Morphological stabilization of the glycocalyces of 23 strains of five Bacteroides species using specific antisera

Cells of five Bacteroides species were examined following treatment with homologous antisera and staining with ruthenium red. They were enveloped by glycocalyces and these extensive fibrous exopolysaccharide matrices were fully retained as an integral "capsule" by some cells, while other cells showed "capsule" as well as detached glycocalyx components forming an intercellular "slime." These extensive glycocalyces collapsed during dehydration for electron microscopy and formed electron-dense accretions on cell surfaces and electron-dense reticula in intercellular spaces when the cells were treated with heterologous antiserum or when antibody stabilization was omitted. The glycocalyces of all strains, both stabilized and unstabilized, were observed outside the outer membranes of cell walls that showed the "classic" gram-negative structural organization. Appropriate modifications of the indirect fluorescent antibody test demonstrated an integral "capsule" on all strains examined; detached glycocalyx and varying amounts of slime were demonstrated after stabilization with homologous, but not heterologous, antiserum.

Detection of the carrier state for classic hemophilia using an enzyme- linked immunosorbent assay (ELISA)

A high proportion of carriers of classic hemophilia can be identified in the laboratory because, in comparison to normal women, the concentration of antigens related to antihemophilic factor (AHF, factor VIII) that are detected in their plasma by heterologous antiserum (factor VIIIR:Ag) is relatively higher than the titer of AHF that is measured in clotting assays (factor VIII:C). Enzyme-linked immunosorbent assay (ELISA) appears to overcome some of the technical difficulties associated with measurement of AHF-like antigens. The results of ELISA correlated closely with those obtained by semiquantitative immunoelectrophoresis, except in patients with von Willebrand's disease. In which ELISA appeared to provide a more quantitative estimate of AHF-like antigen. Utilizing the ELISA technique and a revised method of logarithmic discriminant analysis, we were able to distinguish all of 37 obligate carriers of hemophilia at the level of certainty that would have misclassified 5% of normal women as carriers. The relative simplicity of ELISA suggests its utility in the diagnosis of the carrier state in the female relatives of hemophiliacs.

Detection of the carrier state for classic hemophilia using an enzyme- linked immunosorbent assay (ELISA)

A high proportion of carriers of classic hemophilia can be identified in the laboratory because, in comparison to normal women, the concentration of antigens related to antihemophilic factor (AHF, factor VIII) that are detected in their plasma by heterologous antiserum (factor VIIIR:Ag) is relatively higher than the titer of AHF that is measured in clotting assays (factor VIII:C). Enzyme-linked immunosorbent assay (ELISA) appears to overcome some of the technical difficulties associated with measurement of AHF-like antigens. The results of ELISA correlated closely with those obtained by semiquantitative immunoelectrophoresis, except in patients with von Willebrand's disease. In which ELISA appeared to provide a more quantitative estimate of AHF-like antigen. Utilizing the ELISA technique and a revised method of logarithmic discriminant analysis, we were able to distinguish all of 37 obligate carriers of hemophilia at the level of certainty that would have misclassified 5% of normal women as carriers. The relative simplicity of ELISA suggests its utility in the diagnosis of the carrier state in the female relatives of hemophiliacs.

Physiological Effects of Nonimmune Platelet Associated Immunoglobulin G

SummaryThe function of nonimmune IgG associated with platelets is unknown. In a series of experiments we have investigated this problem, relating amount of platelet-associated IgG (PAIgG) to platelet volume, serotonin release, adherence of platelets to monocytes and platelet senescence. Most of these studies were performed with human platelets. Platelets freed of preexisting PAIgG by incubation at 22° C were incubated with IgG in a series of concentrations ranging from 0.4 — 27.0 X10-6 M. The IgG preparations used were demonstrably free of aggregated forms of the protein. The amount of PAIgG bound to platelets was determined by the use of fluorescein isothiocyanate-conjugated anti-IgG antibody (F-anti-IgG antibody) which was quantified in a fluorospectrophotometer. Newly bound IgG was assayed similarly by the use of F-IgG. A dose-dependent increase in platelet volume was associated with the binding of nonimmune IgG by platelets. The process which leveled off at an IgG concentration of 1.2 —1.5 X10-5 M was almost fully reversible and was not due to platelet shape change or aggregation. Release of serotonin from IgG-treated platelets was relatively small but to the extent that it occurred was positively related to the IgG concentration to which platelets were exposed. Adherence to autologous monocytes studied quantitatively by the use of formaldehyde-fixed cells was also positively related to the amount of IgG on the platelets. Normal or IgG-defident serum had a potent inhibitory (noncompetitive) action on the binding of F-IgG and F-anti-human IgG antibody to human platelets. Cohorts of platelets prepared in rabbits during the recovery phase of immunological thrombocytopenia induced by injection of heterologous antiserum, showed an age-dependent increase of PAIgG and of IgG binding. These results suggest that PAIgG plays a role in the clearance of senescent platelets.

Immunoradiometric Assay Of VIII:CAG, A Potential Tool To Detect Human Anti-VIII:C Antibodies

A modification of a two-site, solid-phase immunoradiometric assay (IRMA) for Factor VIII coagulant antigen (VIII: CAg) has been evaluated for its potential to detect antibodies against Factor VIII coagulant activity (VIII: C) in patient plasma samples. For this purpose the assay system comprised four steps: 1) coating of test tubes with human anti —VIII:C , 2) incubation with normal Factor VIII— VWF complex, 3) incubation with test sample, and 4) binding of radiolabeled human anti–VIII: C as marker.Of eight hemophilic plasma samples containing antibodies against VIII: C (as detected in a clotting assay) five were able to prevent binding of radiolabel partially and three prevented this completely. One of these three (which actually was the antibody used in the IRMA), was effective even at very high dilutions. Two hemophilic plasma samples without detectable antibodies in the clotting assay, a severe Von Willebrand’s disease plasma and normal plasma samples showed no significant interference with binding of radiolabeled human anti-VIII: C. Also, a plasma sample containing a high titer of spontaneous human antibody to VIII: C as well as a heterologous antiserum against Factor VIII—VWF complex did not interfere with binding of radio-activity.It is concluded that the test system described is a sensitive tool to detect antibodies of the same specificity as those used in the IRMA. It may also detect antibodies of differing specificity. The lack of crossreactivity with some antibodies points to interindividual differences in specificity of antibodies against VIII: C.

Role of pili in adherence of Pseudomonas aeruginosa to mammalian buccal epithelial cells

Adherence of Pseudomonas aeruginosa organisms to the upper respiratory epithelium of seriously ill patients in vitro is correlated with subsequent colonization of the respiratory tract by this opportunistic pathogen. The role of pili in the attachment to epithelial cells of P. aeruginosa was studied in an in vitro system employing human buccal epithelial cells and P. aeruginosa pretreated by various means. Pretreatment of the bacteria with proteases, heat, or Formalin caused a significant decrease in adherence. A decrease when compared with controls was also noted in the adherence of P. aeruginosa organisms to buccal epithelial cells preincubated with purified pili prepared from the strain used for adherence testing; however, pili prepared from a heterologous strain failed to block adherence. Similar results were obtained in serological studies when antisera to purified pili prepared from the strain used for adherence testing decreased adherence, whereas heterologous antiserum to pili did not decrease adherence. From these results it appears that pili mediate the adherence of P. aeruginosa organisms to human buccal epithelial cells.