Determination of antibody from typhoid patients against lipopolysaccharide and protein antigens of Salmonella typhi.

Although the Widal test is simple, inexpensive and the most widely used for serodiagnosis of typhoid fever, the sensitivity and specificity of the test is sometimes doubtful. In this study, an enzyme-linked immunosorbent assay (ELISA) was developed for the detection of serum IgG and IgM antibodies to protein and lipopolysaccharide (LPS) antigens of Salmonella typhi which was compared with the Widal test in various groups of subjects. In typhoid patients with hemocultures positive for S. typhi (TP group), ELISA positivity was found on 100% for IgG antiprotein, 94.44% for IgG anti-LPS and 88.89% for IgM to both the protein and LPS antigens. In contrast, the Widal test was positive in only 61.11% for anti-O and 83.33% for anti-H antibodies. In healthy control subjects (HC group), only 5% of serum samples were positive for IgG anti-protein and none was positive for IgG anti-LPS or IgM to either the protein or LPS. In contrast, the Widal test was positive in 7.5% of HC group for anti-O and 17.5% for anti-H antibodies. In blood bank donors (BB group), both ELISA and Widal tests were positive in 23-40% of sera. Since the hospital records of BB group were incomplete. It might be possible that some of these subjects had recently been infected with S. typhi. Our data indicate that the standard Widal test was associated with false negative reactions in 16-39% of blood culture positive subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Separation of human suppressor and helper T cells by concanavalin A-coated sheep erythrocytes.

Normal human peripheral blood mononuclear leukocytes (PBML) were activated by concanavalin A (Con A). Con A-activated and non-activated T cells were separated by E (AET) rosettes (2-aminoethylisothiouronium hydrobromide treated sheep erythrocyte rosettes). Purified T cells were rosetted with Con A-coated sheep red blood cells (Con A-SRBC) at 37 degrees C resulting in Con A-SRBC rosetted and non-rosetted T cells. The Con A-SRBC rosetted T lymphocytes in the T lymphocytes from Con A-activated and non-activated PBML were 44.4 +/- 5.4 percent and 16.0 +/- 7.5 percent (Mean +/- S.D.) while the Con A-SRBC non-rosetted T lymphocytes were 55.6 +/- 5.4 percent and 84.0 +/- 7.5 percent respectively. The Con A-SRBC rosetted and non-rosetted T cells were separated by Ficoll-Hypaque gradient centrifugation. Functional studies of Con A-SRBC rosetted and non-rosetted T cells were performed by in vitro tests using pre-amplified reverse hemolytic plaque assay for measuring numbers of immunoglobulin G (IgG) secreting cells and ELISA quantitation of IgG concentration. Both techniques were used to assess the suppressor and helper functions of the Con A-SRBC rosetted and non-rosetted T cells. The Con A-SRBC rosetted cells obtained from T cells of Con A-activated PBML showed strong suppressor activities to normal PBML in both pre-amplified reverse hemolytic plaque assay and sandwidh ELISA of IgG concentration, while the Con A-SRBC non-rosetted T cells demonstrated strong helper activities to normal PBML in both assay systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Agar plating technique for enumeration of IL-2 producing cells in human peripheral blood mononuclear leukocytes.

An agar plating technique for detection and enumeration of IL-2 producing cells from human peripheral blood mononuclear leukocytes (PBML) has been developed. This method is based on the principle that PHA-stimulated PBML, as effector cells, secrete interleukin 2 (IL-2) into soft agar containing mouse 3-day Con A blasts as IL-2 dependent responder cells. The IL-2 dependent Con A blasts proliferating around the IL-2 producing cells form colonies or clusters of cells and are easily visualized under a dissecting microscope. The development of IL-2 producing cells was optimum when 1 X 10(6) cells/ml PBML were stimulated with 2 micrograms/ml PHA-P for 4 hours, and when 2.5 X 10(5) cells were co-cultured with 6 X 10(6) Con A blasts in soft agar for 5 days. The average number of IL-2 producing cells in 10 normal healthy controls were 754 +/- 94 (mean +/- S.E.M.) cells/10(6) PBML. The numbers of IL-2 producing cells and the levels of IL-2 produced were highly correlated (r = 0.929). The subpopulation of lymphocytes in the colonies was shown to be mostly murine T-cells, since they were mostly Thy 1.2 positive, CD3 negative and surface immunoglobulin negative. This technique is very simple to perform and provides an accurate and straightforward means to enumerate IL-2 producing cells from human PBML in a variety of human immunologic disorders.

Enumeration of interleukin 2-producing cells from rat spleen.

A method for the enumeration of IL2-producing cells from rat spleen has been developed. Rat spleen cells were stimulated with concanavalin A (Con A), washed, then mixed with IL2-dependent cells (3 day Con A blasts) and plated in soft agar. Clusters of IL2-dependent cells formed around IL2-producing cells, giving colonies which were easy to count under a dissecting microscope. All experimental factors influencing development of colonies of IL2-producing cells surrounded by IL2-dependent cells were evaluated and set up. Optimum number of effector cells was 2.5 x 10(5) cells/culture, while optimum number of IL2-dependent cells was 6 x 10(6) cells/culture. Optimum concentration of Con A for IL2 stimulation was 40 micrograms/ml with an optimal stimulation time of 10 hours. Optimum incubation time for development of IL2-producing cell colonies was 5 days. The number of IL2-producing cells by this technique had a good correlation with the level of IL2 in the cell culture fluid (r = 0.885). When colonies were aspirated from agar and stained by Wright stain, a big purple stained cell at the center was surrounded by small cells in almost all colonies examined. All cells from colonies were fluoresed with anti-mouse Thy 1.2-fluorescein conjugate. However, they were negative with anti-mouse Ig-fluorescein conjugate. The number of IL2-producing cells was 816-2080 cells/10(6) of rat spleen cells with mean +/- S.E.M. = 1404 +/- 154/10(6) cells.

Regulation of immunoglobulin secretion by T lymphocytes in human malaria.

In vitro studies were carried out on the nature of immunoglobulin synthesis and secretion by peripheral blood mononuclear leukocytes (PBMLs) and on the function of T and B cells from malaria patients. The mean values of secreted IgG and IgM concentrations of 22 malaria patient PMBLs were significantly higher than those of 20 normal PBMLs. When the suppressor T cell activity and the function of B cells in response to suppressor T cells were assayed by the cell co-culture technique, it was found that there was a decrease in suppressor T cell activity and the B cell function in response to normal suppressor T cells in malaria patients. The defects of these T and B cell functions may play some role in the immunological abnormalities seen in some malaria patients.

Lymphocyte transformation in lepromatous leprosy: a study of the influence of disease activity and symptom duration.

The lymphocyte hyporesponsiveness to M. leprae of patients with active lepromatous leprosy has been well described. This immune defect is less well understood in terms of its time of origin, possible reversibility and specificity. To further examine the persistence and specificity of this abnormality, lymphocyte transformation tests of 93 leprosy patients to lepromin, BCG and PHA were studied. Among lepromatous patients, a decreased response to M. leprae was seen, whether the disease was active or inactive. Decreased responses to BCG were found in lepromatous patients with active disease, but not in those with inactive disease. The duration of patient symptoms was not associated with differences in LTT responses among the active lepromatous patients.