Biomedical subjects
Publications and source records attributed to M E Klegerman.
Three substrains of Mycobacterium bovis BCG which appeared to differ in biological activity could not be differentiated on the basis of fatty acids less than C20, but could be differentiated from six nonpathogenic mycobacterial species.
The ATP content of viable cells in a single lot of freeze-dried Tice-substrain Mycobacterium bovis-BCG vaccine was determined after washing of organisms with isotonic buffer, using four extraction methods: boiling in 0.2 M potassium phosphate buffer at pH 7.4 for 12 min; boiling in 0.1 M Tris-EDTA buffer at pH 7.75 for 2 min; n-butanol/1.9% sodium glutamate-0.01 M sodium arsenate buffer, pH 7.0; and n-butanol/0.01 M Tris-EDTA buffer, pH 7.0. Liberated ATP was assayed with a Lumac biocounter by integrated measurement of light produced by firefly luciferin/luciferase. The dose response of internal standards paralleled that of ATP standards in water, and the response of endogenous ATP in BCG was not significantly different from a composite linear internal standard curve above 10 pg ATP (corresponding to about 10(5) viable organisms per ml), the sensitivity of the assay. When the ATP content of BCG was calculated from the composite curve, the n-butanol/Tris-EDTA method was found to be the most precise (CV less than 10%). Butanol extraction procedures were about twice as efficient as boiling methods and yielded an ATP value of about 3.4 fg/CFU, similar to ATP/CFU factors previously reported for other BCG substrains. However, when results were corrected for quench and ATP recovery, which varied with extraction method, the conversion factor increased nearly threefold.
The zeta potential of three lots of Tice substrain BCG organisms was measured over a pH range of 2.0 to 11.0 at low electrolyte concentration. For two lots, the cells were cationic at pH 4.2-4.4 and anionic above this isoelectric point. Washing the cells twice with water lowered the isoelectric point to 2.7. The cationic/anionic profile was retained in all three lots, although the third lot had an isoelectric point of 3.0 initially. Cells of the Glaxo substrain, on the other hand, were anionic over the entire pH range and are evidently unaffected by the washing process. It appears that cells of the Glaxo strain have only electronegative phosphate groups at their surface whereas the Tice substrain may possess a loosely adhering cell-surface protein.
A model 447 Coulomatic K-F titrimeter was used to determine the water content of seventeen lots of freeze-dried Tice-substrain BCG vaccine. The results were compared with corresponding moisture contents determined by a standard gravimetric method at the time of manufacture. The advantages of the titrimetric method include simplicity, rapidity, convenience, sensitivity, reproducibility and specificity, whereas the gravimetric method is tedious and time-consuming. Although moisture content determined by the K-F titrimeter tended to be higher than that determined by the gravimetric method, the results correlated significantly (r = 0.882, P less than 10(-5]. Alteration of national and international regulations to permit use of the K-F titrimeter is recommended.
The zeta potential of cells of an attenuated vaccine Mycobacterium bovis, BCG, Tice substrain, was measured over a pH range of 1.5-11.0 at low electrolyte concentration. There was a marked electro-positive charge at low pH, the zero charge point, pH 4.4, being similar for viable and heat killed vaccine. At pH 6.5 the addition of sodium dodecyl sulphate or sodium tauroglycolate made little difference to the zeta potential. However, progressive addition of cetylpyridinium chloride reduced and finally reversed the charge to a maximum of +60 mV at a concentration of 1.5 x 10(-2) mol dm-3 surfactant. Higher concentrations reduced the charge although it remained positive. The nature of the adsorbing species on the cell surface is discussed since the Tice substrain has both cationic and anionic surface charges whereas the Glaxo strain is reported to only possess electro-negative phosphate surface groups. Some hydrophobic interaction involving lipid within the surface may also be involved.
We determined the serum concentration of human IgG antibody to the native capsular polysaccharide of group B Streptococcus (GBS) type III needed to passively protect mice against lethal homologous challenge. Antibody was measured by an ELISA, standardized by two methods, and corrected for nonprecipitating antibody. A concentration of 1.3 micrograms of IgG antibody to GBS type III/ml protected 126 (97%) of 130 mice from an 80%-96% lethal dose bacterial challenge. Concentrations of IgG antibody to GBS type III in sera from 42 infected infants were less than or equal to 0.3 micrograms/ml. Concentrations of antibody ranged from less than 0.02 to 21.7 micrograms/ml in sera from 102 unselected pregnant women (median, 0.05 microgram/ml); 13% had concentrations greater than or equal to 1.3 microgram/ml. Levels in 25 women colonized with GBS type III who gave birth to normal infants were significantly higher and ranged from 0.1 to 10.7 microgram/ml (median, 0.78 micrograms/ml). In a study of transplacental passage of antibody, protective levels were found in a number of infants with gestational ages between 28 and 36 weeks.
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Strain differences have been postulated to explain the observation that group B Streptococcus type III (GBS III) late-onset disease occurs in only a fraction of colonized infants. To determine the distribution of type-specific polysaccharide antigen (Ag) in GBS III, Ag was measured by rocket immunoelectrophoresis in both supernatant fluids and EDTA extracts and by radial immunodiffusion in multiple HCl extracts of the pellet from cultures of 10 strains of GBS III. Capsular Ag was defined as the sum of Ag in EDTA extracts + Ag in multiple HCl extracts. Both Ag in EDTA extracts and Ag in supernatant fluids correlated with capsular Ag (r = 0.94). GBS III strains were obtained from the blood of 19 infants with late-onset sepsis, from the cerebrospinal fluid or blood of 22 infants with late-onset meningitis, and from mucosal surfaces of both 18 infants and 12 mothers of infants with low levels of type-specific antibody and asymptomatic colonization. Mean values of Ag in supernatant fluids in strains from infants with late-onset sepsis (1.50 +/- 0.08 micrograms/ml) and late-onset meningitis (1.67 +/- 0.09 micrograms/ml) were significantly greater than those in asymptomatic colonization strains (1.14 +/- 0.05 micrograms/ml; P less than 0.001). The number of organisms required for a 50% lethal dose in the chick embryo, determined in 29 strains, was inversely related to Ag in supernatant fluids (r = -0.60). The demonstration that the quantity of capsular Ag produced by GBS III strains is related to their virulence in chick embryos and to their invasiveness in susceptible infants supports the hypothesis that Ag is a virulence factor in humans.
We studied the concentration of circulating human immunoglobulin G (IgG) antibody to the native capsular polysaccharide of group B streptococcus (GBS) type Ib necessary to protect mice against lethal challenge by laboratory and clinical GBS Ib strains. Antibody was measured by an enzyme-linked immunosorbent assay in which native polysaccharide antigen coupled to human serum albumin was used. The assay was standardized by a quantitative precipitation test, using native antigen and specific human IgG antibody purified by affinity chromatography. IgG anti-GBS Ib antibody level-protection curves for 90% lethal dose challenge of mice were sigmoidal. The curves of whole serum and affinity-chromatographed IgG anti-GBS Ib were superimposable. The serum concentrations of human antibody required for complete protection of mice varied with the infecting strain and ranged from 0.038 to 0.175 microgram/ml. Protective levels of human IgG anti-GBS Ib were lower than those we found previously for homologous protection against GBS Ia challenge (range, 0.25 to 1.0 microgram/ml).
Human IgG antibody to the type-specific polysaccharide antigen of group B Streptococcus type Ia in the sera of mice was measured by enzyme-linked immunosorbent assay, previously standardized by quantitative precipitation, 24 hr after passive immunization with human serum or affinity-chromatographed antibody. The concentrations of antibody needed to protect mice against 90% lethal dose challenge varied with the bacterial inoculum and ranged from 0.25 to 1 microgram/ml using five strains of group B Streptococcus type Ia. Affinity-chromatographed antibody gave results comparable to serum, indicating the specificity of the antibody and the absence of other humoral factors in protection with this serum. Sera from 11 infected infants and their mothers had concentrations of antibody of less than or equal to 0.17 micrograms/ml, below the protective level delineated in the experimental model. Twelve percent of 50 adult women and 36% of 25 women colonized with group B Streptococcus type Ia had antibody levels of greater than or equal to 1 microgram/ml.
An ELISA was developed to measure human IgG antibody to the native polysaccharide antigen of GBS serotype Ia. Because the polysaccharide binds poorly to polyvinyl chloride, its adherence was enhanced by activation with cyanogen bromide and coupling to HSA in a molar ratio of polysaccharide to albumin of 1:4.5. There was minimal loss of sialic acid during coupling, and the coupled antigen showed identity with uncoupled native antigen by Ouchterlony analysis. OD values obtained by ELISA showed a log-linear relation to concentration of specific antibody in whole and affinity-chromatographed human sera measured by quantitative precipitation over a range of 0.25 to 3.5 microgram/ml. In replicate ELISA experiments using serially diluted human serum, dilutions with antibody content as low as 0.016 microgram/ml could be reliably differentiated from PBS or agammaglobulinemic serum. The concentration of antibody in 98 selected human sera measured by ELISA correlated well (r = 0.89, p less than 0.001) with results obtained by indirect IF assay. This quantitative ELISA for GBS antibody is rapid, convenient, economical, and suitable for routine use.
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A case of Behcet syndrome with immunologic evaluation, including screening of a vulvar ulcer for IgG, IgM, IgA, and fibrinogen by direct fluorescent microscopy is presented. Attempts were made to demonstrate cellular and humoral immune responses to mucosal antigens by lymphoblast transformation in the presence of cadaver esophageal mucosal extracts and indirect immunofluorescence using autologous serum and mucosal tissue. Serial measurements of percentages of total T, active T, and B lymphocyte populations, and lymphocyte response to phytohemagglutinin (PHA) stimulation during the course of Behcet syndrome are also presented. Clinical evaluation, histology of a Behcet vulvar ulcer, and a 2-year followup with good response to chlorambucil are reviewed.
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A quantitative in vivo assay for BCG anticancer efficacy was developed to maximize detection of tumor-antagonistic mechanisms. Cultured S180 sarcoma cells admixed with various quantities of Mycobacterium bovis-BCG organisms were injected subcutaneously into CFW Swiss-Webster mice and response was measured as tumor incidence 14 days after injection. Assay of various BCG substrains, lots and killed preparations revealed characteristic patterns of BCG dose-dependent tumor inhibition and enhancement that suggest the existence in the vaccine of multiple active components, the relative concentrations of which vary among cultures. Inhibition of tumor growth by high doses of BCG (greater than 10 micrograms dry weight) was found to be a function of total cell mass and not of the bacterial viability, suggesting that this activity is dependent upon one or more heat-stable components.
A purified hot-water extract from Mycobacterium bovis (BCG vaccine) has been found to have significant antitumor activity against a murine sarcoma in vivo, but not in vitro, suggesting that the active compound is behaving as an immunostimulant. The material, termed PS1, has an average molecular weight of 22.4 kDa, is freely soluble in water, but has low solubility in acetone or ethanol, and is remarkably heat-stable, as is the parent BCG vaccine in terms of high-dose antitumor activity. PS1 contains at least 50% carbohydrate, consisting mainly of glucose, galactose and mannose, and about 10% lipid that may correspond to phosphatidylinositol. It shares chemical and biological properties with an arabinomannan isolated from M. tuberculosis, but it contains only trace quantities of lipoarabinomannan (LAM). Crossed immunoelectrophoresis indicated that PS1 contains the mycobacterial antigen 89, but only a single, non-migrating precipitin arc appeared on immunoelectrophoresis against a standard anti-BCG serum. PS1 appears to be non-toxic in mice up to a dose of 5 mg/kg, while as little as 70 micrograms/kg is sufficient to inhibit tumor formation significantly.