Peripheral blood T-lymphocytes of patients with chronic B-cell lympholeukemia express Ia-like antigens

15.7 +/- 3.5% of sheep rosette-forming cells (SRFC) were isolated from the peripheral blood of 11 patients with B-cell chronic lympholeukemia (B-CLL). SRFC did not express surface immunoglobulins, antigens of nondifferentiated blasts and antigens of early T-cell precursors, while NK-cell antigen expression was low. 6 of 11 patients revealed 44.6 +/- 19.6% of Ia-like antigens in SRFC. Ia-like antigen expression was 4.8 +/- 0.6% in SRFC isolated from the peripheral blood of 19 healthy donors. The expression of Ia-like antigens in T cells of patients with B-CLL is suggested to be related to the activation of regulatory T-lymphocyte subpopulation.     

Ultra-fast and sensitive detection of non-typhoidal Salmonella using microwave-accelerated metal-enhanced fluorescence ("MAMEF")

Certain serovars of Salmonella enterica subsp. enterica cause invasive disease (e.g., enteric fever, bacteremia, septicemia, meningitis, etc.) in humans and constitute a global public health problem. A rapid, sensitive diagnostic test is needed to allow prompt initiation of therapy in individual patients and for measuring disease burden at the population level. An innovative and promising new rapid diagnostic technique is microwave-accelerated metal-enhanced fluorescence (MAMEF). We have adapted this assay platform to detect the chromosomal oriC locus common to all Salmonella enterica subsp. enterica serovars. We have shown efficient lysis of biologically relevant concentrations of Salmonella spp. suspended in bacteriological media using microwave-induced lysis. Following lysis and DNA release, as little as 1 CFU of Salmonella in 1 ml of medium can be detected in <30 seconds. Furthermore the assay is sensitive and specific: it can detect oriC from Salmonella serovars Typhi, Paratyphi A, Paratyphi B, Paratyphi C, Typhimurium, Enteritidis and Choleraesuis but does not detect Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae or Acinetobacter baumanii. We have also performed preliminary experiments using a synthetic Salmonella oriC oligonucleotide suspended in whole human blood and observed rapid detection when the sample was diluted 1:1 with PBS. These pre-clinical data encourage progress to the next step to detect Salmonella in blood (and other ordinarily sterile, clinically relevant body fluids).     

Volatile metabolites in sera of normal and diabetic patients

The profiles of volatile metabolites in serum samples from normal individuals and from individuals with diabetes mellitus with varying degrees of polyneuropathy have been studied. The transevaporator procedure was used to obtain sample extracts which were chromatographed on a highly efficient glass column coated with Silar 10C (106 m × 0.25 mm I.D.). Differences in profiles between normal subjects and diabetic subjects on no drug therapy were noticed. However, correlations between the severity of the neuropathy and the concentrations of certain ketones could not be established. Compounds present both in diabetic and normal sera have been identified by mass spectrometry.     

Antibodies to bacterial and tumor-derived antigens in sera from normal guinea pigs.

Antibodies that react with radiolabeled antigens derived from guinea pig line-10 tumor cells and Mycobacterium bovis (BCG) were detected in sera from normal tumor-free strain-2 guinea pigs (NGPS). Binding by NGPS to the two antigens was inhibited by extracts of either line-10 cells or BCG. Binding by NGPS to the line-10 antigen was inhibited by a number of other bacterial extracts. NGPS was tested after absorption with a variety of cells including line-10, line-1, normal guinea pig spleen, normal adult and fetal liver cells. Results indicated that some of the antibodies in NGPS were directed to line-10-specific determinants. The specific stimulating antigen for these antibodies was not identified but because of the antigenic relationship between BCG, line-10 cells and other bacteria, antibodies to line-10-associated antigens might have been induced by exposure to environmental microorganisms.     

Detection of Rotavirus from stool samples using a standardized immuno-PCR ("Imperacer") method with end-point and real-time detection

Immuno-PCR (IPCR) has been studied to increase the detection sensitivity of current enzyme-linked immuno-sorbent assays (ELISA) as a novel approach for the early detection of Rotavirus infection, a major source for serious diarrhoea for susceptible risk groups. IPCR utilizes specific antibody-DNA conjugates with subsequent amplification of the marker-DNA. An antibody-DNA conjugate specific for Rotavirus antigen VP6 was synthesized and used in combination with a commercially available Rotavirus-ELISA kit. IPCR was carried out using reagents and protocols of the standardized Imperacer system. Real-time PCR monitoring of the marker-DNA amplification was compared to endpoint quantification of amplified haptene-labeled PCR products, using a microtiterplate-based PCR-ELISA. In spiked calibration samples, as few as 100 virus particles/ml could be clearly detected using the IPCR method and either real-time or end-point quantification compared to about 100,000 virus particles/ml in ELISA. Rotavirus positive and negative stool samples were correctly identified by IPCR with a clear separation even of a 10,000-fold dilution of the positive stool samples from the negative control.     

Identification of immune complex antigens in sera of Indian kala-azar patients

Level of circulating immune complex (IC) in visceral leishmaniasis is much higher than that in control sera. In immunoblot experiment, treatment of kala-azar IC with patient sera showed at least 6 bands of which the band around 55 kDa region was most prominent. The band at 55 kDa is primarily due to the presence of an antigen recognized by its corresponding antibody present in the patient sera. This was confirmed by using radiolabelled antibody from kala-azar patient serum and antipromastigote serum. The heavy chain of IgG originating from IC is also present in the same region which was detected by its recognition with antihuman IgG. The IC gave a band at 55 kDa region with sea-urchin antitubulin. Kala-azar sera also reacted with purified rat brain tubulin. The present results suggest that a tubulin like protein is present at 55 kDa region along with the heavy chain of IgG.     

Immunoradiometric assay for detection of filarial antigens in human serum

The immunoradiometric assay (IRMA) for detection of filarial antigens in the serum of patients infected with Brugia malayi (Bm) or the closely related filarial parasite Wuchereria bancrofti (Wb) was investigated, and its performance and clinical utility were examined. Reference sera prepared by the addition of crude Bm antigen (BmA) to negative control human sera provided a reproducible reference curve. The IRMA displayed acceptable precision and reproducibility. Agreement between dilutions (parallelism) was good in sera without specific antibody, but the presence of even modest levels of antibody resulted in nonparallelism in about one-half of the tested sera from endemic areas. Significant reduction in detectable BmA occurred when low levels of specific antibody (less than 1 microgram/ml) were added to BmA containing sera. Thus, antibody interference limited absolute quantitation of antigen in the IRMA. Results were therefore expressed in a semi-quantitative manner by using the mean + 3 SD of the binding of nonexposed human sera as the positive threshold. The frequency of reliable filarial antigen detection in individuals from the Wb endemic areas of India and the South Pacific was the following: microfilaremia, 15 out of 15; elephantiasis, 2 out of 18; tropical pulmonary eosinophilia, 2 out 8. These findings show clearly that a two-site IRMA can effectively detect circulating antigen (and thus be diagnostic of infection) in a great many patients with filariasis, but to enhance the sensitivity of the assay to the point where all patients can be diagnosed, a number of suggested modifications will be necessary.     

Serological detection and immunogold localization of cross-reactive antigens shared by Camellia sinensis and Exobasidium vexans

AIMS: Pathogenicity of Exobasidium vexans, causal agent of blister blight of tea, was studied in 30 commercially cultivated tea varieties by analysing the antigenic patterns of host and pathogen using immunological techniques. METHODS AND RESULTS: Whole plant inoculation of tea varieties with E. vexans showed that T-78 and T-17/1/54 were most susceptible and most resistant respectively. Antigen preparations from tea varieties, pathogen, nonpathogen (Fusarium oxysporum) and of nonhosts (Glycine max, Leucaena leucocephala and Oryza sativa) were compared by indirect enzyme-linked immunosorbent assay and dot-immunobinding assay using polyclonal antibodies raised against the pathogen, nonpathogen, susceptible and resistant tea varieties. Cross-reactive antigens (CRA) were found among susceptible varieties and E. vexans isolates but not in resistant varieties, nonhosts or nonpathogen. Indirect staining of antibodies using fluorescein isothiocyanate indicated CRA were concentrated mainly around epidermal and mesophyll cells in compatible host (T-78). This was substantiated by ultrastructural studies using gold-labelled antibodies through transmission electron microscopy which showed specific localization in the chloroplasts and host cytoplasm. CONCLUSION: Pathogenicity of E. vexans to different tea varieties is therefore related to the level of antigenic similarity between host and pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: Immunological methods proved to be valuable in screening commercially cultivated tea varieties against E. vexans.     

Transplantation in minature swine. IV. Chemical characterization of MSLA and Ia-like antigens

Immunochemical analyses of radioactively labeled lymphocyte antigens from miniature swine of three different homozygous major histocompatibility (MHC) types, AA, CC, and DD, have been performed. Anti-MHC sera were incubated with lentil lectin purified Nonidet P-40 swine lymphocyte extracts. Antigen-antibody complexes were then precipitated with protein A bearing staphylococci, eluted, and electrophoresed on sodium dodecyl sulfate polyacrylamide gels. Analysis of antigens from AA or DD cells revealed peaks of 42,000, 31,000, 25,000, and 11,000 dalton m.w. Platelet absorption of the anti-MHC sera yielded antibodies that only precipitated the intermediate m.w. molecules and lysed a subpopulation of swine peripheral blood lymphocytes, suggesting that these molecules were the miniature swine analogues of murine Ia antigens. Antibodies eluted from platelets lysed all lymphocyte populations and precipitated only the 42,000 and 11,000 dalton peaks, indicating that these molecules represent the analogue of murine H-2 histocompatibility antigens, containing a heavy chain and putative swine beta2-microglobulin.