Incidence of autoimmune thyroiditis in interferon-alpha treated and untreated patients with chronic hepatitis C virus infection.

OBJECTIVES: To clarify the relationship between interferon-alpha (IFN-alpha) therapy and autoimmune thyroiditis in chronic hepatitis C virus (HCV) infection, we investigated a selected number of patients without basal thyroid dysfunctions. MATERIALS AND METHODS: 130 patients (average age: 20-70), with chronic HCV infection and without basal clinical and laboratory signs of autoimmune thyroiditis were divided into two groups: IFN-alpha treated (A) and untreated (B) patients. Group A received IFN-alpha (three million U.I./3 times a week) for six months; group B was followed for the same period. Thyroid peroxidase and thyroglobulin autoantibodies were measured by radioimmunoassay; thyroid function was measured by radioimmunoassay (free thyroxine and triiodothyronine) and immunoradiometric assay (thyroid stimulating hormone). RESULTS: After a 6-month period, thyroid autoantibodies positivity was documented in 21.1% of group A and in 10.3% of group B patients, both statistically relevant (p<0.001 and p<0.011, respectively). The comparison between the two groups was not statistically relevant (p=0.142). CONCLUSIONS: Our study showed a prevalence of de novo thyroid autoimmunity in chronic HCV patients treated with IFN-alpha, confirming previous data in literature. The lack of a significant difference between treated and untreated patients strongly suggests that the anti-thyroid autoimmune response is linked to the HCV infection itself. Moreover, IFN-alpha therapy probably does not represent a risk factor in renewing the autoimmune processes of the thyroid gland. Thyroid function and autoantibodies must be systematically monitored in patients with HCV infection, especially in female and IFN-alpha treated population, not only to verify the possible thyroid abnormalities but also to rule out concomitant autoimmune diseases.

Hashimoto's disease during interferon-alpha therapy in a patient with pre-treatment negative anti-thyroid autoantibodies and with the specific genetic susceptibility to the thyroid disease.

OBJECTIVES: The authors described a case of Hashimoto's disease during interferon-alpha (IFN-alpha) treatment for chronic viral C hepatitis in a patient with the specific genetic susceptibility associated with the thyroid disease. RESULTS: A 60-year-old woman with chronic active viral C hepatitis (HCV genotype = 3a) started IFN-alpha therapy in November '96. Before treatment thyroid function tests were normal and anti-thyroid (anti-thyroglobulin and anti-thyroid peroxidase) Abs were negative. During IFN therapy, serum aminotransferases fell within the normal range and viremia (serum HCV-RNA) became negative after one year. After 20 months, the patient presented clinical features of primary hypothyroidism. Anti-thyroid Abs were found positive. Hormonal, ultrasonographic, radioiodine scanning and fine needle aspiration findings were consistent with the diagnosis of Hashimoto's thyroiditis. The tissutal typing of the patient showed the presence of Human Leukocyte Antigen (HLA) DRB1*11 gene (corresponding to DR5 antigen). IFN-alpha therapy was suspended and a treatment with l-T4 started. Chronic viral infection relapsed after the suspension of the IFN-alpha therapy. CONCLUSIONS: This case report showed that the clinical appearance of Hashimoto's disease after IFN-alpha therapy for chronic C hepatitis in our patient was associated with a specific genetic predisposition (DR5) for this pathology. Further studies are necessary to evaluate whether the study of HLA antigens may be a very useful tool to detect the patients with a predisposition to develop autoimmune thyroiditis, in order to make a early diagnosis of thyroid disorders during the IFN-alpha treatment.

CAMP is involved in transcriptional regulation of delta9-desaturase during Histoplasma capsulatum morphogenesis.

We have characterized the promoter region of the delta9-desaturase gene from two different strains of the dimorphic fungus Histoplasma capsulatum. Desaturase transcription is regulated in the two phases of growth: it is transcribed in the yeast phase at 37 degrees C, while it is inactive in the mycelial phase at 25 degrees C. Phase transition can be induced by shifting the temperature from 25 to 37 degrees C or by adding cAMP to the growth medium. We have identified a stress-responsive cis element (STRE) responsive to cyclic AMP (cAMP)-signaling pathway and demonstrated that this element acts in H. capsulatum. We have also identified an element, hereafter called DRE (Desaturase Regulatory Element), present in the promoters of the H. capsulatum and S. cerevisiae delta9-desaturase gene. We show that this element is necessary but not sufficient to regulate transcription of the H. capsulatum delta9-desaturase gene.

An AP1 element is involved in transcriptional regulation of delta9-desaturase gene of Histoplasma capsulatum.

We have characterized a region of the promoter of a cloned delta9-desaturase gene (Ole1) of Histoplasma capsulatum, a dimorphic pathogenic fungus of humans. The product of the delta9-desaturase gene is involved in regulating membrane fluid state in animal cells and microorganisms. To identify sequences critical for Ole1 expression in both the saprobic mycelial and parasitic yeast phases of this organism, we performed a deletion analysis. Evidence is presented that a 240 nt region of the proximal promoter is involved in a phase-specific binding in vitro. By sequence analysis we have identified one likely regulatory element that coincides with an AP1 binding site (TGACTAA) that is located at -740 nt of 5'-upstream from the ATG. Using gel mobility shift assays, we show that this cis-acting element binds nuclear proteins extracted from the yeast and mycelial phases of H. capsulatum that may participate in control of expression of the delta9-desaturase gene.

Integration of an Epstein-Barr virus episome 3' into the gene encoding immunoglobulin heavy-chain alpha 1 in a lymphoblastoid cell line.

For the first time we have characterized an unoccupied site of Epstein-Barr (EBV) virus integration in a lymphoblastoid cell line, RGN1. The site of integration is about 1.5 kb downstream from the gene encoding the heavy chain constant alpha 1, specifying immunoglobulin A (IgA). Sequence and Southern analysis allowed us to hypothesize that integration occurred via a double exchange involving the viral latent origin of DNA replication (oriP) and the human DNA. The region involved in the integration is transcribed into poly(A)+ RNA in all the tested lymphoid lines, but not in the RGN1 line. We suggest a mechanism of integration primed by interactions between oriP and cell ori and its potential role in the establishment and/or evolution of EBV-carrying lines.

The transcriptionally competent U2 gene is necessary and sufficient for adenovirus type 12 induction of the fragile site at 17q21-22.

Adenovirus type 12 induces four fragile sites upon infection of human cells. The U2 locus, consisting of up to 20 tandem repeats of a 5.8-kbp monomer, maps at the most sensitive of these sites at 17q21-22. We have previously shown that an artificial U2 locus integrated into the human genome generates a new virus-induced fragile site. To determine which elements within the U2 monomer are responsible for fragility, we constructed loci consisting of tandem repeats of subfragments of the U2 monomer. With this approach, we demonstrate that a transcriptionally competent U2 gene is necessary and sufficient for virus-induced fragility and that no other element within the 5.8-kbp monomer contributes to this effect.

A temperature-sensitive strain of Histoplasma capsulatum has an altered delta 9-fatty acid desaturase gene.

We have isolated and characterized the delta 9-desaturase gene (Ole1), which codes for a key enzyme involved in regulating membrane fluidity in animal cells and microorganisms, from two strains of Histoplasma capsulatum, one that is temperature-tolerant (G217B) and the other temperature-susceptible (Downs). These pathogenic fungi are dimorphic in that they undergo a morphologic transition from the mycelial to yeast-like form when the temperature of incubation is switched from 25 to 37 degrees C or when they infect a susceptible host. The coding sequences of the two genes, both containing an intron of 93 nucleotides, are virtually identical and analogous to the delta 9-desaturase gene of Saccharomyces cerevisiae and those of the rat, mouse and human. Ole1 transcription of the thermotolerant G217B and thermosensitive Downs strains is similar in yeast phase cells and during the temperature shift down from 34, 37, or 40 to 25 degrees C (yeast-to-mycelia transition). Nevertheless, the delta 9-desaturase gene is transcriptionally inactive in mycelia of G217B at 25 degrees C while it is actively transcribed in the Downs strain at the same temperature. These results are in agreement with the finding that membranes of the Downs strain have a higher level of oleic acid. The differential expression of delta 9-desaturase genes is discussed in relationship to differences in thermosensitivity in the fungal isolates and in regulating the level of expression of heat shock genes.

Bacteriophage T4 gene 28.

The complete nucleotide sequence of bacteriophage T4D gene 28 has been determined. Gene 28 product is a structural component of the viral baseplate for which an enzymatic activity has also been proposed.

The Histoplasma capsulatum cdc2 gene is transcriptionally regulated during the morphologic transition.

To understand the molecular mechanisms that control the reversible morphologic transition from mycelia to yeast in dimorphic fungi, we have isolated and characterized a cdc2 gene from Histoplasma capsulatum. This organism is a dimorphic pathogenic fungus that grows as a filamentous saprobic mold in soil and as a unicellular pathogenic yeast in human tissue. The cloned gene, whose protein product has a high degree of homology with other members of the cdc2 family, is split into four exons and three introns of 95, 52 and 85 nucleotides. Analyses of cDNA clones confirm the presence of the eukaryotic splice donor (GT) and acceptor (AG) sites. The spliced gene codes for a protein of 324 amino acids (aa) with a predicted molecular mass of 36.9 kDa. The H. capsulatum cdc2 product has 71% aa identity with Saccharomyces cerevisiae and 70% with Schizosaccharomyces pombe. The deduced protein contains the sequence, PSTAIRE, that is normally found in most p34cdc2 proteins. H. capsulatum cdc2 is transcriptionally regulated during the morphologic mycelium<==>yeast transitions and is more actively transcribed in the yeast than in the mycelial phase.

The search for a gene involved in the determination of limited duplicative capacity in human cells.

On the assumption that EBV integration into the genome of human B lymphocyte might lead to the inactivation of a hypothetical gene determining the limited duplicative capacity and consequently participate to the cell immortalization, a search for the human-virus junction was done. This led to the identification of a site of integration in the central part of the heavy chain of the immunoglobulin region. The coincidence of the involvement of the site in lymphomatogenesis with the first complete characterization of an integration site led to the speculation that the heavy chain gene itself might be an important controller of cell duplication in the B lymphocyte.

The DNA polymerase-encoding gene of Bacillus subtilis bacteriophage SPO1.

The bacteriophage SPO1 DNA polymerase-encoding gene, which contains a self-splicing intron, has been sequenced and its amino acid (aa) sequence has been deduced. The aa sequence of SPO1 DNA polymerase shows a high degree of similarity with that of DNA polymerase I from Escherichia coli (Po1I). Alignment with the sequences of Po1I, and the phi 29 and SPO1 DNA polymerases indicate that the aa residues that have been implicated in 3'----5' exonuclease activities are conserved.

Molecular cloning and expression of hsp82 gene of the dimorphic pathogenic fungus Histoplasma capsulatum.

We have cloned a nucleotide sequence from Histoplasma capsulatum G222B corresponding to a heat inducible hsp82 gene, and determined its entire sequence and the flanking regions. During the temperature-controlled mycelium-to-yeast phase transition the gene is more actively transcribed at 37 degrees C in the temperature tolerant and mouse-virulent G222B strain, while 34 degrees C is the optimum for transcription in the temperature sensitive and mouse-avirulent Downs strain.

Different localization of Epstein-Barr virus genome in two subclones of the Burkitt lymphoma cell line Namalwa.

The Epstein-Barr virus genome contained in the Burkitt lymphoma line Namalwa was previously localized to the short arm of chromosome 1. Analysis of a different subline of the same Namalwa line by means of Southern analysis carried out on genomic DNA, as well as in situ hybridization, showed a localization on the X chromosome.

The intron-containing hsp82 gene of the dimorphic pathogenic fungus Histoplasma capsulatum is properly spliced in severe heat shock conditions.

We have isolated and characterized a heat-inducible gene, hsp82, from the dimorphic pathogenic fungus Histoplasma capsulatum, which is a filamentous mold at 25 degrees C and a unicellular yeast at 37 degrees C. This gene, which has a high degree of homology with other members of the hsp82 gene family, is split into three exons and two introns of 122 and 86 nucleotides, respectively. Contrary to what has been demonstrated in Drosophila melanogaster, Saccharomyces cerevisiae, and other organisms, hsp82 mRNA in H. capsulatum is properly spliced during the severe heat conditions of 37 to 40 degrees C in the temperature-sensitive Downs strain. Splicing accuracy was also observed at 42 degrees C in the temperature-tolerant G222B strain, which showed no evidence of accumulation of primary transcripts. Furthermore, the intron containing the beta-tubulin gene is also properly spliced at the upper temperature range, suggesting that the lack of a block in splicing may be a general phenomenon in this organism.

Two bacteriophage T4 base plate genes (25 and 26) and the DNA repair gene uvsY belong to spatially and temporally overlapping transcription units.

The bacteriophage T4 DNA recombination-repair gene uvsY located at or near an origin of DNA replication and adjacent to the late base plate genes 25 and 26. Our present results reveal a complex transcription pattern in the region encompassing these genes. Most significantly, uvsY and two ORFs, downstream of it, all of which are transcribed from a middle promoter before the onset of DNA replication, are also part of a larger late transcription unit which includes the base plate genes 25 and 26. The late genes 25 and 26 are transcribed not only late, but also early from one or several early promoters further upstream. Translation, however, is inhibited by secondary structures which sequester the ribosome binding site in the early transcript. We discuss possible advantages of these transcriptional patterns for T4 DNA recombination, replication, and repair. The predicted and in vivo-expressed 23.9-kDa product of gene 26 is smaller than the reported size of gene 26 protein isolated from base plates, suggesting that nascent gp26 might be processed to a larger protein during assembly.

Bacteriophage T4 late gene expression: overlapping promoters direct divergent transcription of the base plate gene cluster.

Eight 5' ends of RNA molecules which encompass the bacteriophage T4 base plate late genes 51 to 26 region have been mapped by S1 nuclease protection and reverse transcription within a 246-bp DNA segment. Two of eight 5' ends are initiated at two absolutely conserved late promoter sites, P51 and P26a, that direct RNA synthesis on opposite strands. These two promoters share four of eight promoter sequence base pairs. A third 5' end arises from another promoter, P26b, which shows one base pair mismatch with respect to the absolutely conserved -10 sequence. All the other 5' ends arise from RNA processing and/or degradation. Since no other late transcription promoter sites were found within the base plate cluster sequence, we propose that the two overlapping late promoters, P51 and P26a, direct the expression of the T4 base plate gene cluster, included between map coordinates 114,000 and 121,038: P51 directs the transcription of genes 51, 27, 28, 29, 48, and 54 on the rDNA strand and P26a the transcription of genes 26 and 25 on the /DNA strand. This peculiar promoter configuration might account for the low level of transcription of these late genes.

Symmetric transcription of bacteriophage T4 base plate genes.

Dot-blot and Northern-blot experiments, using strand-specific RNA probes, show that part of the bacteriophage T4 DNA that codes for six of the base plate structural genes (gp 51, 27, 28, 29, 48 and 54), is transcribed in vivo from both DNA strands. The r DNA strand transcripts contain sequences which are translated into structural proteins. Antisense l strand RNA is about 100 fold less abundant than RNA molecules transcribed from the r DNA strand.

The pro alpha 2(V) collagen gene is evolutionarily related to the major fibrillar-forming collagens.

A number of overlapping cDNA clones, covering 5.2 kb of sequences which code for the human pro alpha 2(V) collagen chain, have been isolated. Analysis of the structural data have indicated a close evolutionary kinship between the pro alpha 2(V) chain and the major fibrillar collagen types. Isolation and analysis of an 8 kb genomic fragment has further supported this notion by revealing a homologous arrangement of nine triple-helical domain exons. These studies have therefore provided conclusive evidence which categorizes the Type V collagen as a member of the Group 1 molecules, or fibrillar-forming collagens.

Antibiotic prophylaxis in high-risk biliary operations: multicenter trial of single preoperative ceftriaxone versus multidose cefazolin.

A prospective, double-blind trial of a single preoperative dose of ceftriaxone, a new long-acting cephalosporin, versus one preoperative and three postoperative doses of cefazolin was carried out in 81 patients at high risk of infection after biliary surgery. Indications for antibiotic prophylaxis included recent or ongoing cholecystitis (52 patients), common duct stones (14 patients), common duct obstruction (3 patients), and age greater than 70 years (22 patients). Intraoperative bile cultures were positive in 7 of 41 patients (17.1 percent) given ceftriaxone and 12 of 40 patients (30 percent) given cefazolin, but there were no wound infections in either group. Neither regimen was associated with significant antibiotic resistance. Side effects, such as proteinuria and elevated liver transaminases and alkaline phosphatase levels, were transient and not definitely related to the antibiotics. We conclude that a single preoperative dose of ceftriaxone is as effective as multiple perioperative doses of cefazolin in the prophylaxis of infection associated with biliary tract surgery.