Search PubMed⌕ Search

Biomedical subjects

A Vitelli

Publications and source records attributed to A Vitelli.

At least 19 recordsLinked to original sources
PubMed2006

Early impairment of hepatitis C virus specific T cell proliferation during acute infection leads to failure of viral clearance.

BACKGROUND AND AIMS: Cellular mediated immunity (CMI) is thought to play a key role in resolution of primary hepatitis C virus (HCV) infection. However, CD4+ and CD8+ T cell responses are also generated during acute infection in individuals who become chronic, suggesting that they developed a defective CMI. The aim of this study was to verify if and when such immune dysfunction is established by measuring the breadth, magnitude, function, and duration of CMI in a large cohort of subjects during the natural course of acute HCV infection. METHODS: CMI was comprehensively studied by prospective sampling of 31 HCV acutely infected subjects enrolled at the onset of infection and followed for a median period of one year. RESULTS: Our results indicated that while at the onset of acute HCV infection a measurable CMI with effector function was detected in the majority of subjects, after approximately six months less than 10% of chronically infected individuals displayed significant CMI compared with 70% of subjects who cleared the virus. We showed that progressive disappearance of HCV specific T cells from the peripheral blood of chronic patients was due to an impaired ability to proliferate that could be rescued in vitro by concomitant exposure to interleukin 2 and the antigen. CONCLUSION: Our data provide evidence of strong and multispecific T cell responses with a sustained ability to proliferate in response to antigen stimulation as reliable pharmacodynamic measures of a protective CMI during acute infection, and suggest that early impairment of proliferation may contribute to loss of T cell response and chronic HCV persistence.

Acute Disease↗
PubMed2004

The cytoarchitecture of sulcal folding in Heschl's sulcus and the temporal cortex in the normal brain and schizophrenia: lamina thickness and cell density.

Developmental and psychiatric disorders, including schizophrenia, may be associated with altered cortical thickness and folding. Two studies were performed: (1) to assess cortical layering around a sulcus; cortical thickness, relative thickness of the supragranular (I-III):infragranular (IV-VI) layers, and cell density were assessed at anatomically defined points around Heschl's sulcus in tissue from 10 controls and 10 schizophrenia patients. (2) To sample sulci of contrasting prominence; sulcal depth, width, lamina thickness, and cell density from laminae II-VI were taken from various sulci within the temporal lobes from another group of 6 controls and 10 patients. Reduced cell density was found in the fundi of sulci in schizophrenia. Independent of diagnosis; increased sulcal prominence in temporal cortex accompanies reduced lamina thickness (particularly layers V and VI), deep layers show negative relationships between cell density and layer thickness, and total cortex width in Heschl's sulcus reduces by half at the bottom compared to the top. Furthermore, compared to the supragranular layers, the infragranular division is relatively thicker at the top of a gyrus, equal in the wall of the sulcus and relatively thinner at the bottom. Many effects of sulcal folding on laminar proportions in controls are similar in schizophrenia. However, cell density is less at the bottom of some sulci in the temporal lobe in schizophrenia. Sampling methods should consider that cortical folding affects cell and lamina distribution in the sampled region in a highly localised manner.

Aged↗
PubMed2004

Multispecific T cell response and negative HCV RNA tests during acute HCV infection are early prognostic factors of spontaneous clearance.

BACKGROUND/AIMS: Hepatitis C virus (HCV) infection results in a high frequency of chronic disease. The aim of this study was to identify early prognostic markers of disease resolution by performing a comprehensive analysis of viral and host factors during the natural course of acute HCV infection. METHODS: The clinical course of acute hepatitis C was determined in 34 consecutive patients. Epidemiological and virological parameters, as well as cell mediated immunity (CMI) and distribution of human leukocyte antigens (HLA) alleles were analysed. RESULTS: Ten out of 34 patients experienced self-limiting infection, with most resolving patients showing fast kinetics of viral clearance: at least one negative HCV RNA test during this phase predicted a favourable outcome. Among other clinical epidemiological parameters measured, the self-limiting course was significantly associated with higher median peak bilirubin levels at the onset of disease, and with the female sex, but only the latter parameter was independently associated after multivariate analysis. No significant differences between self-limiting or chronic course were observed for the distribution of DRB1 and DQB1 alleles. HCV specific T cell response was more frequently detected during acute HCV infection, than in patients with chronic HCV disease. A significantly broader T cell response was found in patients with self-limiting infection than in those with chronic evolving acute hepatitis C. CONCLUSION: The results suggest that host related factors, in particular sex and CMI, play a crucial role in the spontaneous clearance of this virus. Most importantly, a negative HCV RNA test and broad CMI within the first month after onset of the symptoms represent very efficacious predictors of viral clearance and could thus be used as criteria in selecting candidates for early antiviral treatment.

Adult↗
PubMed2000

Biochemical characterization of a hepatitis C virus RNA-dependent RNA polymerase mutant lacking the C-terminal hydrophobic sequence.

The RNA-dependent RNA polymerase activity of hepatitis C virus is carried out by the NS5B protein. The full-length protein was previously purified as a non-fusion protein from insect cells infected with a recombinant baculovirus. The characterization is now described of a C-terminal hydrophobic domain deletion mutant of NS5B purified from E. coli. In addition to increased solubility, deletion of this sequence also positively affected the polymerase enzymatic activity. The efficiency of nucleotide polymerization of both the full-length and the C-terminal truncated enzymes were compared on homopolymeric template-primer couples as well as on RNA templates with heteropolymeric sequences. The largest difference in the polymerase activity was observed on the latter. On all the templates, the increased activity could be ascribed, at least in part, to enhanced template turnover of the deletion mutant with respect to the full-length enzyme. The elongation rates of the two enzyme forms were compared under single processive cycle conditions. Under these conditions, both the full-length and the deletion mutant were able to incorporate about 700 nt/min.

Animals↗
PubMed2000

Binding of hepatitis C virus E2 glycoprotein to CD81 does not correlate with species permissiveness to infection.

Hepatitis C virus (HCV) glycoprotein E2 binds to human cells by interacting with the CD81 molecule, which has been proposed to be the viral receptor. A correlation between binding to CD81 and species permissiveness to HCV infection has also been reported. We have determined the sequence of CD81 from the tamarin, a primate species known to be refractory to HCV infection. Tamarin CD81 (t-CD81) differs from the human molecule at 5 amino acid positions (155, 163, 169, 180, and 196) within the large extracellular loop (LEL), where the binding site for E2 has been located. Soluble recombinant forms of human CD81 (h-CD81), t-CD81, and African green monkey CD81 (agm-CD81) LEL molecules were analyzed by enzyme-linked immunosorbent assay for binding to E2 glycoprotein. Both h-CD81 and t-CD81 molecules were able to bind E2. Competition experiments showed that the two receptors cross-compete and that the t-CD81 binds with stronger affinity than the human molecule. Recently, h-CD81 residue 186 has been characterized as the critical residue involved in the interaction with E2. Recombinant CD81 mutant proteins were expressed to test whether human and tamarin receptors interacted with E2 in a comparable manner. Mutation of residue 186 (F186L) dramatically reduced the binding capability of t-CD81, a result that has already been demonstrated for the human receptor, whereas the opposite mutation (L186F) in agm-CD81 resulted in a neat gain of binding activity. Finally, the in vitro data were confirmed by detection of E2 binding to cotton-top tamarin (Saguinus oedipus) cell line B95-8 expressing endogenous CD81. These results indicate that the binding of E2 to CD81 is not predictive of an infection-producing interaction between HCV and host cells.

Amino Acid Sequence↗
PubMed2000

In vivo selection of protease cleavage sites by using chimeric Sindbis virus libraries.

Identifying protease cleavage sites contributes to our understanding of their specificity and biochemical properties and can help in designing specific inhibitors. One route to this end is the generation and screening of random libraries of cleavage sites. Both synthetic and phage-displayed libraries have been extensively used in vitro. We describe a novel system based on recombinant Sindbis virus which can be used to identify cleavage sites in vivo, thus eliminating the need for a purified enzyme and overcoming the problem of choosing the correct in vitro conditions. As a model we used the serine protease of the hepatitis C virus (HCV). We engineered the gene coding for this enzyme and two specific cleavage sites in the Sindbis virus structural gene and constructed libraries of viral genomes with a random sequence at either of the cleavage sites. The system was designed so that only viral genomes coding for sequences cleaved by the protease would produce viable viruses. With this system we selected viruses containing sequences mirroring those of the natural HCV protease substrates which were cleaved with comparable efficiencies.

Antibodies↗
PubMed1999

A high-throughput radiometric assay for hepatitis C virus NS3 protease.

A novel radiometric in vitro assay for discovery of inhibitors of hepatitis C viral protease activity, suitable for high-throughput screening, was developed. The NS3 protein of hepatitis C virus (HCV) contains a serine protease, whose function is to process the majority of the nonstructural proteins of the viral polyprotein. The viral NS4A protein is a cofactor of NS3 protease activity in the cleavage of NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions. To establish an in vitro assay system we used NS3 proteases from different HCV strains, purified from Escherichia coli and a synthetic radiolabeled peptide substrate that mimics the NS4A-NS4B junction. Upon incubation with the enzyme the substrate was separated from the radiolabeled cleavage product by addition of an ion exchange resin. The assay was performed in a microtiter plate format and offered the potential for assaying numerous samples using a laboratory robot. Taking advantage of these features, we used the assay to optimize reaction conditions by simultaneously varying different buffer components. We showed that physicochemical conditions affect NS3 protease activity in a strain-specific way. Furthermore, the sensitivity of the assay makes it suitable for detection and detailed mechanistic characterization of inhibitors with low-nanomolar affinities for the HCV serine protease.

Buffers↗
PubMed1998

Enhanced inflammatory response to coronary angioplasty in patients with severe unstable angina.

BACKGROUND: Systemic markers of inflammation have been found in unstable angina. Disruption of culprit coronary stenoses may cause a greater inflammatory response in patients with unstable than those with stable angina. We assessed the time course of C-reactive protein (CRP), serum amyloid A protein (SAA), and interleukin-6 (IL-6) after single-vessel PTCA in 30 patients with stable and 56 patients with unstable angina (protocol A). We also studied 12 patients with stable and 15 with unstable angina after diagnostic coronary angiography (protocol B). METHODS AND RESULTS: Peripheral blood samples were taken before and 6, 24, 48, and 72 hours after PTCA or angiography. In protocol A, baseline CRP, SAA, and IL-6 levels were normal in 87% of stable and 29% of unstable patients. After PTCA, CRP, SAA, and IL-6 did not change in stable patients and unstable patients with normal baseline levels but increased in unstable patients with raised baseline levels (all P<0.001). In protocol B, CRP, SAA, and IL-6 did not change in stable angina patients after angiography but increased in unstable angina patients (all P<0.05). Baseline CRP and SAA levels correlated with their peak values after PTCA and angiography (all P<0.001). CONCLUSIONS: Our data suggest that plaque rupture per se is not the main cause of the acute-phase protein increase in unstable angina and that increased baseline levels of acute-phase proteins are a marker of the hyperresponsiveness of the inflammatory system even to small stimuli. Thus, an enhanced inflammatory response to nonspecific stimuli may be involved in the pathogenesis of unstable angina.

Acute-Phase Proteins↗
PubMed1996

Elevated levels of interleukin-6 in unstable angina.

BACKGROUND: Elevated plasma levels of C-reactive protein have been found in the majority of patients with unstable angina. The evidence of elevated levels of acute-phase proteins in unstable angina is in line with a growing body of evidence that suggests that inflammation plays a role in this syndrome and is an indirect sign of increased production of interleukin-6, which is the major determinant of acute-phase-protein production by the liver. However, in unstable angina, there is no direct proof of the role played by interleukin-6. METHODS AND RESULTS: We measured levels of interleukin-6 in 38 patients with unstable angina at the time of their admission to the coronary care unit and in 29 patients with stable angina. In the same groups of patients, we also measured C-reactive protein. Interleukin-6 (undetectable, ie, < 3 pg/mL, in healthy volunteers) was detectable in 23 (61%) of 38 patients with unstable angina but in only 6 (21%) of 29 with stable angina (P < .01). Median interleukin-6 levels were 5.25 pg/mL (range, 0 to 90 pg/mL) in patients with unstable angina but were below the detection limit of the assay in patients with stable angina (range, 0 to 7 pg/mL). A significant correlation was observed between interleukin-6 and C-reactive protein levels (r = .4, P = .013). CONCLUSIONS: Our study demonstrates that raised levels of interleukin-6 are common in unstable angina, correlate with C-reactive protein, and are associated with prognosis, thus confirming the importance of the cytokine pathway for the production by the liver of acute-phase proteins and strengthening the importance of inflammation in this syndrome. Further studies are required to elucidate better the role of interleukins in unstable angina.

Aged↗
PubMed1993

The two C/EBP isoforms, IL-6DBP/NF-IL6 and C/EBP delta/NF-IL6 beta, are induced by IL-6 to promote acute phase gene transcription via different mechanisms.

The promoter regions of three IL-6 inducible genes, hemopexin (Hpx), haptoglobin (Hp) and C-reactive protein (CRP) contain cis-acting IL-6 responsive elements (IL-6REs) which are necessary and sufficient to induce IL-6 transcription activation. Transcription factors of the C/EBP family interact with IL-6REs. Among these, IL-6DBP/NF-IL6 plays a key role in IL-6 signal transduction because its trans-activation potential is induced by IL-6 in the human hepatoma cell line Hep3B. We show here that a different C/EBP-related factor, C/EBP delta/NF-IL6 beta, is the major IL-6 induced protein interacting with IL-6REs in the nuclei of Hep3B cells. In contrast to IL-6DBP/NF-IL6, whose activity in Hep3B cells is modulated by IL-6 via a post-translational mechanism, C/EBP delta/NF-IL6 beta is transcriptionally induced by IL-6. Another contrasting feature is that the C/EBP delta cDNA transfected in Hep3B cells activates transcription from an IL-6RE synthetic promoter in a constitutive manner which is not further enhanced by IL-6. Therefore, in Hep3B cells, two distinct members of the C/EBP family are recruited in the IL-6 signal transduction pathway via different mechanisms.

Acute-Phase Proteins↗
PubMed1991

Identification of regulatory residues of the yeast adenylyl cyclase.

We have attempted to identify amino acid residues of the yeast adenylyl cyclase that are involved in the regulation of its activity, by isolating adenylyl cyclase-linked spontaneous mutations capable of suppressing the temperature-sensitive phenotype of ras1- ras2-ts1 strains. We previously identified a mutated adenylyl cyclase in which a single point mutation, called CR14, led to the replacement of threonine 1651 with isoleucine. We have now investigated the biological effects of CR14, and of other mutations that cause the replacement of threonine 1651 by distinct amino acids. We have observed that the response of adenylyl cyclase to Ras can be either enhanced or attenuated, without significant effects on the steady-state level of the former enzyme in vivo, depending on the amino acid side chain at position 1651. Therefore, this residue identifies a regulatory region on the adenylyl cyclase molecule. We have also taken advantage of the attenuation of adenylyl cyclase function caused by the replacement of threonine 1651 with aspartic acid to isolate intragenic suppressor mutations. We have identified several point mutations, leading to single amino acid substitutions, individually capable of reactivating the attenuated adenylyl cyclase. The corresponding amino acid changes are located within a relatively small region, including residues 1331, 1345, 1348 and 1374. This region could be physiologically involved in the negative control of the carboxy-terminal catalytic domain.

Adenylyl Cyclases↗
PubMed1989

The origin of DNA single strand breaks induced by ethylating agents in mammalian cells.

Chinese hamster ovary (CHO) cells were treated with two ethylating agents, N-ethyl-N-nitrosourea (ENU) and diethylsulfate (DES), and the kinetics of DNA single strand break (ssb) induction and rejoining were determined in parallel with DNA adduct formation and removal. In the case of DES, DNA ssb as determined by alkaline elution (AE) were repaired very slowly with more than 50% of the lesions still present on DNA 3 h after treatment. In contrast, 45% of ENU-induced ssb were repaired within 10 min. From the relative concentration of the different ethylated products and their repair rates as measured by high performance liquid chromatography (HPLC) analysis of the ethylated DNA, a theoretical function was constructed that describes the number of ssb expected at each time point after exposure to the mutagen. DES-induced ssb are explained by excision repair processes active on the ethylated purines, mainly 3-ethyladenine (3-EtAde) and 7-ethylguanine (7-EtGua). On the same basis, the rapidly repaired ENU-induced ssb remain unexplained. These results are also discussed in relation to the sensitivity of the two techniques, AE and HPLC, for detecting DNA damage.

Alkylating Agents↗
PubMed1988

Yeast mutants temperature-sensitive for growth after random mutagenesis of the chromosomal RAS2 gene and deletion of the RAS1 gene.

Saccharomyces cerevisiae strains with a disrupted RAS1 gene and with an intact RAS2 gene (ras1- RAS2 strains) grew well on both fermentable and nonfermentable carbon sources. By constructing isogenic mutants having a disrupted RAS1 locus and a randomly mutagenized chromosomal RAS2 gene, we obtained yeast strains with specific growth defects. The strain TS1 was unable to grow on nonfermentable carbon sources and galactose at 37 degrees C, while it could grow on glucose at the same temperature. The mutated RAS2 gene in TS1 cells encoded a protein with the glycines at positions 82 and 84 replaced by serine and arginine respectively. Both mutations were necessary for temperature sensitivity. We also isolated a mutant yeast that was unable to grow on nonfermentable carbon sources both at 30 and 37 degrees C, while growing on glucose at both temperatures. This phenotype was caused by a single chromosomal mutation, leading to the replacement of aspartic acid 40 of the RAS2 protein by asparagine. A ras1- yeast strain with a chromosomal RAS2 gene harbouring the three mutations together did not grow at any temperature using non-fermentable carbon sources, but it was able to grow on glucose at 30 degrees C, and not at 37 degrees C. The mutated proteins were much less effective than the wild-type RAS2 protein in the stimulation of adenylate cyclase, but were efficiently expressed in vivo. The possible roles of residues 40, 82 and 84 of the RAS2 protein in the regulation of adenylate cyclase are discussed.

Adenylyl Cyclases↗
PubMed1988

Relationship between specific alkylated bases and mutations at two gene loci induced by ethylnitrosourea and diethyl sulfate in CHO cells.

DNA adduct formation and induction of mutations at 2 gene loci, hypoxanthine-guanine-phosphoribosyltransferase (HPRT) and Na,K-ATPase, were determined simultaneously in Chinese hamster ovary (CHO) cells after treatment with 2 ethylating agents, ethylnitrosourea (ENU) or diethyl sulfate (DES). Doses of DES and ENU, which resulted in equal levels of O6-ethylguanine (O6-EtGua) and O4-ethylthymine (O4-EtThy) in the DNA, were found to induce very similar frequencies of 6-thioguanine-resistant (6-TGr) mutants. Formation of these DNA adducts might therefore be correlated with mutations induced at the HPRT locus. When, however, the same analysis was applied to ouabain-resistant (ouar) mutants, it was found that, at similar levels of O6-EtGua and O4-EtThy, DES induced many more ouar mutants than ENU. This result supports the notion that primary DNA lesions other than O6-EtGua and O4-EtThy are involved in the fixation of ENU- and DES-induced mutations at the Na,K-ATPase gene locus.

Alkylating Agents↗
PubMed1987

Induction kinetics of mutations at two genetic loci, DNA damage and repair in CHO cells after different exposure times to N-ethyl-N-nitrosourea.

Ouabain resistance (ouar) and 6-thioguanine resistance (6-TGr) mutation frequencies were measured in Chinese hamster ovary cells after treatment with N-ethyl-N-nitrosourea (ENU) for varying periods of time. Maximal mutation frequency at the Na+/K+ ATPase gene locus (ouar mutations) was attained within 5 min of exposure, whereas the mutation frequency at the hypoxanthine guanine phosphoribosyltransferase locus (6-TGr mutations) continued to increase up to 60 min, following the theoretical curve for exponential decay of ENU with time. Detection of DNA single strand breaks (ssb) by alkaline elution showed that maximal levels were attained within 5 min of treatment with ENU. Fast repair of DNA ssb occurred early after exposure (greater than 50% repair within 10 min). Analysis of DNA ethylation products by h.p.l.c. showed initially rapid removal of O2-ethylcytosine (25% in the first hour), slow removal of 7-ethylguanine, 3-ethyladenine and 3-ethylguanine and no removal at all of O6-ethylguanine, O4-ethylthymine and ethylphosphotriesters. These time-course studies reveal different target gene responses in the fixation of DNA damage into mutations.

Animals↗
PubMed1986

Suppression of defective RAS1 and RAS2 functions in yeast by an adenylate cyclase activated by a single amino acid change.

We have constructed the yeast strain TS1, with the RAS2 gene replaced by mutant allele encoding a partially defective gene product, and with an inactive RAS1 gene. TS1 cells accumulate as unbudded cells upon temperature shift from 30 to 37 degrees C, thus showing that the RAS1 and RAS2 gene functions are important for progression through the G1 phase of the cell cycle. After the isolation of revertants able to grow at the nonpermissive temperature, we have found that a chromosomal point mutation can bypass the G1 arrest of TS1 and cdc25 cells, and the lethality of ras1 ras2 mutants. The mutation predicts the replacement of threonine by isoleucine at position 1651 of yeast adenylate cyclase. The RAS-independent, as well as the RAS-dependent adenylate cyclase activity, is increased by the mutation. Like the wild-type enzyme, the RAS-dependent activity of the mutant adenylate cyclase is turned on by the GTP-bound form of the RAS2 protein. The amino acid sequence surrounding the threonine 1651 shows similarity with protein kinase substrates. Possible implications for the function of adenylate cyclase are discussed.

Adenylyl Cyclases↗
PubMed1986

Continuous subcutaneous insulin infusion versus intensive conventional insulin therapy in type I and type II diabetic pregnancy.

Two groups of pregnant diabetic women, fifteen with type I and fourteen with type II diabetes, were randomly assigned either to CSII or to ICT and the subgroups compared with respect to glycaemic control, insulin requirement and perinatal out-come. Ten non-diabetic pregnant women served as controls for the variations in the metabolic parameters considered (24-hour mean blood glucose and glycosylated hemoglobin). Strict glycaemic control was achieved and maintained by both regimens before week 13 in all patients with type I and in 57.1% of patients with type II diabetes. The mean insulin requirements in the type I group increased up to week 34-36 and then stabilized to term in patients receiving CSII and rose progressively to term in those receiving ICT. In the type II group insulin requirements rose up to week 36 in patients receiving CSII and up to week 32 in those receiving ICT, stabilizing thereafter on both regimens. No significant differences in mean insulin requirement at the different stages of gestation were found between the patients receiving CSII and those receiving ICT of either group. Perinatal outcome was satisfactory in both groups, although control of foetal growth was better with ICT than with CSII. CSII is a practical, safe and effective method of maintaining maternal normoglycemia in pregnancy but for the present we cannot consider it superior to ICT in the treatment of pregnant diabetic women.

Adolescent↗