Daniele Primi

Genomic and Molecular Evolutionary Analysis of a Newly Identified Infectious Agent (SEN Virus) and Its Relationship to the TT Virus Family

A new group of transmissible single-stranded (ss) DNA viruses (SENV) distantly related to the large TT virus (TTV) family was recently identified. Eight different SENV isolates have been found, some with an association with posttransfusion hepatitis. A phylogenetic analysis of near-complete open-reading frame 1, including conserved motifs and excluding recombinant regions, was performed. The analysis used TTV-like minivirus as an outgroup, to determine a root of the phylogenetic tree, and compared 8 SENV isolates, 6 prototype TTV isolates, and 7 TTV variants (including SANBAN, TUS01, PMV, and YONBAN). Four distinct clusters separated by a bootstrap value of 100% were observed. YONBAN isolates formed a distinct outer group, representing the earliest recognized phylogenetic divergence (group 1). Prototype TTV formed group 2, PMV formed group 3, and SENV, SANBAN, and TUS01 isolates formed group 4, the most recently evolved group. This taxonomic classification suggests that these circular ssDNA viruses probably evolved from a common ancestor virus.

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.

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.

Analysis of the hepatitis B virus genome and immune response in HBsAg, anti-HBs positive chronic hepatitis

Although the development of antibodies against the hepatitis B virus surface antigen generally leads to the clearance of the infecting virus, anti-HBs reactivity has been reported in patients with chronic hepatitis. In the present study we analyzed the viral genome and the antibody specificity in a series of serum samples collected from a patient who seroconverted to anti-HBs during interferon therapy without clearing HBsAg. The appearance of an anti-HBs response was accompanied by the emergence of a pre-S1 defective viral genome. However, the wild-type adw2 molecular species remained largely dominant during follow up. The patients antibody response to the surface viral antigens was directed towards the heterologous y subdeterminant and the pre-S1 fragment deleted in the variant hepatitis B virus. These results suggest that the selection of the escape viral mutant does not play a major role in viral persistence.

HLA-DQB1 typing of north east Italian IDDM patients using amplified DNA, oligonucleotide probes and a rapid DNA-enzyme immunoassay (DEIA)

We report on HLA-DQB1 typing in IDDM patients of north east Italian region using an enzymatic method based on the detection of hybridization reaction between PCR amplified DNA from whole blood and allele specific oligonucleotides by an antibody directed against double stranded DNA (DNA-enzyme immunoassay or DEIA). The method is reliable, simple and sensitive as the classical radioactive method with the advantage of using a universal non radioactive detection reagent. Nineteen families, each including one subject with juvenile insulin-dependent diabetes mellitus (IDDM) were analyzed. A strong association between absence of an aspartic acid (Asp) in position 57 of DQB1 beta chain in homozygous conditions and susceptibility to IDDM was found. In contrast with some previous observations, however, no significant association was found between Asp/non-Asp heterozygous genotype and IDDM. No patients were found with an homozygous Asp/Asp genotype, known to be protective in caucasoid population. Of particular interest was the DQB1 allelic distribution in our population sample. The non-Asp allele most frequently found in IDDM subjects was the DQB1 0201 allele and this finding was statistically significant (Pc value < 0.05, relative risk = 5.01). No significant association was found for any other allele including the DQB1 0302 (Pc value = not significant although with relative risk = 3.28) previously reported as the most frequent allele in IDDM caucasoid patients.

Detection of BCR/ABL transcripts in chronic myeloid leukaemia by polymerase chain reaction and DNA enzyme immunoassay: a DNA probe assay without DNA labelling

The detection of the t(9;22) translocation, typical of chronic myeloid leukaemia (CML), can be accomplished by cytogenetical detection of Philadelphia (Ph1) chromosome or by molecular analysis of the bcr/abl fusion gene with nucleic acid probes after amplification by polymerase chain reaction (PCR). PCR‐based approaches are now widely used for follow up of CML patients during therapy or after bone marrow transplantation (BMT). We describe here a microtitre, colorimetric assay (DNA Enzyme Immunoassay, DEIA) for analysis of t(9;22) translocation after enzymatical amplification of RNA from CML patients. This assay is based on the use of a monoclonal antibody specifically reacting with double stranded DNA, i.e. with hybridized DNA. The assay represents a nonisotopic alternative to other current hybridization assays and requires no modifications of primers, probe or target DNA.

Superantigen-reactive human T cells express a biased repertoire of T-cell receptor Vβ joining regions

A major characteristic of superantigens is their ability to stimulate T cells based predominantly on the type of variable segment of the T-cell receptor (TCR) V beta chain. Recently, however, reports from several laboratories have also implied a role for non-V beta elements in superantigen binding. The goal of the present study was to determine whether TCR V beta-D beta-J beta joining sequences may influence the interaction of superantigens with their target cells. To ascertain how the actual TCR repertoire of superantigen-triggered cells deviates from the theoretical one, we generated a large panel of joining region sequences from TCR carrying the TCR V beta 12 and TCR V beta 5,1 regions. The 245 sequences analysed represent transcripts of T cells from the same donor triggered either with an anti-CD3 monoclonal antibody or with the Staphylococcus aureus enterotoxins. Comparison of the joining sequences of these different groups demonstrates a skewed J beta usage in the sequences derived from superantigen-triggered cells and also provides evidence that ascribes to the putative CDR3 region of V beta segments a role in superantigen recognition. Finally, the data presented give some hints of the regions of the putative CDR3 loop that may play a major role in this function.

Expression and combinatorial diversity of germ line-encoded T cell receptor V genes in human peripheral blood T cells

The potential diversity of the T cell receptor (TcR) is defined by the combinational expression of variable segments and by mechanisms that insert or delete nucleotides at the junctional regions. The available repertoire is strongly influenced by negative and positive selection events. To study whether the diversity of the human T cell receptor of peripheral T cells is further restricted by the interaction between the TcR alpha and beta chains, we compared the level of transcription of different V alpha elements in human T cell blasts expressing either restricted or unrestricted sets of V beta genes. Our data establish that in some individuals, but not in others, the transcription of a given V alpha element is independent from the presence of particular V beta transcripts. Furthermore, our data also suggest that, in contrast to mouse, major TcR V gene deletions are absent in humans. Taken collectively, these results indicate that the diversity of the peripheral human TcR repertoire can benefit from the combinatorial expression of all the V elements present in the genome.

Diagnosis of viral hepatitis with a nonisotopic hybridization assay

The DNA enzyme immunoassay is an efficient method for the screening of PCR products derived from different hepatitis virus genomes, and allows to bypass both agarose gel electrophoresis and Southern blot hybridization with radioactively labeled probes. A wider application of this method will disclose new perspectives for the introduction of PCR in clinical laboratories.

Hepatitis B virus (HBV) genomic variations in chronic hepatitis

Using PCR we have examined the sequence of the pre-C/C region of HBV from sera of anti-HBe positive, chronically HBV-infected patients. The large majority of sera tested contained a mixture of heterogeneous pre-C sequences with 1-3 non-randomly located point mutations. Some of the resultant variant viruses are incapable of synthesizing immunogenic proteins and may be involved in viral persistence in chronic carrier states.