Definition of discrete signals involved in human T-cell activation.

Mitogenic activities of monoclonal antibodies directed at defined receptor structures expressed on the surface of mature human T lymphocytes were employed to study, in detail, signals involved in primary T-cell activation. Based on differential requirements for stimulation, two discrete pathways of human T-cell activation can be defined: the antigen-induced mode of activation initiated through the Ti-T3 antigen-receptor complex and an alternative pathway which can be triggered by monoclonal antibodies directed at the T11 glycoprotein. Perhaps more importantly, the approach taken here allows the definition of stable intermediate cellular stages within the activation cascade and, thus, to analyze the signalling capabilities of individual receptor molecules.

Semiquantitative assessment of pre-core stop-codon mutant and wildtype hepatitis B virus during the course of chronic hepatitis B using a new PCR-based assay.

In most patients with chronic hepatitis B positive for antibodies (anti-HBe) to HBe antigen (HBeAg), a pre-core mutant hepatitis B virus (HBV) with a point-mutation at nt. 1896 can be isolated. Clinical significance of the mutant virus in chronic hepatitis B is not proven yet, and screening of large numbers of sera during different clinical courses of numerous patients is necessary. We therefore aimed to develop a fast and reliable assay, that allows to discriminate wildtype from nt. 1896 G-->A mutant HBV and to determine the ratio of mutant and wildtype HBV in patients' sera. A mutation specific polymerase chain reaction (ms PCR) with new primers served to distinguish nt. 1896 G-->A mutant from wildtype HBV. This msPCR proved to be more sensitive and specific than similar assays described previously. When compared to a dilution series of a cloned HBV-DNA standard, the amount of wildtype and nt. 1896 G-->A mutant HBV could be determined semiquantitatively. 10(2) to 10(7) copies of each HBV-DNA (equivalent to 10(5) to 10(10) copies of HBV-DNA/ml patients' serum) could be amplified with steadily increasing signals. MsPCR proved to be specific as 10(7) copies did not give an amplification signal if they did not match the respective primer pair used. In a mixed population of mutant and wildtype virus, msPCR allows to detect even a low amount of the minor HBV strain (0.1-0.01%, of the viral population) and to determine the ratio of wildtype and mutant HBV. MsPCR is as fast and convenient to perform as an unmodified PCR. It requires careful performance to avoid contamination but no specific equipment. Clinical usefulness of msPCR was demonstrated when the ratio of wildtype to mutant HBV was determined in 86 sera collected during 3 to 7.5 years follow up of 9 patients suffering from anti-HBe positive chronic hepatitis B. We conclude that this assay conveniently allows to study patients with chronic hepatitis B in order to detect and follow-up the emergence of pre-core stop-codon mutant HBV in correlation to the clinical course.