activity.

Animals↗
PubMed2000

Anaemia in systemic lupus erythematosus: aetiological profile and the role of erythropoietin.

OBJECTIVE: To study the prevalence of different causes of anaemia in patients with systemic lupus erythematosus (SLE) and their associations with immunological and clinical parameters and to evaluate the contribution of erythropoietin (Epo) and anti-erythropoietin (anti-Epo) autoantibodies to the development of SLE anaemia. METHODS: 132 SLE patients with anaemia (defined as haemoglobin of 12 g/dl or less for women and 13.5 g/dl or less for men) from among a total of 345 consecutive SLE patients were prospectively enrolled into the study. Standard haematological and immunological tests were performed and serum Epo and anti-Epo antibodies were assayed. RESULTS: The identified causes were anaemia of chronic disease (ACD) n=49 (37.1%), iron deficiency anaemia (IDA) n = 47 (35.6%), autoimmune haemolytic anaemia (AHA) n = 19 (14.4%) and other causes n = 17 (12.9%). There was significant heterogeneity in the severity of anaemia between the four groups (p<0.01) with AHA cases being on average more severe. The proportion of patients with anticardiolipin antibodies, low complement levels and anti-dsDNA differed significantly among the four groups; these markers were particularly common in patients with AHA, and uncommon in patients with IDA. Twenty one of 100 tested patients had anti-Epo antibodies. Such antibodies were seen practically only in patients with ACD (odds ratio 3.1, p = 0.041) and in patients with high lupus activity (ECLAM) scores (odds ratio 1.27 per point, p = 0.055). Epo response was inadequate in 42.4% and 41.2% of patients with ACD and AHA, respectively. CONCLUSIONS: Anaemia in SLE usually takes the form of ACD and IDA, however autoimmune haemolysis is not uncommon. SLE patients with different causes of anaemia differ in regard to several immunological parameters. Epo response is blunted in anaemic SLE patients, particularly those with ACD and AHA.

Adult↗
PubMed1984

Mechanisms of genetic control of immune responses. I. Evidence for distinct multi-step helper T-cell pathways in cellular and humoral responses to GAT.

We examined multiple genetically regulated humoral and cell-mediated immune (CMI) responses to poly( glu60ala30tyr10 ) (GAT) using a panel of mouse strains. We show that assignment of responder/nonresponder status depends upon the assay method. In addition, two distinct categories of nonresponder mice were found: (1) those which are unresponsive by all parameters tested (H-2q and H-2s haplotypes) and (2) those which are partially nonresponsive [H-2bm12 mutant strain--a low/nonresponder by splenic plaque-forming cell (PFC) and delayed-type hypersensitivity (DTH) responses, but exhibits B6 parental levels of high GAT-specific T-cell proliferation ( Tprlf ) and interleukin-2 production]. The distinction between these two nonresponder types was confirmed by complementation tests in which significant GAT-specific PFC and DTH responses were seen in (H-2q X H-2bm12)F1 hybrids, but not in (H-2q X H-2s)F1 hybrids. Suppressor T cells (Ts) also play a selective role in nonresponsiveness to GAT. Cyclophosphamide treatment of nonresponders (to eliminate Ts activity) as well as immunization with GAT coupled to the immunogenic carrier MBSA result in the development of GAT-specific humoral, but not CMI responses. Our results indicate that the T cell is the cellular site of Ir gene expression and that Tprlf responses do not correlate with functional helper T-cell activity and suggest distinct, multi-step Th/Ts regulatory pathways in the development of humoral and CMI effector functions.

Animals↗
PubMed1982

Human natural killer-like activity against mouse spleen cells.

Unstimulated human peripheral lymphocytes were cytotoxic for normal mouse spleen cells and suppressed the in vitro antibody plaque-forming cell (PFC) response of these cells to sheep red blood cells and dinitrophenylated Ficoll. The cells in the lymphocyte population that were responsible for the immunosuppression had properties of natural killer (NK) or NK-like cells in that they were: (a) non-E-rosetting, (b) nonadherent, (c) unaffected by treatment with anti-human immunoglobulin plus complement, (d) cytotoxic against an established human NK target, K562 leukemic cells, and (e) partially inactivated by mitomycin C. Addition of the human NK-like cells to mouse spleen cell cultures at the time of antigen addition and at an effector cell to target cell ratio as low as 0.67:1 resulted in 85 to 96% suppression of the PFC response. Addition of NK-like cells to cultures 18 h before harvesting in 5-day cultures required higher concentrations and ratios (2.7:1) of effector to target cells to significantly suppress the PFC response. The data suggest that human NK-like activity in suppression of the mouse PFC response is due to killing of the targets. The mouse spleen cell PFC system represents a potential model for assessment of human NK activity that is quite dramatic in its effect and can be used in addition to the well known 51Cr-release assay. Also, since the mouse spleen cell is a normal cell, it provides a model in the PFC system for studying the mechanism of NK regulation of normal cellular function. An additional finding of this study was the observation that E-rosetting T cells significantly enhanced the mouse PFC response. Thus, human peripheral lymphocytes contain discrete cellular population that either enhance or suppress the mouse PFC response.

Animals↗
PubMed1984

Recent progress in the diagnosis of brucellosis.

Progress since 1975 in the development of methods for the diagnosis of brucellosis is summarized. Standard serological diagnosis improved with increased use of acidified antigen agglutination and complement fixation tests. Immunoassays, tests based on the lysis of lipopolysaccharide coated erythrocytes and tests using new antigens have increased the sensitivity and specificity of serological results. The field of cellular immunology has seen the development and field evaluation of a skin test using refined antigens and the assessment of in vitro assays of cellular activity using purified protein and crude brucella antigens. Potential diagnostic uses of these methods are discussed. Bacteriological procedures were improved by introduction of stomachers and improved culture media. The isolation of new Brucella phages and development of a thin layer chromatography method for the determination of oxidation metabolic profiles were advanced in the characterization of Brucellae. Progress was also made in the development of immunoassays for the detection of Brucella antigens in host tissues. The selection of control groups, quality control studies and problems of standardization are areas that require greater attention in future methods development work. Major achievements of the period were 1) demonstrations that diagnosis sensitivity can be increased by new assays for antibody and cellular responses, 2) new methods to discriminate between anti-Brucella abortus and anti-Yersinia enterocolitica 0:9 responses, 3) a radial immunodiffusion test that detects most actively infected cattle, and 4) the simplification and extension of oxidative metabolic and phage typing tests. Advances in clinical microbiology and molecular biology have created new opportunities to improve diagnostic methods in the next decade.

Animals↗
PubMed1973

In vitro antibody-enzyme conjugates with specific bactericidal activity.

IgG with antibacterial antibody opsonic activity was isolated from rabbit antisera produced by intravenous hyperimmunization with several test strains of pneumococci, Group A beta-hemolytic streptococci, Staphylococcus aureus, Proteus mirabilis, Pseudomonas aeruginosa, and Escherichia coli. Antibody-enzyme conjugates were prepared, using diethylmalonimidate to couple glucose oxidase to IgG antibacterial antibody preparations. Opsonic human IgG obtained from serum of patients with subacute bacterial endocarditis was also conjugated to glucose oxidase. Antibody-enzyme conjugates retained combining specificity for test bacteria as demonstrated by indirect immunofluorescence. In vitro test for bactericidal activity of antibody-enzyme conjugates utilized potassium iodide, lactoperoxidase, and glucose as cofactors. Under these conditions glucose oxidase conjugated to antibody generates hydrogen peroxide, and lactoperoxidase enzyme catalyzes the reduction of hydrogen peroxide with simultaneous oxidation of I(-) and halogenation and killing of test bacteria. Potent in vitro bactericidal activity of this system was repeatedly demonstrated for antibody-enzyme conjugates against pneumococci, streptococci, S. aureus, P. mirabilis, and E. coli. However, no bactericidal effect was demonstrable with antibody-enzyme conjugates and two test strains of P. aeruginosa. Bactericidal activity of antibody-enzyme conjugates appeared to parallel original opsonic potency of unconjugated IgG preparations. Antibody-enzyme conjugates at concentrations as low as 0.01 mg/ml were capable of intense bactericidal activity producing substantial drops in surviving bacterial counts within 30-60 min after initiation of assay. These in vitro bactericidal systems indicate that the concept of antibacterial antibody-enzyme conjugates may possibly be adaptable as a mechanism for treatment of patients with leukocyte dysfunction or fulminant bacteremia.

Agglutination Tests↗
PubMed2004

Anti-lipoprotein lipase antibodies: a new player in the complex atherosclerotic process in systemic lupus erythematosus?

OBJECTIVE: The novel description of antibodies to lipoprotein lipase (anti-LPL) associated with dyslipoproteinemia prompted us to analyze the association of anti-LPL with clinical and serologic features in patients with systemic lupus erythematosus (SLE) and its link to markers of inflammation that are known to be involved in atherogenesis. METHODS: Enzyme-linked immunosorbent assay was used to test for the presence of anti-LPL antibodies in 66 consecutive patients with SLE. Clinical and laboratory evaluation, including a fasting lipid profile, autoantibody screening, an assessment for markers of inflammation (C-reactive protein [CRP], erythrocyte sedimentation rate [ESR]), and the SLE Disease Activity Index (SLEDAI) were performed at the time of inclusion in the study. Exclusion criteria were any conditions that affect the lipid profile. SLE patients were categorized into 2 groups according to detection of these anti-LPL antibodies, as follows: anti-LPL+ and anti-LPL-. RESULTS: Anti-LPL antibody IgG was detected in 25 SLE patients (37.8%). Triglyceride levels were significantly higher in the anti-LPL+ group (112.4 +/- 50.2 versus 89.9 +/- 54.5 mg/dl in the anti-LPL- group; P = 0.033), but no significant differences between the 2 groups were detected for total, high-density lipoprotein, and low-density lipoprotein cholesterol levels. A higher frequency of elevated CRP levels and ESRs was observed in the anti-LPL+ group compared with the anti-LPL- group (44% and 17.1%, respectively [P = 0.023] and 52% and 19.5%, respectively [P = 0.013]). Moreover, SLE patients with anti-LPL antibodies also had significantly higher levels of CRP (11.1 +/- 16.4 versus 2.4 +/- 2.6 mug/ml; P = 0.036) and higher ESRs (33.4 +/- 29.8 versus 16.5 +/- 11.8 mm/hour; P = 0.020). Anti-LPL titers had a significant positive correlation with the CRP level (r = 0.56, P < 0.001), the ESR (r = 0.55, P < 0.001), the SLEDAI score (r = 0.45, P < 0.001), anti-double-stranded DNA (anti-dsDNA; r = 0.52, P < 0.001), and anticardiolipin IgG antibodies (r = 0.25, P = 0.04), and a significant negative correlation was detected with total hemolytic complement activity (CH100) (r = -0.34, P = 0.005). Reinforcing these findings, multiple regression analysis also revealed a significant association of anti-LPL with the CRP level (P = 0.025) and anti-dsDNA (P < 0.001). Importantly, a comparison of positive and negative anti-dsDNA sera revealed similar mean CRP levels (P = 0.56) and ESRs (P = 0.102), contrasting with the SLEDAI score (P = 0.004) and CH100 (P = 0.008). CONCLUSION: These data support the link between inflammation, immune response, and dyslipoproteinemia in SLE, introducing anti-LPL as a possible new player that may ultimately help in understanding the complex events of atherogenesis in this disease.

Adult↗
PubMed1980

MLC-derived human helper factor(s) that promote B cell differentiation: induction, functional characterization, and role of Ia antigens.

Utilizing a PFC assay to quantitate the polyclonal activation of human peripheral blood B lymphocytes, we have investigated the induction and functional activity of MLC-derived human helper factor(s). Our data demonstrate that highly purified responder T cells, but not B or null cells, are required for the elaboration of MLC helper factor(s) that trigger the in vitro differentiation of B lymphocytes into PFC. Helper factor can trigger B cell maturation in the absence of helper T cells, since complement- (C) mediated lysis of the small (less than 5%) fraction of T cells present in anti-F(ab)2 immunoabsorbent column purified B cell population eliminates the PWM induced, but not the helper factor-induced PFC response. Responder T cells required for helper factor production do not bear surface membrane Ia, since alpha p23,30 + C treatment of this population does not affect helper factor generation. In contrast, alpha p23,30 + C treatment of the allogeneic stimulator cell population eliminates helper factor production. Taken together, these results demonstrate that interaction between Ia-bearing stimulator cells and Ia- responder T cells is required for the production of MLC-derived helper factor. In additional experiments, we determined that alpha p23,30, in the absence of C, totally abrogates the PFC response triggered by MLC helper factors. This result suggests an important role for Ia antigens in the functional activity of preformed helper factor molecules.

Antibodies↗
PubMed1983

H-2-linked murine factor B phenotypes.

A hemolytic assay has been developed which is specific for Factor B (B) activity in murine EDTA-plasma. Three discrete levels of B activity were observed among B10-congenic strains. Mice with standard H-2 haplotypes, b, d, k, r, f, q, s, and u, all exhibited the same mean level of activity. However, plasma from H-2v (B10.SM) mice contained only 0.25 of that level, and those with standard haplotype H-2ja (B10.WB) or wild haplotype H-2wr7 (B10.WR) exhibited 2.5 times the H-2b (B10) basal level of activity. These differences among B10 congenic lines suggested that the activity is H-2 controlled; further tentative mapping with intra-H-2 recombinants indicated that the gene is located in the S region. A fourth phenotype was found among progeny of backcross generations between B10.BR (H-2k) and mice of subspecies Mus musculus molossinus and M. m. bactrianus. This ultra-high activity was found also to be governed by a gene very closely linked to Ss, the primary S region marker. F1 generations between disparate phenotypes yielded progeny with activity levels intermediate between the parents; progeny of parents of different strains with the same phenotype expressed B hemolytic titres equal to those of the parental strains. No differences in antigenic levels of the protein among the strains of different phenotypes could be detected by radial immunodiffusion. In mixing experiments, resultant activity levels were intermediate between the higher and the lower phenotype, ruling out independent inhibitors or activators of the reaction. These studies indicate that an H-2-linked S region-located single gene governs structural differences in allelic B molecules that lead to differences in specific activities.

Alleles↗
PubMed1980

Presence of a tumour-inhibiting factor (TIF) in sera from normal but not tumour-bearing mice.

Some plasmacytomas produce myeloma proteins with known antibody specificities and the secretion of these proteins by individual tumour cells can be determined using haemolytic plaque assay. After a 3 day culture of mouse plasmacytoma cells in medium containing 10% normal mouse serum, the number of plaques was reduced to less than 10% when compared to that of tumour cells incubated with either foetal calf serum or normal rabbit serum. However, tumour cells incubated with sera from mice bearing TEPC-15, McPC-603, or MOPC-315 plasmacytomas displayed control levels of plaques. The production of plaques paralleled the viability of tumour cells suggesting that the reduction of plaque formation is due to the decreased viable cell number. The tumour-inhibiting activity was recovered from the fraction of apparent molecular weight of 300,000-400,000 after a partial purification using an agarose (A 0.5 M) column. This fraction, however, did not suppress in vitro induction of antibody production. Kinetic experiments using sera obtained sequentially from individual mice receiving either TEPC-15 or MOPC-315 plasmacytomas further indicated that the tumour-inhibiting activity is severely reduced during a 2 week period after tumour inoculation. The inhibition of tumour cells did not appear to be specific since tumour cells of three plasmacytomas (TEPC-15, MOPC-167 and MOPC-315), a mastocytoma (P815) and a lymphoma (EL-4) displayed a similar susceptibility to normal serum.

Animals↗
PubMed1976

The xenogeneic effect. II. Requirement for unactivated murine T cells during restoration of immune responsiveness with xenogeneic reconstitution factor.

Studies were conducted on the mechanism of action of a soluble mediator which was generated in human mixed lymphocyte cultures and assayed for helper activity in T cell-deficient murine spleen cell cultures. The mediator, termed xenogeneic reconstitution factor or XFR, restored the anti-sheep erythrocyte plaque-forming cell response of spleen cells from thymectomized, lethally irradiated, and syngeneic bone marrow transplated (TxB) mice. It was found that in order to obtain a maximal antibody response, the XRF had to be present sometime during the first 24 to 40 hr of the induction process. XRF was also required during the last 24 hr of the incubation period. A striking synergistic effect was obtained by combined exposure of the T cell-deficient spleen cell cultures to XRF during the first and last days of culture. These data suggested a bi-modal mechanism of action of XRF, and raised the possibility that in addition to providing a signal to the B cells, XRF-mediated activation of the residual TxB splenic T cells was crucial to the successful restoration of the antibody response. Treatment of the nonadherent splenocytes from the TxB mice with anti-T cell serum and guinea pig complement completely abrogated the antibody response of these cells in the presence of adherent spleen cells, sheep erythrocytes, and the helper factor XRF. The antibody response of this combination of cells, antigen, and XRF was reconstituted by a population of unimmunized murine T cells which, in the absence of XRF, were totally unable to restore responsiveness. These results suggest that B cell activation to the formation of antibody involves the mandatory co-participation of two functionally distinct helper activities.

Animals↗
PubMed1987

Generation of an activated form of human C5 (C5b-like C5) by oxygen radicals.

Treatment of purified human C5 with hydrogen peroxide leads to a reduction of functional activity in the classical immune haemolysis assay. The effect of H2O2 is enhanced by traces of iron-EDTA, and becomes even stronger when ascorbic acid is present in addition. The enhancement by iron and protective effects of various radical scavengers indicate that the conversion of C5 is brought about by hydroxyl radicals mainly, generated from H2O2 during its decomposition. By the conversion C5 acquires a new functional property: it becomes capable of binding C6, and the resulting complex C56 lyses non-sensitized red cells in cooperation with the late components C7, C8, and C9 (reactive lysis). In this respect, H2O2-treated C5 resembles the activation fragment of C5, C5b. However, unlike C5b, C5(H2O2) is not fragmented but comprises the whole molecule, according to polyacrylamide gel electrophoresis. In accordance with the lack of cleavage, no chemotactic activity indicating formation of C5a, became apparent after H2O2 treatment of C5. In whole human serum, however, the H2O2 system induces additional processes leading to the generation of C5a activity and indicating C5 cleavage. The pathways to this reaction are as yet not clear. The effect of oxygen radicals on C5 may be the basis of the enhancing effect of stimulated leukocytes on C5 activation in body fluids. Activated leukocytes are known to produce H2O2 and oxygen radicals.

Complement Activation↗
PubMed1976

Triggering of human peripheral blood B cells: polyclonal induction and modulation of an in vitro PFC response.

In the present studies we utilized polyclonal activators to analyze the signals required to induce antibody synthesis by subpopulations of human peripheral blood lymphocytes. The in vitro synthesis of antibody directed against sheep erythrocytes was measured by a modification of the Jerne plaque assay. The plaques observed contained central lymphoid cells, could be inhibited by anti-human immunoglobulin, and were strictly dependent on both the addition of complement and viable B lymphocytes actively synthesizing protein. Kinetic analysis showed that the PFC response developed over time during cell culture with peak activity occurring between days 6 and 7. It was found that human peripheral blood B lymphocytes, isolated by Sephadex G-200 anti-Fab column chromatography, were nonspecifically induced by either PWM or soluble products of antigen-activated human T cells to differentiate into PFC. In contrast, unfractionated peripheral blood lymphocytes. Ig- cells isolated from immunoabsorbant columns, and B cells enriched by nylon wool adherence were not triggered to antibody synthesis even in the presence of polyclonal activators. B cells isolated by nylon wool adherence and further fractionated by binding and elution from anti-Fab columns were then readily triggered by PWM to differentiate into PFC. These results suggest that the interaction of human B cells with anti-Fab columns, presumably at the Ig receptor, serves as a signal important in the differentiation events leading to PFC activity. The anti-Fab interaction alone, although critical, was not sufficient to induce the differentiation of precursor cells into PFC since subsequent triggering by antigen-activated T cell supernatants or PWM was required for the development of an optimal PFC response. Additional experimental data demonstrated that the PFC response could be regulated by autologous peripheral Ig- lymphoid cells in vitro. At low concentrations of PWM, Ig- cells consistently augmented the B cell PFC response whereas at high concentrations of PWM, suppression was observed. Depletion of E rosetting cells from the Ig- population eliminated the subset responsible for augmentation but did not eliminate the suppressor cell activity.

Animals↗
PubMed1992

A calcium-binding monoclonal antibody that recognizes a non-calcium-binding epitope in the short consensus repeat units (SCRs) of complement C1r.

C1r is a Ca(2+)-binding serine protease that interacts with two other plasma proteins, C1q and C1s, to form C1, the first component of the complement cascade. A monoclonal antibody, BG6, has been produced which binds to C1r only in the presence of Ca2+, requiring 3-5 microM Ca2+ for half-maximal binding. The antibody reacts with native and heat-denatured C1r, and with zymogen C1r, but does not cross-react with C1s or C1q. BG6 did not significantly affect the esterolytic activity of C1r toward a synthetic thioester substrate nor the hemolytic activity of C1 reconstituted from subcomponents in the presence of the antibody. A tryptic fragment of C1r which consists of the C-terminal gamma region of the A chain disulfide-linked to the B chain (gamma B) binds in a Ca(2+)-dependent manner to BG6-Sepharose. Western blotting experiments have further localized the epitope to the gamma region of the A chain, which is composed of two short consensus repeat (SCR) units. The N-terminal alpha region contains the only previously determined Ca(2+)-binding site in the C1r molecule. Equilibrium dialysis experiments confirmed that C1r-gamma B does not bind Ca2+, and showed that antibody BG6 and the gamma B/BG6 complex do bind Ca2+. Thus, the Ca(2+)-dependent nature of this interaction is due exclusively to binding of the metal ion to the antibody. Equilibrium dialysis and immunoblotting have further localized the Ca(2+)-binding site to the Fab fragment of BG6, indicating that the metal-induced conformational change residues in or near the variable region of the IgG. BG6 may set a precedent for the preparation of Ca(2+)-dependent antibodies to non-Ca(2+)-binding epitopes in other proteins.

Antibodies, Monoclonal↗
PubMed1993

Protein engineering studies on C1r and C1s.

1. C1r and C1s cDNAs were placed downstream the strong polyhedrin promoter in the Autographa californica nuclear polyhedrosis virus and the recombinant proteins were expressed in insect cells, in biologically active form. The yield of expression is high enough to get recombinant components for chemical and functional studies (5 micrograms/ml cell culture supernatant). 2. The biological activity and the post-translational modifications of the recombinant subcomponents were checked. The rC1r and rC1s proved to be biologically active in the hemolytic assay, although their glycosylations were different compared to that of the serum proteins. The insect cells are able to beta-hydroxylate the Asn residue of the EGF domain in the C1r but with a low efficiency. It is clear now, that this post-translational modification does not play a role in the Ca2+ dependent C1r-C1s interaction. 3. Two deletion mutants of C1r cDNA were constructed in order to clarify the role of domain I and II. The results show that both, domain I, and II are absolutely necessary for the tetramer formation and both have a regulatory role in the autoactivation. The autoactivation of the mutants is accelerated significantly. 4. Hybrid cDNA constructions were also made, and one of them was expressed. In the C1s alpha R hybrid the C1s alpha part cannot dimerize in presence of Ca2+, but it can form a tetramer with C1r2, that can bind to C1q. This observation indicates that the function of the C1s alpha part in the hybrid is modulated by the C1r part (gamma B) of the molecule. 5. In order to control the autoactivation process point mutant cDNAs were constructed through altering the Arg-Ile bond in the catalytic domain of the C1r. The Gln-Ile construction is a stable zymogen while the Arg-Phe mutant has a lower rate of autoactivation. These results do justify our approach of using domain-domain interchange, domain deletion and point mutations in combination, to reveal the structural background of C1 function at intramolecular level.

Animals↗