Further observations and characterization of monoclonal antibodies reacting with B cell alloantigens associated with rheumatic fever and rheumatic heart disease.

Elevated levels of B lymphocytes with a unique surface alloantigen have been reported to be characteristic of patients with acute rheumatic fever or rheumatic heart disease. Mouse monoclonal antibodies (mAbs) to this alloantigen have been proposed as being useful in identifying individuals at risk for the development of these sequelae of group A streptococcal infection. However, previous studies have suggested that the discriminating ability of the mAbs was highest when the mAbs were made by using lymphocytes from the same ethnic population. To confirm and extend this observation, additional mouse mAbs were developed and their properties defined. These three mAbs-PG-12A, PG-13A, and PG-20A-reacted with B cells from more than 90% of North Indian patients with acute rheumatic fever or rheumatic heart disease. Each of these three new mAbs identified the highest levels of reactive B cells in patients with active acute rheumatic fever. Lower levels of positive reacting lymphocytes were found in individuals with quiescent chronic rheumatic heart disease, and markedly reduced percentages of reactive cells were observed in normal control subjects. The proportion of reactive lymphocytes in individual patients varied according to which of the three was tested, suggesting the possibility of a spectrum of "rheumatic" epitopes in susceptible individuals. The data further suggested that enhanced discriminatory ability for identifying "at-risk" susceptible patients could be obtained by testing with a combination of mAbs. If reduction in the incidence of acute rheumatic fever can be facilitated by early identification of susceptible individuals, accurate and sensitive detection of a marker antigen would result in more cost-effective public health measures. Additional population studies are required to more precisely define and confirm these detection techniques.

Ethnic differences in expression of susceptibility marker(s) in rheumatic fever/rheumatic heart disease patients.

The ability of monoclonal antibodies (MAb) against a human B lymphocyte alloantigen has been suggested to discriminate between rheumatic fever "susceptible" individuals and persons with a lower risk of developing RF. However, while such MAb have been reported to identify a majority of RF/RHD patients in some populations, a reduced discriminatory ability has been observed in others. Antigenic variation in the RF marker(s) may exist among ethnic groups which reduce the discriminatory ability of these monoclonal antibodies. We developed MAb using B lymphocytes from RF patients of North Indian ethnic origin. In this same population we compared the new MAb (PGI/MN II) with a previously described MAb of Caucasian ethnic origin (D8/17). In three groups: acute rheumatic fever patients (no evidence of previous attacks of rheumatic fever), patients with chronic rheumatic heart disease and normal controls from the same population, we found a greater discriminating ability of PGI/MNII MAb to identify Indian RF/RHD patients than with the D8/17 MAb. Further, sixty percent of 142 siblings of the RF/RHD patients were "positive" when tested with PGI/MN II. The data from these studies suggest that before such MAb can be used for identification of RF "susceptibles" in public health programs, variation among ethnic populations must be assessed.

Cytokines and immunoglobulin in rheumatic heart disease: production by blood and tonsillar mononuclear cells.

Rheumatic fever and rheumatic heart disease are considered to result from abnormal immune responses after Group A streptococcal pharyngitis. Production of interleukin 1 (IL-1), tumor necrosis factor-alpha (TNF), interleukin 2 (IL-2) and immunoglobulin (Ig) by blood and tonsillar mononuclear cells from rheumatic or healthy children was measured after stimulation in vitro by pokeweed mitogen (PWM) or the streptococcal extracellular product, blastogen A (BLA). Tonsillar cells from patients with rheumatic heart disease produced significantly less IL-1, TNF, IL-2, and Ig than control tonsillar cells. In contrast, blood mononuclear cell cultures from rheumatic children produced more TNF and IL-2 than controls. Our findings suggest that abnormal regulation of cytokine and Ig production may contribute to the pathogenesis of acute rheumatic fever and rheumatic heart disease.

Distribution of cells bearing "rheumatic" antigens in peripheral blood of patients with rheumatic fever/rheumatic heart disease.

Cell surfaces of some peripheral blood cells from individuals with a history of rheumatic fever/rheumatic heart disease (RHD) have been demonstrated by the use of monoclonal antibodies to be antigenically distinct from the majority of the population. Our study examines the distribution of cells bearing these "rheumatic" antigens in 23 subjects with rheumatic fever/RHD of Maori, Polynesian and Caucasian ancestry and 182 members of their families (rheumatic fever/RHD families) as well as in 46 members of families in which no member had been demonstrated to have had rheumatic fever/RHD (control families). Mononuclear cells from the blood of all cooperating family members were prepared and non-T cells isolated by sheep red blood cell rosette depletion. The binding of monoclonal antibodies 83S19.23 and D8103 to non-T cells was measured using an immunoperoxidase technique. Subjects with rheumatic fever/RHD had a significantly higher proportion of cells binding the antibodies than the unaffected members of all families. Unaffected members of rheumatic fever/RHD families had significantly higher levels of such rheumatic cells than control families. An increase in the proportion of rheumatic cells with age was noted in unaffected members of rheumatic fever/RHD families but not in rheumatic fever/RHD subjects of control families. A level of 13% 83S19.23 positive non-T cells optimally discriminated between rheumatic and nonrheumatic individuals. The relative risk for rheumatic fever/RHD with 13% or greater positive cells was 9.48. The negative predictive value of having less than 13% positive cells was 98.3%. In the population studied, 83S19.23 seems especially capable of identifying those with low risk for rheumatic fever/RHD.

Group A streptococcal pyrogenic exotoxin (scarlet fever toxin) type A and blastogen A are the same protein.

Group A streptococcal pyrogenic exotoxins A, B, and C (also known as scarlet fever toxins and erythrogenic toxins) were evaluated for relatedness to another streptococcus-derived lymphocyte mitogen, blastogen A. Streptococcal pyrogenic exotoxin A and blastogen A were immunologically cross-reactive and shared the same molecular weight, N-terminal amino acid sequence, and capacity to stimulate rabbit splenocyte proliferation nonspecifically.

Augmentation of cytotoxic activity by mitogens in rheumatic heart disease.

Natural killer cell activity and alterations in cytotoxicity after culture with streptococcal blastogen A and phytohemagglutinin (PHA) were examined in patients with inactive rheumatic heart disease (RHD) and control patients. Natural cytotoxic activity of mononuclear cells (MNC) did not differ between RHD and control patients with either peripheral blood or tonsils. In cultured blood MNC the level of cytotoxic activity stimulated by blastogen A was significantly greater in patients with RHD at all effector:target cell ratios. These differences in cytotoxic activity were not observed with cultured tonsillar MNC. In similar experiments with a different group of patients, culture with PHA or blastogen A both produced a significantly greater increase in cytotoxic activity in blood MNC from patients with RHD. The increase was significantly lower with PHA than with blastogen A. The ability of mitogens to differentially augment cytotoxic activity in cells from the blood of patients with RHD implies that a population of cells exists in these patients that could be activated during acute rheumatic fever to play a role in pathogenesis.

Multiple forms of rat kidney L-arginine:glycine amidinotransferase.

The relative amount of L-arginine:glycine amidinotransferase (transamidinase) protein in kidneys from rats fed a complete purified diet with and without the addition of creatine and/or glycine was determined by a monoclonal antibody-immunosorbent inhibition assay. Kidneys from the creatine-fed rats had 10% of the transamidinase activities and 78% of the monoclonal antibody immunoreactive transamidinase protein as kidneys from the control rats. An excellent correlation between transamidinase activities and protein was reported previously when the amounts of enzyme protein were determined by immunotitration with polyclonal antibodies. One possible explanation for the contrasting results was that multiple forms of transamidinase are present in rat kidneys. If so, the monoclonal antibody may have recognized forms of the enzyme that were not decreased in amounts commensurate with the decrease in enzyme activities as a result of creatine feeding. Evidence is presented in this report that multiple forms of transamidinase are present in rat kidneys. The distribution of the isoelectric points of the individual forms of transamidinase in kidneys of the control rats appeared to be dissimilar from that in the creatine-fed rats. Therefore, an alteration in the distribution of the individual forms of the enzyme may be a factor in the alteration of transamidinase activities in creatine-fed rats.

Cell surface markers and cellular immune response associated with rheumatic heart disease: complex segregation analysis.

A series of functional and cell surface markers associated with a significantly increased risk of rheumatic heart disease were analyzed for the contribution of genetic factors in their presence. Peripheral blood lymphocytes from nine large kindreds from the New Zealand Maori, Polynesian, and Caucasian populations were isolated, purified, and evaluated with lymphocyte surface markers (monoclonals 83S.19.23 and D8103), as well as studied for blastogenic response to a purified group A streptococcal extracellular product, blastogen A. Segregation analysis of blastogenic response and percent of cells positive for these cell surface markers was consistent with genetic control by single major genes; however, the contribution by polygenes varied by marker, indicating heterogeneity of genetic control of identification of cell surface glycoproteins and blastogenic response to streptococcal products.

Antibody response to bacteriophage hyaluronidase in acute glomerulonephritis after group A streptococcal infection.

In a test of the hypothesis that lysogeny of group A streptococci by a temperate bacteriophage might confer nephritogenicity, 283 sera from 69 patients were examined for IgG and IgM antibodies to M 49 streptococcal bacteriophage hyaluronidase. The IgG and IgM response to bacteriophage hyaluronidase was greatest in M 49 streptococci-infected individuals with nephritis, but M 49 streptococci-infected subjects without nephritis also had a greater immune response than did subjects infected with serotypes other than M 49. Although antibody to bacterial hyaluronidase was detected in all Streptococcus-infected groups, antibody to M 49 streptococcal bacteriophage hyaluronidase usually was found in only M 49 streptococci-infected patients. Although the greatest IgG and IgM antibody response to bacteriophage hyaluronidase can be demonstrated in individuals with glomerulonephritis, the antibody response does not indicate a direct relation of lysogeny and nephritis because subjects with and without nephritis after M 49 streptococcal infection all had a significant rise in antibody titer.

Lymphocyte subpopulations in rheumatic heart disease.

Monoclonal antibodies and indirect immunofluorescence techniques were used to compare the distribution of lymphocyte subpopulations of tonsil and peripheral blood from patients with rheumatic heart disease and age and socioeconomically matched patients undergoing tonsillectomy for chronic recurrent tonsillitis, but who had no evidence of rheumatic fever or rheumatic heart disease. The proportions of B cells (BA-1+), total T cells (Lyt-3), inducer/helper T cells (T4+) and cytotoxic/suppressor T cells (T8) were determined. No significant differences were apparent between rheumatic heart disease and control groups in resting cells from tonsils or blood. Cells undergoing proliferation in response to streptococcal blastogen A were identified by similar techniques. These tonsillar preparations from patients with rheumatic heart disease generated a smaller proportion of T8+ cultured cells and a greater T4/T8 ratio of cultured cells in response to group A streptococcal blastogen A than did nonrheumatic subjects.

Compartmentalization of cells bearing "rheumatic" cell surface antigens in peripheral blood and tonsils in rheumatic heart disease.

Monoclonal antibodies that recognize "rheumatic" antigens of peripheral blood non-T cells were used to study the compartmentalization of such cells in peripheral blood and tonsils of individuals with rheumatic heart disease (RHD) and suitable control subjects. The peripheral blood of most (71%) of the 42 individuals with RHD contained cells reacting with monoclonal antibody 83S19.23 or 256S.10, whereas these cells were present in only 17% of the 41 control subjects (P less than .02). However, none of 21 individuals with RHD had such cells in their tonsils, although they were present in the tonsils of 50% of the 40 control subjects (P less than .03). These results may reflect a failure in RHD or organ-specific homing of cells with the epitopes recognized by the antibodies. The presence of these cells in tonsils may be important in the immune response to streptococcal pharyngeal infection, and their absence in RHD may be involved in the unusual immune responses characteristic of this disease.

Interference with granulocyte function by Staphylococcus epidermidis slime.

The interaction of Staphylococcus epidermidis slime with human neutrophils (PMN) was examined by using isolated slime and allowing bacteria to elaborate slime and other extracellular products in situ. S. epidermidis slime was found to contain a chemoattractant. Incubation of PMN with 50 micrograms or more of slime per ml inhibited subsequent chemotaxis of the PMN to n-formyl-methionyl-leucyl-phenylalanine by 27% and to zymosan-activated serum by 44 to 67% with increasing slime concentrations. S. epidermidis slime stimulated little degranulation of untreated PMN. After pretreatment of PMN with 5 micrograms of cytochalasin b per ml, slime predominantly induced release of specific granule contents (33.8% lactoferrin release by 250 micrograms of slime per ml versus 10% myeloperoxidase release by 250 micrograms of slime per ml). By a surface phagocytosis assay, PMN uptake of radiolabeled S. epidermidis which were incubated for 18 h on a plastic surface for slime expression was less than that for S. epidermidis adhered to the plastic for 2 h or grown in unsupplemented nutrient broth. These results suggest that S. epidermidis slime interaction with PMN may be potentially detrimental to host defense and may contribute to the ability of this organism to persist on surfaces of foreign bodies in the vascular or central nervous system.

Enzyme-linked immunosorbent assay for identification and measurement of antibodies to group A streptococcal bacteriophage.

A sensitive enzyme immunoassay (ELISA) was developed to identify and measure antibodies to group A streptococcal bacteriophage hyaluronidase. With a purified preparation of bacteriophage hyaluronidase as the solid-phase antigen, the ELISA was shown to be as specific as and more sensitive than the standard bacteriophage neutralization test for measurement of antibody to bacteriophage. In rabbits immunized with bacteriophage, the ELISA detected antibody earlier than the neutralization assay (7 vs. 11 days) and was able to distinguish IgG and IgM class antibodies. A strong correlation was demonstrated between antibody titers measured by ELISA and bacteriophage neutralization (r = 0.88; P less than 0.001). Preliminary data using the ELISA, modified to measure human antibody to bacteriophage hyaluronidase, indicated that an antibody response of both IgG and IgM classes occurred in humans after group A streptococcal infection. This ELISA provided a sensitive method for detection and measurement of antibody to a specific bacteriophage antigen, which will be useful in the investigation of the role of bacteriophage in the pathogenesis of group A streptococcal infections.

Investigation on extracellular slime substance produced by Staphylococcus epidermidis.

The extracellular slime substance produced by Staphylococcus epidermidis was investigated. Slime production was assessed by bacterial agglutination in the presence of concanavalin A (Con A) or poly-L-lysine and by bacterial adherence to polyethylene. Media for slime production was optimized using these criteria. A phenol-saline extract of crude slime was separated into four fractions on a DEAE-sepharose column. Total protein and sugar content and the monosaccharide constituents were determined. Crude slime and the phenol-saline extract showed a strong precipitation reaction with Con A and poly-L-lysine (double diffusion). Fractions I and II containing mannose as the most abundant sugar reacted with Con A and two other mannose-specific lectins (Lens culinaris, Pisum sativum). This reaction could be inhibited by mannose. Fractions III and IV were precipitated by poly-L-lysine, probably due to a reaction with glucuronic acid which was only present in these fractions. Precoating of polyethylene with crude slime, phenol-saline extract and fractions III and IV resulted in a marked inhibition of attachment of staphylococcal cells. Production of the extracellular slime substance was completely inhibited by subinhibitory concentrations of the glycosylation inhibitor tunicamycin, whereas penicillin had no influence. Extracellular slime substance produced by S. epidermidis seems to be a complex of glycoconjugate character and plays an important role in the attachment to synthetic polymers. The production of slime by staphylococci can be easily determined using mannose specific lectins and poly-L-lysine.

Effect of extracellular slime substance from Staphylococcus epidermidis on the human cellular immune response.

Staphylococcus epidermidis infection of plastic catheters is often associated with heavy deposits of slime. To test whether this slime affects the human cellular immune response, its effect on the lympho-proliferative response of mononuclear cells to polyclonal stimulators was measured. Slime drastically reduces this response. Its inhibitory action was not immediate but took place over a few days and resulted in destruction of affected cells. The effect is dose related. This inhibition of cellular response may contribute to S epidermidis infection of implanted prostheses.

Functional alterations in non-T cells in rheumatic heart disease.

The mediation of the T cell lymphoproliferative response to streptococcal blastogen A by non-T mononuclear cells was studied in patients with rheumatic heart disease (RHD) and control subjects. Non-T cells are essential for T cell response to blastogen A. Non-T cells from RHD patients were less effective in enabling the T lymphocyte response to blastogen A than control non-T cells though no consistent difference was observed in the response to phytohaemagglutinin. The results suggest that a functional alteration is present in the non-T cells from RHD patients which might be related to the pathogenesis of the disease.

Measurement of immunoglobulin G and M antibodies to type 3 pneumococcal capsular polysaccharide by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay was developed to measure immunoglobulin G (IgG) and IgM type 3 antipneumococcal capsular polysaccharide antibodies. The use of Fab2 fragments of rabbit antipneumococcal IgG antibody in the antibody-antigen sandwich increased the sensitivity for measuring IgM antibodies and decreased background activity in antigen-free cuvettes. This methodology detected type 3 IgM antibody responses in six of six subjects vaccinated with polyvalent pneumococcal vaccine and detected type 3 IgG antibody responses in three subjects. Results of the enzyme-linked immunosorbent assay and radioimmunoassay procedures were concordant, and postvaccination enzyme-linked immunosorbent assay IgM titers showed a stronger correlation with total radioimmunoassay antibody than did postvaccination ELISA IgG titers.

Characterization of the human cellular immune response to purified group A streptococcal blastogen A1.

The human mononuclear cell response to purified extracellular streptococcal protein, blastogen A, was compared to the response of these cells to PHA and tetanus antigen. Blastogen A induced peak uptake of thymidine during day 6 of tissue culture whereas PHA induced peak uptake during day 5 and tetanus during day 8. Like PHA, blastogen A transformed human umbilical cord lymphocytes and those of nonimmune animals. Also like PHA, blastogen A transformed primarily T lymphocytes. However, unlike PHA, the ability of T lymphocytes to respond to blastogen A was almost completely dependent on the presence of viable non-T lymphocytes. Monocytes were not as effective in facilitating the response to blastogen A as they were for PHA. Thus, blastogen A behaves most like a polyclonal T lymphocyte mitogen, although the degree of dependence of the transformation response on the presence of non-T lymphocytes is much greater than that of PHA.

Cellular immune responses to extracellular streptococcal products in rheumatic heart disease.

The lymphocyte transformation responses to purified preparations of two extracellular products of group A streptococci (blastogen A and nuclease B), to phytohemagglutinin, and to Candida albicans antigen were measured in tonsillar and peripheral blood lymphocytes from patients with rheumatic heart disease (RHD) and suitably matched nonrheumatic (control) subjects. The mean phytohemagglutinin dose responses of tonsillar and peripheral lymphocytes from RHD patients were essentially indistinguishable from those of controls. In contrast, the responses of tonsillar and peripheral blood lymphocytes to the two extracellular products of group A streptococci were significantly lower in RHD patients than in nonrheumatic control subjects. Candida antigen produced very little stimulation of lymphocytes in any of the subjects. The geometric means of antibody levels against streptolysin O, nuclease B, and nicotinamide adenine dinucleotidase showed no consistent differences between the control group and the group of RHD subjects. Group A streptococci were isolated from the tonsils of approximately 25% of both groups of subjects. The RHD patients clearly had a depressed cellular immune response to the two purified streptococcal extracellular antigens. The equal frequency in recovery of group A streptococci from tonsils and the absence of consistent difference in titers of humoral antibodies to streptococcal extracellular antigens, particularly nuclease B, suggest that this differential response is not due to a lower level of stimulation by repeated exposure to group A streptococcal products.