Antibody and DNA probes for detection of nitrite reductase in seawater.

A polyclonal antiserum was produced by immunization with nitrite reductase (NiR) purified from Pseudomonas stutzeri (ATCC 14405) and tested for specificity among known denitrifying strains. The antiserum was nearly strain-specific, identifying NiR only in some, but not all, other P. stutzeri strains. Denitrifying isolates from water column and sediment environments were also screened; several isolates from an intertidal microbial mat reacted with the NiR antiserum. Activity assays for NiR in polyacrylamide gels demonstrated that strains with apparently very similar NiR proteins did not react with the antiserum. These results imply that the NiR protein is more variable even among closely related strains than previously suspected. A DNA probe for a 721 bp region of the NiR structural gene was obtained by PCR amplification of P. stutzeri (ATCC 14405) DNA and used to screen denitrifying strains and isolates. The probe hybridized with a greater variety of strains than did the antiserum, implying that the DNA probe may be a more broadly useful and functional probe in environmental samples, whilst the NiR antiserum is nearly strain- or, at most, species-specific. Limits for detection of the enzyme and gene in seawater were estimated and NiR DNA was detected in DNA extracted from natural seawater. The hybridization data imply that in the order of 1-10 in 1000 cells in natural seawater possess homology with the NiR gene probe.

Antibodies to lymphocyte membrane preparations in systemic lupus erythematosus.

IgG and IgM antilymphocyte antibodies were determined in sera from patients with systemic lupus erythematosus (SLE) by an ELISA method using isolated lymphocyte plasma membranes as antigens. Lymphocyte membranes prepared from T or B cell lines or normal peripheral blood showed a high degree of purity by enzyme assay and electron microscopic monitoring. Sera from patients with active SLE showed high IgG and IgM ELISA reactivity against T and B cell plasma membranes compared with normal sera. IgM antibodies to lymphocyte membranes showed a correlation with microcytotoxicity results. Serial studies showed a relationship of antimembrane antibody to disease activity in some patients, but not in others. The ELISA method for antilymphocyte antibody assay provides a sensitive technique for monitoring reactivity against antigens retained in isolated lymphocyte membrane preparations.

Alterations in lymphocyte cell surface markers during various human infections.

Peripheral blood lymphocyte cell surface markers were studied in 146 patients with various forms of acute infection using B cell identification with antisurface immunoglobulin and T cell subset enumeration with hybridoma T cell subpopulation reagents. Significant depression was recorded in total numbers of T cells and T cell helper-inducer and suppressor-cytotoxic subsets in pneumonia, acute pyelonephritis, and severe generalized sepsis. In addition, proportions of T cells being the OKT4 helper-inducer phenotype were reduced only in patients over the age of 60 with pneumonia or sepsis. Patients with severe infection frequently had multiple T cell phenotypic surface marker abnormalities. In some instances, when depressions of total T cell numbers as well as respective helper-inducer or suppressor-cytotoxic T cells were noted in the face of generalized sepsis, lack of improvement in these abnormalities during the course of treatment was associated with rapid clinical deterioration and death. On the contrary, in patients with a successful response to appropriate therapy, initial depressions of total T cell numbers and subsets improved progressively with clinical resolution of sepsis and illness.

Lymphocytes binding C-reactive protein during acute rheumatic fever.

Lymphocytes binding C-reactive protein (CRP) were studied in 31 patients with acute rheumatic fever and 30 controls who were children. Marked elevations in both proportions and absolute numbers of CRP-binding lymphocytes were recorded in rheumatic fever (P less than 0.001). No clear correlation was noted between plasma CRP as quantitated by radioimmunoassay and proportions or numbers of CRP-binding cells. Double-labeling experiments indicated that 60-80% of CRP-binding lymphocytes also showed Fc receptors reacting with fluorescein-conjugated IgG aggregates. Passage of lymphocytes over Ig--anti-IgG columns, removed cells bearing surface Ig but not CRP-binding lymphocytes. Studies of T-cell subpopulations indicated no overlap between Tmicron- and CRP-binding cells; however about half of Tgamma-cells showed concurrent CRP binding. "Active" T-cell rosetting cells did not bind CRP. A 12-15-h incubation of lymphocytes at 37 degrees C in 5% CO2-air showed persistence of CRP binding in substantial proportions of cells particularly in acute rheumatic fever. CRP-binding lymphocytes may represent a marker for immunologically committed cells in acute rheumatic fever.