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Biomedical subjects

D Primi

Publications and source records attributed to D Primi.

At least 37 records · Page 2Linked to original sources
PubMed1994

Hepatitis B virus C gene heterogeneity in a familial cluster of anti-HBc negative chronic carriers.

We studied a familial cluster of adult HBV chronic carriers characterized by serological markers of active viral replication, normal or slightly elevated ALT levels and, in four out of five cases, absence of anti-HBc reactivity. The nucleotide sequence of the pre-C/C region of HBV was analyzed in three patients, showing the presence of wild-type HBV sequences accompanied by different deleted molecules. Some of the variant genomes were able to encode proteins retaining HBcAg and/or HBeAg reactivity, whereas others contained deletions leading to the synthesis of truncated proteins that could not be immunoprecipitated by anti-HBc or anti-HBe mono- and polyclonal antibodies. Defective molecules encoded by the variant C gene sequences might play a role in the determination of the clinical profile observed in the analyzed patients.

Adolescent↗
PubMed1994

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.

Blotting, Southern↗
PubMed1994

Intracellular localization of full-length and truncated hepatitis C virus core protein expressed in mammalian cells.

The putative hepatitis C virus core protein has a predicted molecular weight of about 22 kD and contains two carboxy (COOH)-terminal hydrophobic domains. The cleavages generating the hepatitis C virus structural proteins (core, E1 and E2) are catalyzed by host signal peptidases. In the present study, we investigated the processing and intracellular localization of the hepatitis C virus core protein expressed in mammalian cells. Expression vectors encoding the entire core protein or COOH-terminal deletion mutants under the control of SV40 regulatory sequences were transfected in COS cells. Immunofluorescent staining with either polyclonal immunoglobulin or monoclonal anti-core antibodies showed that fragments containing the COOH-terminal hydrophobic stretch were retained in the cytoplasm of transfected cells, whereas truncated core proteins deleted of 28 or more residues were located in the nucleus. Our results suggest that a putative nuclear targeting sequence is contained in the first 40 residues of the core protein.

Amino Acid Sequence↗
PubMed1994

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 patient's 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.

Adult↗
PubMed1994

Superantigen-reactive human T cells express a biased repertoire of T-cell receptor V beta 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.

Amino Acid Sequence↗
PubMed1994

Differential priming to programmed cell death of superantigen-reactive lymphocytes of HIV patients.

Programmed cell death or apoptosis has been shown to play a central role in CD4+ T cell depletion following HIV infection. Because most apoptotic signals are delivered through T cell receptor stimulation, we investigated whether T cell depletion in AIDS is a stochastic phenomenon or if it preferentially affects T cell subsets defined by their interaction with superantigens. To address this problem we have taken advantage of the exclusive property of superantigens to trigger T cells expressing selective sets of T cell receptor V beta elements. Here we report that CD4+ T cells from HIV-infected patients can proliferate in vitro to T cell receptor mobilization by some superantigens, but not others. Furthermore, the failure of T cells to respond to some superantigens was shown to be due to an active cell death process that differentially affected T cells capable of interacting with different superantigens. The selective programmed cell death priming of T cells responsive to particular superantigens, observed in this study, suggests that T cell depletion in HIV infection is not simply due to the cytopathic effect of the virus. The possible link between programmed cell death and T cell receptor variable regions suggested by the present experiments may help to better define current models of AIDS pathogenesis.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Selection of T lymphocytes in two rheumatoid arthritis patients defines different T-cell receptor V beta repertoires in CD4+ and CD8+ T-cell subsets.

To study the selective pressures responsible for the expansion of T cells in rheumatoid arthritis, we constructed cDNA mini-libraries from purified CD4+ and CD8+ T cells prepared from peripheral blood and from synovial fluids of two rheumatoid arthritis patients. Comparison of these libraries by hybridization with specific probes indicated that V beta 2 and V beta 8 transcripts are selectively enriched in the CD4+ synovial fluid lymphocyte population, while V beta 4 was over-represented among both the CD4+ and CD8+ subsets. The enrichment of V beta 14 and V beta 17 observed in synovial fluid T cells of one patient was, however, selectively confined to the CD8+ T-cell subpopulation. Sequence analysis of several V beta 2, V beta 4 and V beta 8 clones, derived from CD4+ cells, revealed a high degree of heterogeneity in the V beta-D beta-J beta junctions, while a more biased utilization of J segments and a more restricted junctional heterogeneity were observed in V beta 4, V beta 14 and V beta 17 clones derived from CD8+ cells. These data suggest that the disease may be induced by the initial activation of a rather heterogeneous population of T-helper cells that are later responsible for the expansion of a more restricted pool of highly specific effector lymphocytes.

Amino Acid Sequence↗
PubMed1993

T cell repertoire and autoimmune diseases.

Self-reactivity and autoimmunity are processes related to the breakage of self-tolerance that can be distinguished by their different clinical outcome and are widely accepted cornerstones of immunology. The finding that several potentially autoaggressive cells contribute to the repertoire of healthy individuals has stimulated a great deal of experimental work aimed at understanding the mechanisms that prevent autoimmune pathology. In this review we will consider the basic principles, and our present knowledge of the rules that preside over the interplay of the immune system with self-components. One viewpoint stresses the importance of major histocompatibility complex (MHC) and non-MHC genes in determining genetic predisposition to develop autoimmune phenomena. At a different level there is a strong interest in understanding the mechanisms of processing and presentation of self antigens, especially during ontogeny. Another topic of major interest concerns the interaction between MHC genes and the T cell receptor (TcR) complex as well as the identification of TcR V genes that are preferentially expressed by autoimmune T cells. All of these aspects are evaluated in the context of tolerance based on deletion and anergy. Finally we will propose a general model of autoimmunity based on the most recent findings concerning the biological activity of exogenous superantigens.

Animals↗
PubMed1993

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.

Adolescent↗
PubMed1993

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.

Adult↗
PubMed1992

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.

Electrophoresis, Polyacrylamide Gel↗
PubMed1992

Analysis of amplified T cell receptor V beta 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.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1992

Evaluation of hepatitis delta virus RNA levels during interferon therapy by analysis of polymerase chain reaction products with a nonradioisotopic hybridization assay.

We developed a nonradioisotopic assay for detection of hepatitis delta virus RNA in serum by combining reverse transcription of RNA, polymerase chain reaction of the resultant complementary DNA and enzyme linked immunoassay detection of the polymerase chain reaction products using a monoclonal antibody specific for double-stranded DNA. This DNA enzyme immunoassay had a limit of detection of cloned hepatitis delta virus RNA similar to that of standard PCR followed by Southern-blot hybridization (approximately 10 copies/sample) and was 10(3) to 10(4) times more sensitive than direct dot-blot hybridization (approximately 10(5) copies/sample). Serial serum samples from six patients with chronic hepatitis delta virus infection undergoing interferon therapy were analyzed by reverse transcription-polymerase chain reaction followed by both standard hybridization and DNA enzyme immunoassay. The results of both methods were comparable, revealing disappearance of hepatitis delta virus RNA after 3 to 6 mo of therapy in three patients, two of whom had also a significant decrease in ALT activity. The DNA enzyme immunoassay test is therefore a potentially useful method for therapeutic monitoring in chronic hepatitis delta virus infection and may contribute to a wider application of polymerase chain reaction in clinical laboratories.

Adult↗
PubMed1992

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.

Base Sequence↗
PubMed1992

Non-radioisotopic methods for DNA probes.

We have recently developed a new colorimetric method, 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. The molecule does not react with a specific probe immobilized on microwells through an avidin-biotin bridge, nor with non-specific amplified sequences, since they are removed by washing. The antibody reveals the hybridization event, independently of the DNA sequences and, for this reason, the method is broadly applicable and extremely versatile. Although we chose a format based on the immobilization of the probe through an avidin-biotin interaction, DEIA assay can also be applied to other analytical schemes that do not require any modification of the probe. Most importantly, the test has an ELISA format and is rapid and convenient for processing large numbers of samples. This technology has been applied, in our laboratory, to different areas, including virology, genetic and basic immunology. The DEIA assay has been successfully used to detect the presence of hepatitis B (HBV), hepatitis C (HCV) and hepatitis delta virus (HDV) sequences in serum of patients, to discriminate different HLA alleles, to identify mutations in the Cystic Fibrosis gene, and to investigate the role of the T cell receptor in some immunological diseases. The results obtained in all our experiments demonstrated that the advantages offered by the assay do not penalize its analytical performance as compared to conventional Southern blot.

Colorimetry↗
PubMed1991

Selective depletion in HIV infection of T cells that bear specific T cell receptor V beta sequences.

The mechanism of T cell depletion during infection with the human immunodeficiency virus (HIV) is unclear. Examination of the repertoire of T cell receptor V (variable) regions in persons infected with HIV revealed the absence of a common set of V beta regions, whereas V alpha usage was normal. The lack of these V beta segments did not appear to correlate with opportunistic infections. The selective elimination of T cells that express a defined set of V beta sequences may indicate the presence of an HIV-encoded superantigen, similar to those encoded by the long terminal repeat of the mouse mammary tumor virus.

Acquired Immunodeficiency Syndrome↗