Toll-like receptor 4 Asp299Gly and Thr399Ile polymorphisms in gastric cancer of intestinal and diffuse histotypes.

In the present study we investigated the potential role of Toll-like receptor 4 (TLR-4) Asp299Gly and Thr399Ile polymorphisms as risk factors in the development of gastric cancer. TLR-4 Asp299Gly and Thr399Ile polymorphisms were investigated in 171 Italian patients with sporadic gastric cancer and in 151 controls. Unconditional regression (odds ratio and 95% confidence intervals) were used to investigate the association of the studied polymorphisms with gastric cancer. TLR-4 Thr399Ile polymorphism is linked with an increased susceptibility to gastric cancer (P = 0.023 and hazard ratio = 3.62). No significant association for TLR-4 Asp299Gly polymorphism was found. In the subgroup of patients with intestinal-type gastric cancer, a significant risk of gastric cancer was associated with TLR-4 Thr399Ile genotype (P = 0.006). Our results demonstrated that TLR-4 Thr399Ile polymorphism is linked with an increased susceptibility to gastric cancer. An increased risk for intestinal gastric cancer in carriers of the TLR4 Thr399Ile allele was observed. Future epidemiological studies should consider the possible interactions between proinflammatory genotypes (such as TLR and interleukin-1R polymorphisms) and other risk factors for cancer such as dietary habits and/or exposure to environmental carcinogens.

Molecular characterisation and clonal analysis of group A streptococci causing pharyngitis among paediatric patients in Palermo, Italy.

Group A streptococci (n = 123), isolated consecutively from paediatric patients with pharyngitis from Palermo, Italy, were analysed. The emm and sof genes were sequenced, the presence of the speA and speC genes was investigated, and the macrolide resistance phenotypes and genotypes were determined. A limited number of emm/sof genotypes was found, and the most prevalent types were different from those found in a previous study from Rome. Macrolide resistance was found in the most prevalent clones, suggesting that the spread of mobile antibiotic resistance genes among the fittest clones in the community was the main mechanism influencing macrolide resistance rates in different emm types.

Colorectal carcinomas and PTEN/MMAC1 gene mutations.

PURPOSE: PTEN/MMAC1/TEP1 is a tumor suppressor gene encoding a dual-specificity protein phosphatase with homology to the cytoskeleton proteins, chicken tensin and bovine auxilin. PTEN mutations have been described in several types of human cancer. Recently, mutations at an (A)(6) repeat of PTEN exons 7 and 8 in colorectal cancer (CRC) patients with microsatellite instability have been detected. Moreover, an involvement of the transforming growth factor (TGF)-beta pathway in hereditary colorectal syndromes has been proposed. EXPERIMENTAL DESIGN: In this study, we analyzed the frequency of PTEN gene mutations in 36 CRC patients and 5 colon cancer cell lines. Furthermore, in 16 of 36 patients, microsatellite instability and TGF-beta receptor II analysis was possible. The study was performed by PCR and automated sequencing of the entire coding region of the PTEN gene. RESULTS: About 17% of colon cancer patients and one of five (HSR 320) colon cancer cell lines had mutations. Mutations were detected only among patients with locally advanced or metastatic CRC. PTEN mutations were detected in three of five (60%) patients showing both microsatellite instability and TGF-beta receptor II mutations. These patients presented with advanced or metastatic CRC CONCLUSIONS: Overall, these results show that PTEN alteration together with TGF-beta pathway inactivation could contribute to tumorigenesis and metastatic spread of sporadic and microsatellite unstable CRC.

Antibiotic resistance and genotypic characterization by PFGE of clinical and environmental isolates of enterococci.

Fifty-four Enterococcus faecalis and 20 Enterococcus faecium isolates from clinical and non-human sources in Rome, Italy, were characterized by antibiotic resistance and pulsed field gel electrophoresis (PFGE). Resistance to vancomycin, teicoplanin, ampicillin, and ciprofloxacin was more frequent in E. faecium than in E. faecalis, whereas high-level resistance to aminoglycoside was found primarily in E. faecalis. Multi-resistance was found primarily among clinical isolates, but was also observed among environmental isolates. Common genotypes shared among clinical and environmental isolates were observed, however, the majority of isolates occurred as unique, source-specific clones. Several PFGE types were associated with shared features in their antibiotic resistance patterns; evidences of clonal spread between and within wards were also noted. This is the first report indicating clonal relatedness between human and environmental enterococci isolated in Italy.

Group A streptococcal genotypes from pediatric throat isolates in Rome, Italy.

In a study assessing genetic diversity, 114 group A streptococcus (GAS) isolates were recovered from pediatric pharyngitis patients in Rome, Italy. These isolates comprised 22 different M protein gene (emm) sequence types, 14 of which were associated with a distinct serum opacity factor/fibronectin binding protein gene (sof) sequence type. Isolates with the same emm gene sequence type generally shared a highly conserved chromosomal macrorestriction profile. In three instances, isolates with dissimilar macrorestriction profiles had identical emm types; in each of these cases multilocus sequence typing revealed that isolates with the same emm type were clones having the same allelic profiles. Ninety-eight percent of the pharyngeal isolates had emm types previously found to be highly associated with mga locus gene patterns commonly found in pharyngeal GAS isolates.

Routine molecular identification of enterococci by gene-specific PCR and 16S ribosomal DNA sequencing.

For 279 clinically isolated specimens identified by commercial kits as enterococci, genotypic identification was performed by two multiplex PCRs, one with ddl(E. faecalis) and ddl(E. faecium) primers and another with vanC-1 and vanC-2/3 primers, and by 16S ribosomal DNA (rDNA) sequencing. For 253 strains, phenotypic and genotypic results were the same. Multiplex PCR allowed for the identification of 13 discordant results. Six strains were not enterococci and were identified by 16S rDNA sequencing. For 5 discordant and 10 concordant enterococcal strains, 16S rDNA sequencing was needed. Because many supplementary tests are frequently necessary for phenotypic identification, the molecular approach is a good alternative.

Diagnostic value of cytokine assays in cerebrospinal fluid in culture-negative, polymerase chain reaction-positive bacterial meningitis.

Analysis of bacterial DNA using a polymerase chain reaction performed with broad-range eubacterial 16S rDNA primers may yield a diagnosis of bacterial meningitis in cases where Gram staining of cerebrospinal fluid (CFS), antigen detection techniques or culture fail. Since this PCR technique occasionally gives false-positive results due to contamination of samples or laboratory reagents, a study was performed to establish the diagnostic value of assaying concentrations of tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) in 90 CSF samples. A high correlation was found between a positive PCR result and the concentrations of TNF-alpha and IL-10, indicating that cytokine assays may be used as a confirmatory test. The findings suggested that a combination of the PCR technique, amplicon sequencing and assay of TNF-alpha and IL-10 concentrations in CSF is a reliable and cost-effective procedure for diagnosis of culture-negative bacterial meningitis.

Use of oligoprobes on amplified DNA in the diagnosis of bacterial meningitis.

An approach based on the 16 S rDNA polymerase chain reaction (16S PCR) and oligoprobe hybridization was applied to 77 cerebrospinal fluid samples submitted to the clinical microbiology laboratory for culture. Broad-range 16S rDNA primers were selected in conserved regions of the gene. Oligoprobes specific for Neisseria meningitidis, Haemophilus influenzae, Streptococcus spp., and Mycobacterium tuberculosis were selected in specific variable regions of the amplified 600 base pairs (bp) in the 16S rDNA. None of the oligoprobes cross hybridized with DNA from the other bacteria or from common contaminants. There were no false-negative results in culture-positive cerebrospinal fluid samples. Ten cases of meningitis caused by bacteria other than the four probes were not identified by any of the four probes. In culture-negative cerebrospinal fluid samples with some abnormal chemical parameters, there were 14 amplicons -- one of Haemophilus influenzae, three of Streptococcus spp., six of Mycobacterium tuberculosis, and four not identified -- while in normal cerebrospinal fluid samples there were no amplicons.

IgM anti-hepatitis C virus in patients with chronic non-A, non-B hepatitis and their relationship to viral replication.

Patients with hepatitis C virus (HCV) infection may have different patterns of antibody response to various structural and non-structural viral antigens. We have correlated the serological patterns to the clinical features of chronic infection and to viral replication in 68 HCV-Ab-positive patients with chronic liver disease at different stages (19 with cirrhosis-hepatocellular carcinoma, 38 with chronic active hepatitis and 11 with chronic persistent hepatitis). Serum samples from each patient were assayed for HCV-IgM by enzyme immunoassay and for HCV-RNA by the polymerase chain reaction using primer sets derived from the 5'-non-coding region. The prevalence of HCV-IgM was high (54 patients (79.4%)) and the study showed a good correlation between high values of anti-HCV-IgM and the presence of HCV-RNA in serum, since HCV-RNA was detected in 35 of the 54 IgM-positive patients (64.8%) and notably in 19 of the 20 subjects with high levels of specific IgM. Conversely, all the 35 sera containing HCV-RNA were also reactive for HCV-IgM, while none of the HCV-IgM-negative sera was HCV-RNA reactive. Positivity rates for both HCV-RNA and IgM anti-HCV were higher in the more advanced stages of disease; thus, the clinical pattern of HCV chronic hepatitis seems to be strictly related to the serological pattern and the presence of HCV-RNA.

Effects of one-year treatment with rilmenidine on systemic hypertension-induced left ventricular hypertrophy in hypertensive patients.

In patients with essential hypertension, left ventricular hypertrophy (LVH) increases the risk for cardiovascular morbidity and mortality. Thus its reversal represents one of the principal end-points of antihypertensive treatment. We assessed the cardiovascular effects of 1-year antihypertensive treatment with rilmenidine (1 or 2 mg/day orally), a new oxazoline with a potent antihypertensive action that acts selectively through imidazoline-preferring receptors. In 11 hypertensive patients (mean age, 49 +/- 2 years) with LVH, we measured systemic hemodynamics, large artery compliance, cardiac anatomy, and endocrine function. Patients underwent M-mode and 2-dimensional echocardiography as well as Doppler and peripheral pulsed Doppler flowmetry, determination of plasma atrial natriuretic factor (ANF) levels and renin activity (PRA), and of 24-hour urinary electrolyte and creatinine excretion in control conditions (systolic/diastolic blood pressure, 148 +/- 3/102 +/- 1 mm Hg), 4 weeks after blood pressure normalization (131 +/- 2/84 +/- 2 mm Hg; p < 0.01), after 1 year of satisfactory antihypertensive treatment (142 +/- 3/90 +/- 1 mm Hg; p < 0.01) and, finally, 1 month after therapy withdrawal (155 +/- 3/106 +/- 2 mm Hg; difference not significant [NS]). One-year of rilmenidine treatment induced an improvement in brachial artery compliance (from 0.92 +/- 0.06 to 1.16 +/- 0.08 cm4/dyne; p < 0.05), which persisted after withdrawal of treatment (1.17 +/- 0.06 cm4/dyne; p < 0.05). LVH was reversed after 1 year of rilmenidine treatment (from 152 +/- 5 to 131 +/- 4 g/m2 body surface area; p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of indeterminate sera for hepatitis C virus by assays using synthetic peptides.

The development of new immunodiagnostic systems to study antibodies anti-HCV based on the use of synthetic peptides are potentially very important in the evaluation of indeterminate samples. We have compared two immunodiagnostic tests, the 3-RIBA and LIA-HCV test system, to examine the samples rated as indeterminate by the 2-RIBA test. The results showed that the specificity of the new tests has improved, in fact 29/40 of the indeterminate samples studied became positive with both tests. However, the 3-RIBA test showed higher sensitivity, since two samples negative by LIA-HCV, were positive with this system.

Reactivity and protective capacity of a polyclonal antiserum derived from mice immunized with antibiotic exposed Escherichia coli.

The murine immune response to Escherichia coli O6:K-alone or pre-exposed to 0.1 x MIC of aztreonam was investigated. Relative to mice immunized with untreated bacteria, mice immunized with antibiotic-treated microorganisms presented a significantly enhanced protection towards a challenge of 100 x LD50 of viable E. coli O6:K-. Previous injection of 0.1 mL of serum drawn from mice immunized with treated and untreated bacteria protected non-immunized mice towards a challenge of 10 x LD50 of viable E. coli O6:K--. Serum from mice immunized with treated bacteria also protected non-immunized mice towards a lethal challenge of E. coli O111. The antiserum contained high titre of IgG antibodies that cross-reacted with lipopolysaccharide isolated from smooth and rough Gram-negative bacteria. Immunoblotting showed additional bands of reactivity to the untreated E. coli O6:K-. Immunization with antibiotic-treated bacteria led to the production of type specific and cross reactive antibodies that protected animals against viable homologous and heterologous lethal challenges.

Detection of antibodies to hepatitis C virus in dialysis patients.

Dialysis patients are at risk for infection by a variety of blood-borne agents transmitted within dialysis units. The development of the hepatitis C virus (HCV) screening test prompted many studies on the prevalence of anti-HCV among dialysis patients. The authors have evaluated the prevalence of anti-HCV in 405 hemodialysis patients both by Elisa screening and 4-RIBA test system with a follow-up of two years. The study showed a difference in the incidence of antibodies to HCV by year. In 1990, 15.2% were positive with an increase to 20.8% in 1991. There was an increase of 5% in dialysis patients and only 1.9% in the personnel working in the dialysis ward. Another control group of volunteers did not show any positivity. In addition, the correlations of the antibodies against HCV with markers of hepatitis B virus (HBV) and history of transfusion were evaluated. These findings suggest that the patients found to be positive should be dialyzed on separate machines and special precautions must be undertaken to reduce the risk of transmission.

Immunization of mice with antibiotic treated Escherichia coli.

The hypothesis of a different immunogenicity between untreated and antibiotic-treated Escherichia coli was investigated in vivo. Groups of mice were injected weekly for eight weeks with formalin-killed E. coli ATCC 25922 either exposed or not to 0.1 x MIC of aztreonam. A group of mice injected with sterile saline only served as control. IgG production towards whole bacteria was clearly enhanced in the group immunized with antibiotic-treated E. coli as shown in ELISA assays. In the same group, the appearance of additional bands of reactivity in the region of major outer membrane proteins was observed in immunoblot experiments as well as an enhanced protection towards a challenge of 10 x LD50 of live E. coli. These findings seem to support the hypothesis that sub-MICs of antibiotics modify the bacterial surface influencing host-parasite relationships.

Alpha interferon plus thymopentine in treatment of HBV and/or HDV positive patients undergoing liver transplant.

Infection by hepatitis B (HBV) and/or delta virus (HDV), is the most frequent acquired pathology in patients affected by end-stage hepatic disease, candidates for liver transplant. To reduce the risk of virus reactivation after surgery, we used alpha Interferon (IFN) therapy in patients who were HBV-DNA and/or HDV-RNA positives before transplant. Our protocol included alpha IFN at low dosage associated to a thymic hormone that seems to have a synergistic activity with IFN. We have evaluated in four patients, affected by post hepatitic end-stage liver disease, the outcome of HBV and HDV markers in relation to immunological response during treatment. Our interest has been focused on monocyte and natural killer cytotoxic activity. The data show that all patients, before starting therapy, had evidence of active phase viral replication. They also displayed low values of the immunological parameters tested. The study of viral markers showed decrease of HBV and HDV in all patients. The relation between viral markers and natural killer and monocyte cytotoxicity was very interesting; during the treatment we observed a marked increase of both activities. At the same time no relevant modifications in the other immunological parameters tested were found.

Membrane interactions involved in the induction of interferon-alpha by Mycoplasma pneumoniae.

Interferon (IFN) induction by Mycoplasma pneumoniae (MP) in human peripheral blood mononuclear cells (PBMC) has been studied. We show that IFN yields depend on the concentration of both lymphocytes and MP. The effective IFN inducer appears to be the mycoplasma membrane, and IFN production occurs without significant lymphocyte proliferation. Data obtained by both positive and negative selection experiments suggest that the PBMC subpopulation induced by MP is B lymphocyte, as opposed to the main subpopulation induced by NDV that is monocyte. Evidence is also provided suggesting that the membrane interaction between MP and the B cells is mediated by MHC Class II antigens.

[Seroepidemiologic studies in 63 patients given organ transplants in relation to cytomegalovirus, herpes simplex, varicella-zoster and HTLV-III: validity and limits]. / Indagine sieroepidemiologica in 63 pazienti sottoposti a trapianto di organo nei confronti del cytomegalovirus, dell'herpes simplex, del varicella/zoster e dell'HTLV III: validità e limiti.

We carried out a seroepidemiological study on 63 patients undergoing organ transplantation in order to determine viral markers like CMV, HTLV III, HS and V/Z. The antibodies valuation was carried out by enzyme-linked immunosorbent assays (ELISA-Abbott) and complement fixation test. All the tested markers give a high percentage of positivity even if the values relevant to anti-HTLV III antibodies can not be related to infection but, probably, to anti-HLA-Dr antibodies.