A rapid hemi-nested PCR for HTLV-I detection.

A hemi-nested PCR approach was adopted to detect HTLV-1 infection in clinical samples of peripheral blood mononuclear cells (PBMCs) from subjects with positive or indeterminate serological results. Our results showed that the hemi-nested PCR quickly solved the diagnostic query, detecting the presence of proviral HTLV-1 DNA in two of the 252 patients with inconclusive serological results. The main advantage of this method are the typology of DNA extraction, allowing a consistent DNA recovery without amplification problems, the rapidity (4-5 hours), the performance of the assay and its comparable or better sensitivity than other HTLV-1 PCR formats.     

Expression of Intercellular Adhesion Molecule-1 (ICAM-1) on Cultured Human Endometrial Stromal Cells and Its Role in the Interaction With Natural Killers

PROBLEM: Recent evidence emphasizes the role of natural killer cells (NKs) as potential effectors of peritoneal immune surveillance directed against the outgrowth of endometrial cells, refluxed with menstrual debris, in ectopic sites. This NK-mediated cytotoxicity toward autologous endometrial antigens seems to be significantly decreased in endometriosis patients. METHOD: We set up experiments to clarify which molecules are involved in NK-endome-trial cell interaction. In particular, we evaluated the surface expression and functional activity of intercellular adhesion molecule-1 (ICAM-1), a cell surface glycoprotein that has been identified as one of the ligands for lymphocyte function-associated antigen-1 (LFA-1), present on almost all leucocyte cell types. Immunofluorescence flow cytometry was used to assess ICAM-1 expression on resting and IL 1β-activated endometrial stromal cells in culture. Dermal fibroblasts were used as control cells. Cytotoxicity and binding assays by ⁵¹Cr release in presence and absence of a specific monoclonal antibody (mAb) against ICAM-1 were then performed in order to determine the effect of this molecule on NK-mediated cytotoxic and binding activity toward endometrial stromal cells. RESULTS: The results of this study indicated that ICAM-1 expression on endometrial stromal cells seems to be constitutively higher than on dermal fibroblasts and can be up-regulated upon exposure to IL 1β. Furthermore, a mAb against ICAM-1 strongly inhibits the binding but not the cytotoxicity of NKs toward endometrial cells. No difference in the expression of this molecule was observed throughout the cycle. CONCLUSIONS: The presence of ICAM-1 on human endometrium might relate to the action of the immunocompetent cells in human specific reproductive events.     

Molecular pathogenesis of uterine smooth muscle tumors from transcriptional profiling

Uterine smooth muscle tumors range from the very common benign leiomyoma to the uncommon, but frequently lethal, leiomyosarcoma. Morphological and clinical differences between these tumors are presumed to result from differences in gene expression. To test this hypothesis, RNAs from four normal uterine myometria, seven uterine leiomyomas, and nine uterine leiomyosarcomas were profiled using microarrays of oligonucleotides representing about 7,000 unique probe sets. RNAs whose levels distinguished any of the three sample types were selected by analysis of variance (ANOVA). The 153 (2.2% of the total) probe sets representing 146 unique genes with the highest test statistic selected for further analysis met minimum ratio and range thresholds between groups. Cluster analysis distinguished benign and malignant samples at the first node, and myometrium and leiomyoma were resolved in a secondary node. Downregulation of specific genes in uterine leiomyosarcoma was the most common pattern of differential gene expression selected by the three-way ANOVA. Four extrauterine leiomyosarcomas had profiles most similar to that of the uterine leiomyosarcomas. Functional analysis of the 146 genes did not reveal any strong biological theme. These genes were distributed throughout the genome, but there was slight overrepresentation of genes on 1p and 2q. These genes define a tumor signature for uterine smooth muscle neoplasia, and they suggest that the molecular pathways in leiomyoma and leiomyosarcoma are distinct.     

Comparative in vitro study on a ultra-high roughness and dense titanium coating

A new implant surface has been developed with the purpose of avoiding as much stress shielding as possible, and thus prolong the prosthesis lifespan. The aim of this study was to investigate the in vitro effect of this new ultra-high roughness and dense Titanium (Ti) surface (PG60, Ra = 74 microm) in comparison with medium (TI01, Ra = 18 microm) and high (TI60, Ra = 40 microm) roughness and open porous coatings; all the coatings were obtained by vacuum plasma spraying. MG63 osteoblast-like cells were seeded on the tested materials and polystyrene, as control, for 3 and 7 days. Cells proliferated on the material surfaces similarly to the control. Alkaline phosphatase activity had lower values for TI60 than TI01 (p < 0.0005) and PG60 (p < 0.005). Osteocalcin levels measured on TI60 were significantly (p < 0.0005) lower in comparison with TI01 and PG60 at 7 days. Procollagen-I synthesis reduced with increasing roughness and the lowest data was found for PG60. While at 3 days Transforming Growth Factor beta1 levels augmented with increasing roughness, at 7 days TI60, the high roughness surface, was significantly lower than PG60 (p < 0.005) and TI01 (p < 0.001). All tested materials showed significantly higher Interleukin-6 levels than those of polystyrene at both experimental times. Nitric Oxide activity on TI01 was significantly (p < 0.05) higher than on TI60 and polystyrene. In conclusion, the new ultra-high roughness and dense coating PG60 provided a good biological response, even though, at least in vitro, it behaved similarly to the coatings already used in orthopaedics.     

Emilin genes are duplicated and dynamically expressed during zebrafish embryonic development.

Emilins are a family of extracellular matrix proteins with common structural organization and containing a characteristic N-terminal cysteine-rich domain. The prototype of this family, Emilin-1, is found in human and murine organs in association with elastic fibers, and other emilins were recently isolated in mammals. To gain insight into these proteins in lower vertebrates, we investigated the expression of emilins in the fish Danio rerio. Using sequence similarity tools, we identified eight members of this family in zebrafish. Each emilin gene has two paralogs in zebrafish, showing conserved structure with the human ortholog. In situ hybridization revealed that expression of zebrafish emilin genes is regulated in a spatiotemporal manner during embryonic development, with overlapping and site-specific patterns mostly including mesenchymal structures. Expression of certain emilin genes in peculiar areas, such as the central nervous system or the posterior notochord, suggests that they may play a role in key morphogenetic processes.     

p53 Immunoreactivity in inflammatory and neoplastic diseases of the uterine cervix

Immunoreactivity for the tumour suppressor gene product p53 is commonly found in many different human malignancies and few premalignant lesions. Data on cervical neoplasms, however, are still lacking. We retrospectively investigated p53 immunoreactivity in 92 lesions of the uterine cervix, including 44 cases of chronic cervicitis, 29 squamous intraepithelial lesions (SILs), and 19 invasive carcinomas. p53 immunoreactivity confined to the basal cell layer, was detected in 74 per cent of cases showing chronic cervicitis and in all cases with low-grade SILs. Conversely, suprabasal and/or diffuse p53 immunoreactivity was exclusively demonstrated in 25 per cent of high-grade SILs and in 74 per cent of invasive carcinomas. The results of this investigation document a high prevalence of p53-immunoreactive malignant tumours of the uterine cervix. In high-grade SILs, p53-immunoreactive cells paralleled the height of involvement by dysplastic changes within the squamous epithelium. A prolonged half-life of the protein is the most likely explanation for the occurrence of p53 immunoreactivity in neoplastic cells. The unexpected finding of p53-immunoreactive cells in inflammatory lesions, though possibly related to an increased proliferation rate of the basal cell compartment, requires further study and underlines the need for a careful approach to p53 immunocytochemistry.     

Factor analysis in gated cardiac studies

Factor analysis of dynamic structures (FADS) can automatically provide "physiological" factors related to anatomical structures that have different temporal behavior, even if these structures overlap; it also yields images corresponding to the factors' spatial distributions. In normal patients, two significant cardiac factors, corresponding to the atria and the ventricles, may be extracted. A third significant factor can be obtained when additional dynamic structures exist. However, the method does not provide an estimate of the background. It becomes part of the factors, but it does not modify their shapes. FADS has been applied to 45 gated cardiac studies. Results obtained by FADS were compared with those obtained from the amplitude and the phase of first-harmonic Fourier analysis (FA). The joint results were compared with the final diagnosis, established by real-time echocardiography and/or ventriculography. In normal patients, good agreement was obtained between the two approaches. On the whole set of patients, FADS was significantly better than FA (by sign test: p less than 5%).     

The Nutraceutical N-Palmitoylethanolamide (PEA) Reveals Widespread Molecular Effects Unmasking New Therapeutic Targets in Murine Varicocele

Varicocele is an age-related disease with no current medical treatments positively impacting infertility. Toll-like receptor 4 (TLR4) expression is present in normal testis with an involvement in the immunological reactions. The role of peroxisome proliferator-activated receptor-α (PPAR-α), a nuclear receptor, in fertility is still unclear. N-Palmitoylethanolamide (PEA), an emerging nutraceutical compound present in plants and animal foods, is an endogenous PPAR-α agonist with well-demonstrated anti-inflammatory and analgesics characteristics. In this model of mice varicocele, PPAR-α and TLR4 receptors’ roles were investigated through the administration of ultra-micronized PEA (PEA-um). Male wild-type (WT), PPAR-α knockout (KO), and TLR4 KO mice were used. A group underwent sham operation and administration of vehicle or PEA-um (10 mg/kg i.p.) for 21 days. Another group (WT, PPAR-α KO, and TLR4 KO) underwent surgical varicocele and was treated with vehicle or PEA-um (10 mg/kg i.p.) for 21 days. At the end of treatments, all animals were euthanized. Both operated and contralateral testes were processed for histological and morphometric assessment, for PPAR-α, TLR4, occludin, and claudin-11 immunohistochemistry and for PPAR-α, TLR4, transforming growth factor-beta3 (TGF-β3), phospho-extracellular signal-Regulated-Kinase (p-ERK) 1/2, and nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) Western blot analysis. Collectively, our data showed that administration of PEA-um revealed a key role of PPAR-α and TLR4 in varicocele pathophysiology, unmasking new nutraceutical therapeutic targets for future varicocele research and supporting surgical management of male infertility.