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Biomedical subjects

G Marchetti

Publications and source records attributed to G Marchetti.

At least 37 records · Page 2Linked to original sources
PubMed2000

Modulation of factor VII levels by intron 7 polymorphisms: population and in vitro studies.

Previous studies have established that factor VII gene (F7) polymorphisms (5'F7 and R353Q) contribute about one-third of factor VII (FVII) level variation in plasma. However, F7 genotyping in patients with cardiovascular disease has produced conflicting results. Population and expression studies were used to investigate the role of intron 7 (IVS7 ) polymorphisms, including repeat and sequence variations, in controlling activated FVII (FVIIa) and antigen (FVIIag) levels. Genotype-phenotype studies performed in 438 Italian subjects suggested a positive relation between the IVS7 repeat number and FVII levels. The lowest values were associated with the IVS7 + 7G allele. The screening of 52 patients with mild FVII deficiency showed an 8-fold increase in frequency (8%) of this allele, and among heterozygotes for identical mutations, lower FVII levels were observed in the IVS7 + 7G carriers. This frequent genetic component participates in the phenotypic heterogeneity of FVII deficiency. The evaluation of the individual contribution of polymorphisms was assisted by the expression of each IVS7 variant, as a minigene, in eukaryotic cells. The novel quantitative analysis revealed that higher numbers of repeats were associated with higher mRNA expression levels and that the IVS7 + 7G allele, previously defined as a functionally silent polymorphism, was responsible for the lowest relative mRNA expression. Taken together, these findings indicate that the IVS7 polymorphisms contribute to the plasmatic variance of FVII levels via differential efficiency of mRNA splicing. These studies provide further elements to understand the control of FVII levels, which could be of importance to ensure the hemostatic balance under pathologic conditions.

Amino Acid Substitution↗
PubMed2000

Detection of T cell receptor circles (TRECs) as biomarkers for de novo T cell synthesis using a quantitative polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA).

Currently, phenotypic markers that distinguish between recent thymic emigrants/de novo T cells and the rest of the peripheral T cell pool are lacking. This distinction is critical in studies aimed at evaluating immune reconstitution following intensive chemotherapy, in immunodeficiency-related therapies, or in the elucidation of the kinetics of thymic function. During V(D)J T cell receptor rearrangement, DNA extrachromosomal excision products are generated. These products, known as T cell receptor excision circles (TRECs), are not replicated during mitosis and are thus diluted with each round of cell division. Therefore, TRECs can be used as an indicator of recent thymic emigrants. Thus far, quantitative competitive-polymerase chain reaction (QC-PCR) and real time PCR were used to measure TREC levels. However, QC-PCR relies on radioactivity, is cumbersome when processing many samples at once and the cost of real time PCR does not make it a viable option for many laboratories. We describe here the development of a quantitative PCR-ELISA method for the measurement of coding joint TRECs generated from ValphaJalpha recombination. Our assay is ultra sensitive, relies on biotin labeling rather than radioactivity, is based on a 96-well format making multiple process sampling relatively easy, and is cost effective. Using this PCR-ELISA method, we evaluated thymic output among 22 normal subjects, ranging in age from 22-53 years, and among HIV-infected individuals following highly active antiretroviral therapy (HAART). We demonstrate that an inverse relationship exists between TREC levels and aging in normal individuals and that, among some HIV patients, HAART treatment leads to enhanced thymic output. Our assay has direct relevance in projects examining normal and abnormal thymic function and in immune reconstitution studies.

Adult↗
PubMed2000

The distribution of neuropeptide Y-immunoreactive neurons and nerve fibers in the forebrain of the carp Cyprinus carpio L.

The present study reports the distribution of Neuropeptide Y (NPY)-immunoreactive neurons and fibers in the forebrain of the adult carp Cyprinus carpio L. Serial Nissl-stained sections were used for cytoarchitecture and identification of anatomical structures. Immunostaining of NPY-containing neurons and fibers was used as neurochemical marker and tool for comparison with other species, including the goldfish. The general outline of the cytoarchitecture of the carp forebrain is similar to that of other Cypriniformes. However, using NPY immunohistochemistry, we found several specific differences with the goldfish, especially in the diencephalon. In the hypothalamus of the carp NPY-immunoreactive (NPYir) neurons were identified in the n. dorsolateralis thalami, and in the n. ventralis lateralis thalami. In the same location, we observed the n. anterior hypothalami and the n. preglomerulosus pars lateralis, described in the goldfish, as parts of n. prerotundus. However, in the carp we were not able to identify a n. preglomerulosus pars medialis, a n. preglomerulosus pars medialis commissuralis and a n. glomerulosus. We describe a n. rotundus, in which we did not find substructures typical of the goldfish. Further differences with the goldfish, trout and salmon were also noted.

Animals↗
PubMed2000

Comparison of classic and molecular approaches for the identification of untypeable enteroviruses.

Members of the family Picornaviridae are the most common viruses infecting humans, and species in several genera also infect a wide variety of other mammals. Picornaviruses have traditionally been classified by antigenic type, based on a serum neutralization assay. However, this method is time-consuming and labor-intensive, is sensitive to virus aggregation and antigenic variation, and requires a large number of antisera to identify all serotypes, even when antiserum pools are used. We developed generic reverse transcription (RT)-PCR primers that will amplify all human enterovirus serotypes, as well as many rhinoviruses and other picornaviruses, and used RT-PCR amplification of the VP1 gene and amplicon sequencing to identify enteroviruses that were refractory to typing by neutralization with pooled antisera. Enterovirus serotypes determined by sequencing were confirmed by neutralization with monospecific antisera. Of 55 isolates tested, 49 were of known enterovirus serotypes, two were rhinoviruses, and four were clearly picornaviruses but did not match any known picornavirus sequence. All four untyped picornaviruses were closely related to one another in sequence, suggesting that they are of the same serotype. RT-PCR, coupled with amplicon sequencing, is a simple and rapid method for the typing and classification of picornaviruses and may lead to the identification of many new picornavirus serotypes.

Animals↗
PubMed2000

A PCR-colorimetric microwell plate hybridization assay for detection of Mycobacterium tuberculosis and M. avium from culture samples and Ziehl-Neelsen-positive smears.

Differentiation between Mycobacterium tuberculosis and M. avium is essential for the treatment of mycobacterial infections. We have developed an easy and rapid detection assay for the diagnosis of mycobacterial diseases. This is a PCR-hybridization assay based on selective amplification of a 16S rRNA gene sequence using pan-Mycobacterium primers followed by hybridization of the amplification products to biotinylated M. tuberculosis and M. avium-specific probes. A total of 55 mycobacterial isolates were tested. For all isolates, results concordant with those of conventional identification methods were obtained. Moreover, we developed a method for extraction of DNA from Ziehl-Neelsen-positive smears which allows the recovery of intact target DNA in our PCR-hybridization assay. Our method was able to confirm all culture results for 59 Ziehl-Neelsen-positive smears from clinical specimens (35 sputum, 11 lymph node biopsy, 6 stool, 4 pus, 2 urine, and 1 pericardial fluid specimens). These data suggest that our PCR-hybridization assay, which is simple to perform and less expensive than commercial probe methods, may be suitable for the identification of M. tuberculosis and M. avium. It could become a valuable alternative approach for the diagnosis of mycobacterial infections when applied directly to DNA extracted from Ziehl-Neelsen-positive smears as well.

Colorimetry↗
PubMed2000

17beta-estradiol corrects hemostasis in uremic rats by limiting vascular expression of nitric oxide synthases.

Conjugated estrogens shorten the prolonged bleeding time in uremic patients and are similarly effective in a rat model of uremia. We have previously demonstrated that the shortening effect of a conjugated estrogen mixture or 17beta-estradiol on bleeding time was abolished by the nitric oxide (NO) precursor L-arginine, suggesting that the effect of these drugs on hemostasis in uremia might be mediated by changes in the NO synthetic pathway. The present study investigated the biochemical mechanism(s) by which conjugated estrogens limit the excessive formation of NO. 17beta-estradiol (0.6 mg/kg), given to rats made uremic by reduction of renal mass, significantly reduced bleeding time within 24 h and completely normalized plasma concentrations of the NO metabolites, nitrites and nitrates, and of NO synthase (NOS) catalytic activity, determined by NADPH-diaphorase staining in the thoracic aorta. Endothelial NOS (ecNOS) and inducible NOS (iNOS) immunoperoxidase staining in the endothelium of uremic aortas of untreated rats was significantly more intense than in control rats, while in uremic rats receiving 17beta-estradiol staining was comparable to controls. Thus 17beta-estradiol corrected the prolonged bleeding time of uremic rats and fully normalized the formation of NO by reducing the expression of ecNOS and iNOS in vascular endothelium. These results provide a possible biochemical explanation of the well-known effect of estrogens on primary hemostasis in uremia, in experimental animals and humans.

Animals↗
PubMed2000

Aortic dissection after superior mesenteric artery percutaneous stenting. Case report.

We report an unusual case of aortic dissection after superior mesenteric artery percutaneous stenting. A 44-year-old patient, who suffered from back pain and fever, was diagnosed as having an aortic dissection. Aortic dissection, extending from the aortic arch (just after left subclavian artery origin) to the aortic carrefour, was successfully diagnosed by means of Duplex scan and CT scan examination. Two pathogenetic hypotheses, malformative and iatrogenic, were discussed.

Adult↗
PubMed2000

[Interactions between physical training and pharmacologic treatment in the prevention of ventricular remodelling in patients with left ventricular dysfunction].

Exercise training is a recommended treatment for chronic heart failure. So far, randomized clinical trials showed that exercise training can improve peak oxygen consumption and reduce neurohormonal and adrenergic activation, nevertheless, the effect on left ventricular remodeling is still controversial. The present study reviewed the randomized clinical trials that investigated the effects of exercise training on left ventricular remodeling. After a first study that showed a worsening of left ventricular function, the following studies showed a neutral effect, and finally, the ELVD-CHF study showed both a reduction in left ventricular dilation and an improvement of ejection fraction. These different results could be explained by the pharmacological treatment before exercise training: in the first study patients did not assume ACE-inhibitors, in the following studies most patients assumed ACE-inhibitors and, finally, in ELVD-CHF, patients assumed ACE-inhibitors and about one fifth of them were on beta-blockers too. In conclusion, exercise training may improve peak oxygen consumption and reduce neurohormonal and adrenergic activation in patients with chronic heart failure. Further studies are necessary to assess if exercise training, associated with ACE-inhibitors and beta-blockers, can reverse or prevent left ventricular remodeling.

Exercise Therapy↗
PubMed2000

[PCA in the control of acute and chronic pain in children].

BACKGROUND: This study was undertaken to verify the efficacy and safety of patient controlled analgesia (PCA) in preschool and older than 6 years children with acute postoperative and chronic oncologic pain. METHODS: Sixty-two children, aged 4-17 yr, with postoperative (n = 37) or chronic (n = 25) pain received PCA with morphine at the following dosages: bolus = 10-20 gamma kg-1; starter dose = 10-30 gamma kg-1; background infusion = 10-50 gamma kg-1 h-1. Lockout was 5 to 15 minutes. In some children with oncologic pathology adjuvant drugs were also used. RESULTS: Analgesia was considered satisfactory in all children, while a mild sedation was observed only in two children. PCA was also successfully used in preschool children (11%), aged 4 to 6 yr. Side effects were observed in 8 patients, but only in one of them a transient respiratory depression was recorded. The mean PCA duration was 5.3 +/- 5.5 days (min.2; max 24). CONCLUSIONS: These results indicate that PCA is an effective and safe method to control acute postoperative pain as well as to manage chronic oncologic pain in children.

Acute Disease↗
PubMed1999

Monitoring of transmission of tuberculosis between wild boars and cattle: genotypical analysis of strains by molecular epidemiology techniques.

An epidemiological survey for the monitoring of bovine tuberculosis transmission was carried out in western Liguria, a region in northern Italy. Fifteen Mycobacterium bovis strains were isolated from 63 wild boar samples (62 from mandibular lymph nodes and 1 from a liver specimen). Sixteen mediastinal lymph nodes of 16 head of cattle were collected, and 15 Mycobacterium bovis strains were subsequently cultured. All M. bovis strains isolated from cattle and wild boars were genotyped by spoligotyping and by restriction fragment length polymorphism (RFLP) analysis with the IS6110 and IS1081 probes. All M. bovis strains showed the typical spoligotype characterized by the absence of the 39 to 43 spacers in comparison with the number in M. tuberculosis. A total of nine different clusters were identified by spoligotyping. The largest cluster included 9 strains isolated from wild boars and 11 strains isolated from cattle, thus confirming the possibility of transmission between the two animal species. Fingerprinting by RFLP analysis with the IS6110 probe showed an identical single-band pattern for 29 of 30 strains analyzed, and only 1 strain presented a five-band pattern. The use of IS1081 as a second probe was useful for differentiation of M. bovis from M. bovis BCG but not for differentiation among M. bovis strains, which presented the same undifferentiated genomic profile. In relation to the epidemiological investigation, we hypothesized that the feeding in pastures contaminated by cattle discharges could represent the most probable route of transmission of M. bovis between the two animal species. In conclusion, our results confirmed the higher discriminatory power of spoligotyping in relation to that of RFLP analysis for the differentiation of M. bovis genomic profiles. Our data showed the presence of a common M. bovis genotype in both cattle and wild boars, confirming the possible interspecies transmission of M. bovis.

Animals↗
PubMed1999

Oral contraceptives highlight the genotype-specific association between serum phospholipids and activated factor VII.

The present analysis was undertaken to study the effect of oral contraceptive (OC) use on activated factor VII (FVIIa) in subjects characterized by FVII genotypes, with the further aim of evaluating the role of lipids in this pharmacological interaction. In OC users (n=42) and nonusers (n=130) of comparable age, we examined the FVII phenotypic variables (FVII coagulant activity [FVIIc], FVII antigen, and FVIIa), FVII genotypes (the 353R/Q and 5'F7 polymorphisms analyzed in combination; alleles M1/M2 and A1/A2, respectively), and a number of lipid and lipoprotein parameters: serum concentrations of total cholesterol (chol), low density lipoprotein and high density lipoprotein-chol, triglycerides, phospholipids (PhLs), apolipoprotein A1, and lipoprotein(a). PhLs, triglycerides, apolipoprotein A1, chol, FVII antigen, FVIIc, and high density lipoprotein-chol levels were shown to be statistically higher in users than nonusers. FVII levels, particularly those of FVIIa and FVIIc, were much higher in homozygotes for the A1 and M1 alleles (A11 M11), especially in OC users. A strong association was found between PhL and FVIIa: in the multiple regression analysis, women taking OCs who had elevated PhL concentrations also had very high levels of FVIIa, but only if their genotype was A11 M11. These results indicate that the increased FVII levels in OC users depend on the FVII genotype and that high PhL concentrations predict very high levels of FVIIa and FVIIc.

Cardiovascular Diseases↗
PubMed1999

Use of N-acetylcysteine in the management of coronary artery diseases.

Thrombolysis after acute myocardial infarction may lead to a number of adverse effects (reperfusion injury) such as myocardial stunning, arrhythmias and even myocardial damage and extension of the infarct size. Some recent clinical studies have demonstrated that the intravenous infusion of N-acetylcysteine during thrombolysis was associated with a decrease in infarct size and better preservation of left ventricular function, probably due to antioxidant and free radical scavenger properties of N-acetylcysteine. Short- and long-term studies indicated that also in patients with unstable angina pectoris and threat of infarct, the intravenous or oral administration of N-acetylcysteine in association with nitroglycerin is highly effective in decreasing the risk of worsening, mainly by preventing the occurrence of acute myocardial infarction.

Acetylcysteine↗
PubMed1998

Molecular mechanisms of FVII deficiency: expression of mutations clustered in the IVS7 donor splice site of factor VII gene.

In three Italian patients, two point mutations and a short deletion were found in the intron 7 of factor VII gene, clustered in the donor splice site and located in the first of several repeats. The mutation 9726+5G-->A, the most frequent cause of symptomatic factor VII deficiency in Italy, as well as the deletion (9729del4) gave rise in expression studies to abnormally spliced transcripts, which were exclusively produced from the cryptic site in the second repeat. The insertion in the mature mRNA of the first intronic repeat caused (9726+5G-->A) a reading frameshift, abolishing most of the factor VII catalytic domain, or produced (9729del4), an altered factor with 11 additional residues, the activity of which was not detectable in the cell medium after mutagenesis and expression studies. Studies of factor VII ectopic mRNA from leukocytes and expression studies indicated that the deleted gene produced 30% of normally spliced transcript. Differently, the 9726+5G-->A mutation permitted a very low level (0.2% to 1%) of correct splicing to occur, which could be of great importance to prevent the onset, in the homozygous patients, of most of the life-threatening bleeding symptoms. The 9726+7A-->G mutation was found to be a rare and functionally silent polymorphism. These findings, which provide further evidence of the interplay of sequence and position in the 5' splice site selection, throw light on the heterogeneous molecular bases and clinical phenotypes of FVII deficiency.

Animals↗
PubMed1998

Two novel mutations (Pro864His, Val867Glu) causing type 2A von Willebrand disease and affecting a single restriction site in exon 28.

We detected two transversions in two unrelated Italian patients with type 2A von Willebrand disease (VWD): a C to A at nucleotide 8821 and a T to A at nucleotide 8830, resulting in the missense mutations Pro864His and Val867Glu respectively. Both mutations were in the heterozygous form and abolished the BstXI restriction site in exon 28 of the VWF gene. In both mutations plasma VWF multimer pattern improved by antiproteases. Moreover, DDAVP normalized plasma VWF multimers in the Pro864His patient, especially when protease inhibitors were present. These new mutations appear to be of the 2A VWD subtype due to the increased susceptibility to proteases.

Adult↗