Alessandra Bettinardi

An immunoassay for specific amplified HCV sequences

The direct detection of viraemia could improve greatly the efficacy of presently available assays. Due to its sensitivity, the polymerase chain reaction represents the method of choice for direct detection of viral nucleic acid. However, the clinical application of this method is hampered by the requirement of hybridization with radioactively labelled probes. In this study we demonstrate that HCV cDNA, amplified by the polymerase chain reaction from both liver tissues and sera, can be detected specifically by a new non-radioisotopic method, DNA enzyme immunoassay, that is based on an antibody that selectively recognizes double, but not single-stranded DNA. The assay reveals the hybridization events, independently from the DNA sequences, and therefore can be used with any combination of primers and probes. Most importantly, the method has a conventional ELISA format and is compatible with standard facilities of clinical laboratories. The availability of this new approach for revealing amplified sequences may facilitate greatly the use of PCR as the method of choice for early diagnosis of HCV infection.

Different TCRBV genes generate biased patterns of V-D-J diversity in human T cells

The aim of this work was to assess whether each T-cell receptor (TCR) BV segment generates a random pattern of junctional diversity or if, alternatively, biased patterns of V-D-J rearrangements limit the number of available TCR specificities. Detailed molecular analysis of T-cell receptors expressed by lymphocytes was obtained by generating a large number of junctional regions sequences from TCRBV3, TCRBV4, TCRBV5S1, TCRBV12, TCRBV13S2, TCRBV17, TCRBV20, and TCRBV22 variable genes. The > 800 sequences analyzed have allowed the characterization of the recombination frequencies of each germline-encoded V,D, and J segments, as well as of the magnitude of exonucleolytic nibbling and of the number of N nucleotides inserted for each group of TCRB segments. The data obtained indicate that the extent of junctional diversity varies considerably depending on the TCRBV gene implicated in the recombination event, due to the occurrence of skewed patterns of J and D region usage. Furthermore, our results show that “illegitimate” rearrangements occur with unexpectedly high incidence, specifically at the level of TCRBD to TCRBJ joining. These findings provide additional information for a more accurate estimation of the size of the TCRBV repertoire and for understanding the well-established biased pattern of TCRBV expression in humans.

Detection of skewed T-cell receptor V-β gene usage in the peripheral blood of patients with multiple sclerosis

The ex vivo analysis of the T-cell receptor V-beta (TCRBV) gene usage by circulating T lymphocytes in Multiple Sclerosis (MS) patients may contribute to understanding disease pathogenesis. In the present study, TCRBV gene usage was analyzed in freshly collected unstimulated peripheral blood mononuclear cells (PBMC) isolated from 40 MS patients and 20 healthy controls. Nine patients presented abnormal repertoires, with expansion of one or more TCRBV segments. Among these patients, six presented expansion of TCRBV9 chain expression, three also having an expansion of TCRBV1, TCRBV11 and TCRBV22 segments. The most frequently observed TCRBV chain expansion, TCRBV9, was further analyzed and identified as polyclonal. Evaluation of clinical variables showed that median disease duration was shorter in patients with TCRBV gene expression abnormalities. Longitudinal evaluation of five patients with a skewed repertoire showed regression of expanded TCRBV chains expression to normal values. These data indicate that certain MS patients have abnormal TCRBV gene expression. Such abnormalities are caused by polyclonal expansions of T lymphocyte subpopulations that use the same TCRBV gene families, are unstable and preferentially observed early in the course of the disease.

Analysis of amplified T cell receptor Vβ transcripts by a non-isotopic immunoassay

We have recently described a new colorimetric DNA enzyme immunoassay (DEIA) for detecting specific hybrids of complementary nucleic acids. This technology is based on an antibody that selectively recognizes double, but not single stranded DNA and therefore reveals the hybridization event independently from the DNA sequences. Most importantly, the test has an ELISA format and is very rapid and convenient for processing large numbers of samples. In the present report we have adapted this method to reveal the specificity of amplified T cell receptor V beta transcripts. V beta genes were amplified by polymerase chain reaction, using family specific primers and the specificity of the amplified products was determined by Southern blot and by DEIA. Our data demonstrate that DEIA had the same degree of sensitivity and specificity of conventional Southern hybridization. The possibility of analyzing amplified products with the simplicity of a conventional immunoassay should greatly facilitate the analysis of complex multigenic systems such as the T cell receptor and the immunoglobulin repertoire.