Outbreak of infection with hepatitis A virus (HAV) associated with a foodhandler and confirmed by sequence analysis reveals a new HAV genotype IB variant

An outbreak of infection with hepatitis A virus associated with a foodhandler and involving 26 subjects occurred in Southern Italy. Sequence analysis of the VP3-VP1 and VP1-P2A junctions confirmed that the outbreak was due to a point source and allowed the identification of a new genotype IB variant. This report confirms the usefulness of sequence-based molecular fingerprinting during outbreaks.     

Detection of clarithromycin-resistant Helicobacter pylori in stool samples

The recognition of the role of Helicobacter pylori in gastric diseases has led to the widespread use of antibiotics in the eradication of this pathogen. The most advocated therapy, triple therapy, often includes clarithromycin. It is well known that clarithromycin resistance is one of the major causes of eradication failure. The development of a rapid noninvasive technique that could easily be performed on fecal samples and that could also provide information about the antibiotic resistance of this microorganism is therefore advisable. Previous findings have demonstrated that clarithromycin resistance is due to a single point mutation in the 23S rRNA. All the mutations described have been associated with specific restriction sites, namely BsaI (A2143G), MboII (A2142C/G), and HhaI (T2717C). On this basis we have developed a new method, a seminested PCR, allowing screening for clarithromycin resistance of H. pylori directly on stool samples. This method furnished a 783-bp fragment of the 23S rRNA, which was subsequently digested by MboII, BsaI, and HhaI, in order to identify single point mutations associated with clarithromycin resistance. Of a total of 283 stool samples examined, 125 were H. pylori positive and two of them were shown to contain clarithromycin-resistant strains due to the presence of a mutation at position 2717, whereas no PCR products contained mutations at position 2142 or 2143. In order to evaluate the reliability of the new system, we compared the results of restriction analysis of the PCR products with the MICs shown by the H. pylori isolates by culturing gastric biopsies from the same patients.     

The Vi capsular antigen of Salmonella enterica serotype Typhi reduces Toll-like receptor-dependent interleukin-8 expression in the intestinal mucosa

Human infections with nontyphoidal Salmonella serotypes, such as S. enterica serotype Typhimurium, are characterized by a massive neutrophil influx in the colon and terminal ileum. In contrast, neutrophils are scarce in intestinal infiltrates of typhoid fever patients. Here, we show that in S. enterica serotype Typhi, the causative agent of typhoid fever, expression of the Vi capsular antigen reduced expression of the neutrophil chemoattractant interleukin-8 (IL-8) in host cells. Capsulated bacteria elicited IL-8 expression in polarized human epithelial cells (T84) and human macrophage-like cells (THP-1) in vitro at significantly reduced levels compared to noncapsulated bacteria. Experiments with a human cell line (HEK293) transfected with human Toll-like receptors (TLRs) demonstrated that in the presence of TLR5 or TLR4/MD2/CD14, a noncapsulated serotype Typhi mutant was able to induce the expression of IL-8, while this host response was significantly reduced when cells were infected with the capsulated serotype Typhi wild type. The relevance of these in vitro observations for the interaction of serotype Typhi with its human host was further studied ex vivo using human colonic tissue explants. Expression of IL-8 was detected in human colonic tissue explants infected with serotype Typhimurium or a noncapsulated serotype Typhi mutant. In contrast, infection with the serotype Typhi wild type did not elicit IL-8 expression in colonic tissue explants. Collectively, these data suggest that the scarcity of neutrophils in intestinal infiltrates of typhoid fever patients is due to a capsule-mediated reduction of TLR-dependent IL-8 production in the intestinal mucosa.     

Auditory brainstem response in premenopausal women taking oral contraceptives

BACKGROUND: The aim of this prospective study was to evaluate the effects of the new monophasic oral contraceptives on the audiological system in premenopausal women. METHODS: The auditory brainstem response (ABR) was measured in 94 women during the follicular, periovular and luteal phases of one menstrual cycle in which ovulation was confirmed using sonography and serum progesterone concentration. The latencies for waves I, III and V were determined, and the inter-peak intervals were calculated for waves I-III, I-V and III-V. All 94 women began taking oral contraceptives: 23 women used 20 microg ethinyl estradiol (EE) plus 150 microg desogestrel, 24 women used 30 microg EE plus 75 microg gestodene, and 47 women used 15 microg EE plus 60 microg gestodene. During the third month of contraceptive intake, each subject was again tested for ABR, as above. RESULTS: The wave latencies and inter-peak intervals showed shorter values during the periovular phase with respect to the luteal phase (P < 0.05), the follicular phase for wave I and for inter-peak interval I-V (P < 0.05) of the menstrual cycle. All of the ABR results in pill users were statistically different from those of the periovular phase (P < 0.05), though similar to those of both the luteal and follicular phases (P = NS). CONCLUSIONS: ABR seems to depend on the variations of ovarian steroids during the menstrual cycle and during oral contraceptive intake.     

Early (Stage A) prostatic cancer

Summary This study was performed in order to elucidate some of the problems of incidence, morphology and natural history concerned with Stage A prostatic cancer or prostatic microcarcinoma (PMC).The prostates of 100 patients, treated by subtotal prostatectomy for benign prostatic hyperplasia (BPH), were studied by comparing both routine and step-section techniques. The incidence of PMC was 41% by the former and 86% by the latter technique. Assessment of the size of PMC, as measured by the sum of the two main diameters, resulted in three groups: A₁, A₂, A₃. The last of these may represent a frankly malignant condition, judged by size and the histological appearance. Radical prostatectomy is strongly suggested as appropriate therapy for this group.Supported in part by a Grant from the Ministry of Education (art. 286 T.U., 1977/78)     

Comparison of Real-Time, Quantitative PCR with Molecular Beacons to Nested PCR and Culture Methods for Detection of Mycobacterium avium subsp. paratuberculosis in Bovine Fecal Samples

An automated PCR with fluorescent probes (molecular beacons) detected Mycobacterium avium subsp. paratuberculosis in bovine feces. When the PCR was compared with culture in testing 41 fecal samples, kappa scores of 0.94 to 0.96, a sensitivity of 93 to 96%, and a specificity of 92% were obtained. Results were quantitated by using a standard curve derived from a plasmid containing IS900. A minimum quantity of 1.7 x 10(-4) pg of DNA, correlating to 1 to 8 CFU, was detected.     

IFNgamma and TNFalpha account for a pro-clonogenic activity secreted by activated murine peritoneal macrophages

In the present study, we found that murine peritoneal macrophages elicited by BCG or Listeria monocytogenes release into the media an activity capable of stimulating the lung colonization as well as the expression of MHC class I antigens in B16 melanoma cells. A similar activity has previously been found in media conditioned by Corynebacterium parvum-elicited macrophages. Analysis by gel filtration chromatography of media conditioned by Corynebacterium parvum-, BCG- or Listeria monocytogenes-elicited macrophages revealed that the material responsible for the pro-clonogenic activity concentrated in chromatographic fractions corresponding to molecular weights (25 to 52 kDa) which are characteristic of certain cytokines. Thus, we challenged the various macrophage-conditioned media with polyclonal antibodies against IFNγand TNFα, and found that the macrophage pro-clonogenic activity was completely abolished in the presence of anti-IFNγantibodies, but only partially inhibited by anti-TNFαantibodies. This finding suggests a cooperative participation of the two cytokines to the pro-clonogenic activity of the media conditioned by Corynebacterium parvum-, BCG- or Listeria monocytogenes-elicited macrophages. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vitro regulation of beta1 and beta3 integrin subunits in endometrial epithelial cells from normal endometrium

OBJECTIVE: To evaluate the effect of prostaglandin E2 (PGE2) and interleukin-1beta (IL-1beta) on integrin expression. DESIGN Cultures of endometrial epithelial cells from normal endometrium. SETTING: All endometrial specimens were obtained from the Obstetrics and gynecology Department of the Catholic University, Rome, Italy. PATIENTS: Four patients were normal menstrual cycles undergoing operative laparoscopy for non-endometrial problems. INTERVENTION: Endometrial samples were collected by uterine courettings. MAIN OUTCOME MEASURE: Immunocytochemistry for beta1 and beta3 integrin subunits. RESULTS: PGE2 clearly enhances both beta1 and beta3 integrin subunit expression. IL-1beta seem to slightly increase only beta3 subunit expression. CONCLUSIONS: In light of the critical role played by eicosanoids in endometrial differentiation, we suggest that PGE2 is also involved in local paracrine regulation of integrin expression.     

Primary osteoblasts response to shock wave therapy using different parameters

Over the past decade extracorporeal shock-wave therapy (ESWT) has been increasingly applied to orthopaedic and musculoskeletal pathologies, the aim of this study was to assess how the energy density of the shock waves and the number of impulses affect viability, differentiation and synthetic activity of osteoblasts. Primary sheep osteoblasts cultures were treated with ESWT with an electro-hydraulic shock wave generator by selecting three different energy levels (14-21-28 kV corresponding at 0.15-0.31-0.40 mJ/mm2) and two different total numbers of impulses (500, 1000) for each level. At the end of treatment, cell counts and viability were recorded. Cells were then cultivated for 48 hours starting from a concentration of 1 x 10(4) cells/ml. The biological activity and viability were evaluated at 24 and 48 hours after treatment. No cytodestructive effects were observed in Group A, while a cytodestructive effect of ESWT was seen in cultures receiving the highest energy treatments. The different shock wave treatment induced differences in MTT assays after 24 and 48 hours, in particular the highest level showed a detrimental effect on cell respiration at both experimental times as compared to the Control Group and the protein metabolism was generally depressed by ESWT with impulses at the highest energy level. After 24 hours such effect further increased with the growing number of impulses. The lowest energy level appeared to significantly improve the metabolic parameter in primary cell cultures as compared to controls when 500 impulses were selected. The current study has demonstrated that one of the most important aspects to be considered is not the total number of impulses used but the energy level of the shock waves, thus confirming that ESWT has a dose-dependent effect on cells.     

Splice variants of the relaxin and INSL3 receptors reveal unanticipated molecular complexity

LGR7 and LGR8 are G protein-coupled receptors that belong to the leucine-rich repeat-containing G-protein coupled receptor (LGR) family, including the thyroid-stimulating hormone (TSH), LH and FSH receptors. LGR7 and LGR8 stimulate cAMP production upon binding of the cognate ligands, relaxin and insulin-like peptide 3 (INSL3), respectively. We cloned several novel splice variants of both LGR7 and LGR8 and analysed the function of four variants. LGR7.1 is a truncated receptor, including only the N-terminal region of the receptor and two leucine rich repeats. In contrast, LGR7.2, LGR7.10 and LGR 8.1 all contain an intact seven transmembrane domain and most of the extracellular region, lacking only one or two exons in the ectodomain. Our analysis demonstrates that although LGR7.10 and LGR8.1 are expressed at the cell surface, LGR7.2 is predominantly retained within cells and LGR7.1 is partially secreted. mRNA expression analysis revealed that several variants are co-expressed in various tissues. None of these variants were able to stimulate cAMP production following relaxin or INSL3 treatment. Unexpectedly, we did not detect any direct specific relaxin or INSL3 binding on any of the splice variants. The large number of receptor splice variants identified suggests an unforeseen complexity in the physiology of this novel hormone-receptor system.