Detection of HCV RNA and antibodies to HCV after needlestick injury.
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Adult↗
Biomedical subjects
D Primi
Publications and source records attributed to D Primi.
Restricted expression of T cell receptor V beta but not V alpha genes in rheumatoid arthritis.
Synovial fluids of patients with rheumatoid arthritis contain activated T lymphocytes that may play an important role in the pathogenesis of the disease. Previous studies have suggested that the T cell receptor (TcR) repertoire of these cells is restricted, reflecting in vivo selection of a limited number of T cell specificities at the site of inflammation. To characterize better these T cell populations we used the polymerase chain reaction technology to estimate the proportion of TcR alpha and beta RNA containing any particular V elements from transcripts directly isolated from the synovial fluid cells and from peripheral blood mononuclear cells of three patients with rheumatoid arthritis. Our data show that, in contrast to peripheral blood mononuclear cells, synovial fluid T cells expressed only few V beta transcripts, one of which was overrepresented in two patients. Peripheral and joint fluid T cells, on the other hand, appeared to express the same set of non-restricted V alpha elements. These results suggest that a major antigen associated with the pathogenesis of rheumatoid arthritis may interact selectively with the V beta component of the TcR.
Arthritis, Rheumatoid↗
Use of variable human V delta genes to create functional T cell receptor alpha chain transcripts.
Previous studies of the human T cell receptor delta genes identified five commonly used V delta segments distinct from any of the known V alpha genes. To define better the relationship between the T cell receptor delta and alpha repertoires we amplified cDNA obtained by polyclonally activated lymphocytes with a common 3' antisense C alpha-specific primer and with five different 5' sense V delta family-specific primers. Amplified products were detected in staphylococcal enterotoxin C2, staphylococcal enterotoxin E, phytohemagglutinin, concanavalin A, anti-CD3 and anti-V beta 8-activated cells, although each cell population expressed a selective pattern of V delta genes. Sequence analysis revealed that each of the known V delta genes can productively rearrange to J alpha segments to produce functional V delta-J alpha-C alpha transcripts. These results argue strongly against the notion that the human V delta and V alpha repertoires are distinct. They further suggest that the restricted delta repertoire observed in many gamma/delta clones results from selection rather than from controlled rearrangements at the T cell receptor alpha/delta locus.
Amino Acid Sequence↗
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.
Adult↗
Sequence heterogeneity of the hepatitis C virus envelope region. Implications for the location of predicted antigenic determinants.
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Animals↗
DNA enzyme immunoassay: a rapid and convenient colorimetric method for diagnosis of cystic fibrosis.
Genetic analysis of inherited diseases has been greatly facilitated by new approaches, involving genomic DNA amplification by the polymerase chain reaction (PCR), followed by hybridization with wild type-specific or mutation-specific oligonucleotide (MSO) probes. The main advantage of these methods is that they allow easy detection of point mutations starting from minimal amounts of biological materials. These techniques, however, require procedures which are not well suited to large-scale screening or use in routine laboratories. The development of dedicated kits to perform these tests efficiently in clinical laboratories is an important current issue. We developed a new non-radioisotopic assay to reveal specifically DNA-DNA hybrids between amplified DNA and MSO probes, and applied it to the detection of two mutations causing cystic fibrosis. The detection of hybrids is achieved by means of an anti double-stranded DNA antibody, in a format which is designed as a colorimetric assay resembling a common enzymatic immunoassay (EIA). The assay detects the hybridization event, independent of the nucleic acid sequences involved in the formation of the specific hybrids, and can be used with any combination of target DNA and probes. Therefore, this test represents a significant improvement for the clinical use of the polymerase chain reaction in the diagnosis of inherited diseases.
Antibodies, Monoclonal↗
Detection of serum hepatitis C virus RNA in acute non-A, non-B hepatitis.
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Acute Disease↗
Heterogeneity of the hepatitis C virus genome.
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Base Sequence↗
DNA enzyme immunoassay: general method for detecting products of polymerase chain reaction.
We developed a new colorimetric method, DNA enzyme immunoassay (DEIA), for detecting specific hybrids of complementary nucleic acids and applied it to the detection of hepatitis B virus (HBV) DNA amplified from serum samples by means of the polymerase chain reaction (PCR) technique. The method is based on the ability of an anti-DNA monoclonal antibody to discriminate between single-stranded and double-stranded DNA. A solid phase was coated with a specific oligonucleotide probe, internal to the amplified region of HBV DNA, via an avidin-biotin bridge. The denatured PCR product was hybridized with the solid-phase probe, and the amplified DNA probe hybrid was then incubated with a monoclonal antibody specific for double- but not single-stranded DNA. Colorimetric detection of the DNA-antibody complex was achieved by adding an anti-mouse Ig antibody labeled with horseradish peroxidase. The combined use of DEIA and PCR can reveal a few HBV genome copies present in a serum sample. This method has several advantages: (a) the sensitivity is adequate for the detection of amplified DNA; (b) the signal is associated with the hybridization event, independently of modifications of the probe or of the amplification primers; and (c) the test is simple and rapid and, most importantly, requires only the standard facilities of a routine clinical laboratory.
Antibodies, Anti-Idiotypic↗
Hepatitis C virus RNA in symptomless donors implicated in post-transfusion non-A, non-B hepatitis.
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Base Sequence↗
On the mechanism of non-allelically excluded V alpha-J alpha T cell receptor secondary rearrangements in a murine T cell lymphoma.
We had previously demonstrated that several subclones derived from a CD3+, CD4-/CD8-, TCR-alpha beta+ murine T cell line have undergone secondary V alpha-J alpha rearrangements at the TCR-alpha locus (1). In an effort to examine the molecular mechanism responsible for these V alpha-J alpha replacements, the structures of TCR-alpha cDNA prepared from both the parental and subcloned T cell lines have been determined. Here we report that: 1) the mechanism whereby the secondary rearrangements occur is a precise deletion event that involves germ-line V alpha genes 5' to the preexisting V alpha-J alpha complex joining to J alpha segments 3' of the preexisting complex deleting the region in between, 2) preexisting productive V alpha-J alpha rearrangements of the parental line do not allelically exclude productive and nonproductive secondary rearrangements, 3) both productively rearranged TCR-alpha alleles of the parental cell line can undergo secondary rearrangements, 4) the presence of unrearranged germline V alpha transcripts in the parental line support an "accessibility" model of regulated lymphocyte receptor gene rearrangement. In addition, we present data which suggests that one of the subcloned lines has undergone a third rearrangement of one of its TCR-alpha alleles. One interpretation of these results is that T cells may have the ability to circumvent allelic exclusion at the TCR-alpha locus early in their ontogeny. This could provide T cells with an additional mechanism for generating an Ag receptor repertoire which is not found in B cells.
Alleles↗
Expression of the human V beta 8 gene product preferentially correlates with class II major histocompatibility complex restriction specificity.
We report that, in peripheral blood T cells, the V beta 8 gene product is much more represented on CD4+CD8- than on CD4-CD8+ lymphocytes. This skewing was observed in all individuals tested and with two independent strategies, one of which allows the selection of V beta 8+ T cells independently from their major histocompatibility complex-antigen specificity. Our data imply that the human V beta 8 gene product confers class II restriction specificity and that both the T cell receptor chains and CD4 molecule are involved in the selection of the T cell repertoire.
Gene Expression↗
High genomic variability in the pre-C region of hepatitis B virus in anti-HBe, HBV DNA-positive chronic hepatitis.
Some chronic HBV carriers have circulating HBV DNA despite the presence of anti-HBeAg antibodies. This observation has recently been related to the presence, in the HBV pre-C region, of a translational stop codon that prevents HBeAg synthesis. In the present study, we analyzed, at the nucleotide level, the pre-C/C region of HBV isolated from the sera of 11 anti-HBe, HBV DNA-positive chronic carriers. Nucleotide sequence analysis of 25 independent clones revealed that the pre-C sequence is highly variable, even among clones derived from the same serum sample. Moreover, our data show that HBeAg synthesis can also be prevented by as yet undescribed T-C substitution at position 1815 that eliminates the start codon of the pre-C transcript. These results suggest that the HBV genome contains segments of high variability that have probably been selected during evolution to favor the segregation of functionally advantaged mutants capable of avoiding host immunity.
Base Sequence↗
Effects of two levels of intake of chloride, potassium, and calcium on mineral and acid-base metabolism in premature infants.
Growth (weight, length, mid-upper arm circumference), acid-base status, serum electrolyte levels, and selected parameters of urine (creatinine and electrolyte levels, urine pH, renal net acid excretion, levels of metabolites of aldosterone) were determined serially from the 10th to the 32nd days for 4 weeks in 21 premature infants (birth weight, 1,100-2,000 g) fed either formula A or formula B (formula B was formula A supplemented with chloride, potassium, and calcium). Premature infants fed formula B showed a higher weight gain (31 versus 28.2 g/day), a higher increment of middle-upper arm circumference (0.31 versus 0.24 cm/week), and a decreased renal net acid excretion (1.24 versus 1.92 mEq/kg/day). Almost all premature infants fed formula A and some with a high growth rate receiving formula B showed hypochloruria corresponding to chloride deficiency. Premature infants fed unsupplemented humanized formulas may have an inadequate intake of minerals.
Acid-Base Equilibrium↗
The joining of germ-line V alpha to J alpha genes replaces the preexisting V alpha-J alpha complexes in a T cell receptor alpha, beta positive T cell line.
To determine whether T cell receptor genes follow the same principle of allelic exclusion as B lymphocytes, we have analyzed the rearrangements and expression of TCR alpha and beta genes in the progeny of the CD3+, CD4-/CD8- M14T line. Here, we show that this line can undergo secondary rearrangements that replace the pre-existing V alpha-J alpha rearrangements by joining an upstream V alpha gene to a downstream J alpha segment. Both the productively and nonproductively rearranged alleles in the M14T line can undergo secondary rearrangements while its TCR beta genes are stable. These secondary recombinations are usually productive, and new forms of TCR alpha polypeptides are expressed in these cells in association with the original C beta chain. Developmental control of this V alpha-J alpha replacement phenomenon could play a pivotal role in the thymic selection of the T cell repertoire.
Alleles↗
Induction of T cell receptor gamma chain transcription in murine T cells by trans-complementation.
As an initial approach to understanding the molecular basis of the developmentally regulated expression of T cell receptor (TcR) genes, we constructed hybrids among T cell clones that differ in their expression of CD4 and CD8 antigens as well as in alpha, beta and gamma mRNA levels. Here we report that the TcR gamma gene becomes transcriptionally active in hybrid cells formed between parental clones that lack TcR gamma mRNA. We also observed negative trans-regulation of TcR beta but not of TcR alpha transcription in one of these hybrids. Positive TcR gamma transcription was observed both with (CD4-CD8- X CD4-CD8-) and (CD4-CD8- X CD4+CD8+) hybrids while negative TcR beta gene regulation was only detected in (CD4-CD8- X CD4+CD8+) cells. These data suggest that the regulation of TcR alpha, beta and gamma genes is mediated by the action of specific transacting factors that become asynchronously active in the various stages of T cell development.
Animals↗
Rearrangement and expression of T cell receptor and immunoglobulin loci in immortalized CD4-CD8- T cell lines.
Injection of newborn mice with mixtures of wild-type moloney murine leukemia (Mo-MuLV) virus and other recombinant retroviruses harboring the myc oncogene alone or in combination with the H-ras oncogene resulted in a 100% incidence of lymphatic leukemias from which permanent cell lines could be established in vitro. These cells are immunoglobulin (Ig)-, Thy-1+BP- and CD8-CD4- indicating that they are early thymocytes. Such transformed pre-T lines lack retroviral myc and ras genes but occasionally possess proviral insertion near to their endogenous myc and pim genes. We show that both Ig heavy chain (Igh) and T cell receptor (TcR) genes are rearranged in most of these lines. In some cases, a primary recombination was followed by a secondary rearrangement at the same locus. We show that VT gamma genes can rearrange outside of their known cluster suggesting that TcR gamma diversification in such pre-T cells may be different to that in more mature T cells. Ig D-JH recombinations may precede TcR gene recombination in these early T cell lines, and some but not all express sterile Cmu transcripts. Some of these lines express surface heterodimers that appear composed of alpha and beta chains that can be immunoprecipitated with a monoclonal anti-T3 antibody but not with the anti-V beta 8 monoclonal antibody F23.1. This established pre-T cell line represents novel biological material for the dissection of T cell development and function analogous to A-MuLV transformed pre-B cells.
Animals↗
Cyclic AMP-modulated potassium channels in murine B cells and their precursors.
A voltage-dependent potassium current (the delayed rectifier) has been found in murine B cells and their precursors with the whole-cell patch-clamp technique. The type of channel involved in the generation of this current appears to be present throughout all stages of pre-B-cell differentiation, since it is detected in pre-B cell lines infected with Abelson murine leukemia virus; these cell lines represent various phases of B-cell development. Thus, the presence of this channel is not obviously correlated with B-cell differentiation. Although blocked by Co2+, the channel, or channels, does not appear to be activated by Ca2+ entry. It is, however, inactivated by high intracellular Ca2+ concentrations. In addition, elevation of intracellular adenosine 3', 5'-monophosphate induces at all potentials a rapid decrease in the peak potassium conductance and increased rates of activation and inactivation. Therefore, potassium channels can be physiologically modulated by second messengers in lymphocytes.
Animals↗