Vlastimil Mayer

Enhanced chemiluminescence-based hybridization analysis for PCR-mediated HIV-1 DNA detection offers an alternative to 32P-labelled probes

The efficiency of enhanced chemiluminescence based on a novel generation substrate for alkaline phosphatase, adamantyl-1,2-dioxetane phosphate, was compared with that of 32P-labelled probe for visualization of human immunodeficiency virus type 1 (HTV-1)-specific DNA-DNA hybrids. The probe used for nonisotopic detection was digoxigenin labelled and targeted by anti-digoxigenin antibody Fab-fragments conjugated to alkaline phosphatase. The dot-blot hybridization analysis performed on a dilution series of HIV-1 proviral DNA demonstrated a lower sensitivity limit of 0.5 pg with the nonisotopic method. However, one order of magnitude less DNA could still be detected by a random-primed 32P-labelled probe. The ability of nonradioactive and radioactive probes to detect 590-bp gag gene-specific target sequences generated by the polymerase chain reaction (PCR)-mediated amplification of HIV-1 DNA was also compared. Analysis of 20 samples from individuals at increased risk for HIV infection by using the two assayed systems produced virtually equivalent signal images on corresponding specimens. Furthermore, complete concordance in the performance was found when HIV-1 proviral DNA was investigated by PCR in additional 50 samples of human blood mononuclear cells.

Immunological alterations in anti-HTLV-III negative haemophiliacs and homosexual men in Hungary

Hungary can be considered as a low risk area for AIDS since no patient with full-blown AIDS or AIDS-related complex has been found in the country. A complex clinical and immunological (T cell subsets, DNCB sensitization test, circulating immune complexes, acid-labile alpha interferon) investigation was performed between November, 1983 and June, 1984 in order to study whether alterations found in symptom-free homosexuals and haemophiliacs in the risk areas for AIDS can be observed in Hungary as well. 38 patients with mild haemophilia, 35 patients with severe haemophilia and 40 homosexual men were investigated in parallel to 37 heterosexual blood donors as controls. Anti-HTLV-III antibodies were measured later in the stored serum aliquots from the same subjects by the indirect membrane immunofluorescence assay. Although specific anti-HTLV-III antibodies were not detected in the haemophiliacs or homosexuals, immunological alterations characteristic for the members of AIDS risk groups in the high risk areas (decrease in the percentage of OKT4 cells and/or decrease of the OKT4/8 ratio) were found in one-third of the homosexuals and haemophiliacs tested. In addition, a significant part of these subjects did not develop delayed type hypersensitivity skin reaction on DNCB rechallenge. These findings indicate that an immunodeficiency independent of HTLV-III infection can be present in two major AIDS risk groups, in homosexual men and haemophiliacs.

A BIOMETRICAL VIEW ON NORMAL VALUES OF CD4 AND CD8 LYMPHOCYTE COUNTS IN PERIPHERAL BLOOD

&NA; Statistical methods have been used to determine an optimal approach to the definition of the reference range for CD4 (“helper”) and CD8 (“suppressor/cytotoxic”) T cell numbers and the CD4/CD8 ratio in the peripheral blood. A graphical presentation of the absolute values for CD4 and CD8 for 85 healthy blood donors showed that a reference ellipse defined by fitting a gaussian distribution to logarithmically transformed data for absolute counts of CD4 and CD8 cells was superior to the fitting of an ellpse to untransformed data. Further analysis for another 147 subjects showed that the 95% tolerance prediction for the CD4/CD8 ratio in health could be stated with 95% confidence as 0.6 to 5.0. This approach allows clear definition of reference ranges for T cell tests in health and would also be applicable to results for patients with a disease such as HIV 1 infection in which a reference range for “well” patients exists and a change in the T cell ratio is of prognostic significance.

Different types of false positive anti-HIV reactions in patients on haemodialysis

Serum samples of 589 haemodialysis patients were screened for HIV antibody by ELISA methods. Of these, 36 samples were found to be repeatedly reactive. None of the 36, however, could be confirmed by competitive enzyme immunoassays and Western blot; therefore, they were considered to be false positive. The sera could be divided in two groups. The sera of Group 1 were designated as the usual type of false positivity, caused most probably by anti-lymphocyte antibodies. In 19 sera, however, a special type of false positivity was found. These sera reacted strongly with the plates coated with the supernatants of HIV-infected cells but not with those of uninfected H9 cells. Three and two sera showed, respectively, positive immunofluorescence reaction with the HIV-infected, but not with the uninfected, H9 and CEM cells. Reactivity to HIV-infected H9 cells could be adsorbed from a part of these samples with lesser amounts of HIV-infected than uninfected H9 cells. This special type of false positivity was observed frequently (7/65) in patients who rejected a kidney graft. These findings suggest that this type of anti-HIV false positivity is due to antibodies reacting with cellular antigens present in HIV-infected but not in uninfected lymphocytes. Their appearance seems to be associated with the immunological activation occurring at graft rejection.

ANTIGEN-SPECIFIC TRANSFER FACTOR INDUCED IN MICE BY AN ATTENUATED VIRUS FROM THE TICK-BORNE ENCEPHALITIS COMPLEX BIOLOGICAL, HISTOLOGICAL AND CONCENTRATION STUDIES

Publisher Summary The transfer factor (TF)-administered inbred C3H mice proved as a satisfactory testing system for induction of T cytotoxic lymphocytes, specific against the neoantigens on the tick-borne encephalitis (TBE) virus-infected chromium-labeled syngeneic L929 cells. According the Tcc induction, a method for TBE virus antigen-specific TF was developed, allowing to characterize some aspects of TF biology, to study the lymphatic organs response by immunomorphological methods, and to obtain data about partial concentration of the dialyzable active substance. This chapter presents data from the investigations on the murine STF, which suggest that it may serve as a model for similarly oriented efforts with the DLL of human origin. It may be of value, especially if a preparation of a certain grade of purity, relatively high potency, and deliberately selected specificity appears as useful.