Immunofluorescent detection of anti-cytoskeleton antibodies using vinblastine-treated mononuclear cells

A reliable and reproducible immunofluorescence method is described for the detection of anti-cytoskeleton antibodies in human sera, based on the use of vinblastine-treated peripheral blood mononuclear cells as substrate. Three immunofluorescence patterns associated with antibodies to microfilaments, intermediate filaments and microtubules are readily identified.     

Cellular and subcellular localization of the small G protein RhoA in the human and rat embryonic and adult kidney

Rho proteins, a subgroup of the Ras GTPase superfamily, control many cellular processes and morphogenetic events by acting as signaling molecules in the transduction pathways of various receptors. Among the "Rho-dependent" receptors are the extracellular matrix- and growth factor-binding sites; these are particularly involved in the modulation of renal development since they control the epithelial-mesenchymal interactions that drive kidney organogenesis. The present study has addressed the immunohistochemical localization of RhoA in developing and adult kidneys of rats and humans because: a) Rho proteins are known to have a morphogenetic role, b) data in the literature on expression of Rho GTPases during mammalian histogenesis and organogenesis are scarce, and c) their involvement in the transduction pathways of receptors is implicated in kidney development. In particular, RhoA peptide was found to be localized in the mesonephric duct and vesicles in both rats and humans; metanephric anlagen were mainly stained in ampullar-derived cells. Periglomerular tubules of fetal and adult kidneys as well as collecting ducts of adult kidneys showed intense staining. Therefore, the present study provides new information on the distribution patterns of RhoA during early stages of mammalian kidney development suggesting that this signaling molecule may take part in epithelial-mesenchymal induction processes that control kidney organogenesis. RhoA expression in adult structures may be linked with renewal of renal epithelial cells and the maintenance of their morphology and polarity.     

Sperm nuclear DNA damage and altered chromatin structure: effect on fertilization and embryo development

In the first part of this report we investigate whether chromatin anomalies in human spermatozoa can influence fertilization after intracytoplasmic sperm injection (ICSI). We have examined the sperm chromatin packaging quality using the chromomycin A3 (CMA3) fluorochrome and the presence of DNA damage in spermatozoa using in-situ nick translation. When comparing the spermatozoa of patients undergoing in-vitro fertilization (IVF) and ICSI distinct differences are evident in that ICSI males have a higher CMA3 fluorescence, indicating spermatozoa with loosely packed chromatin, and more spermatozoa containing endogenous DNA nicks. When examining the unfertilized oocytes of ICSI patients we found that men who had a high percentage of anomalies in their chromatin, i.e. > 30% CMA3 fluorescence and > 10% nicks, had more than double the number of unfertilized oocytes containing spermatozoa that had remained condensed. The observation that failed fertilized oocytes, injected with spermatozoa from patients with a higher percentage of sperm nuclear anomalies, contain more condensed spermatozoa indicates that a selection process against these spermatozoa may be in place at the time of fertilization. In the second part of the study we show that spare ICSI embryos have significantly lower rates of development to the blastocyst stage compared with those developed after routine IVF. These results show that a greater understanding of the molecular basis of male infertility is therefore needed to broaden our knowledge on the effect that abnormal spermatozoa have on fertilization and embryo development.     

Evidence for the involvement of SDF-1 and CXCR4 in the disruption of endothelial cell-branching morphogenesis and angiogenesis by TNF-alpha and IFN-gamma

Vigorous inflammatory responses are associated with tissue damage, particularly when toxic levels of inflammatory cytokines are produced. Despite proangiogenic factors being present early at sites of inflammation, vascular repair occurs toward the end of the inflammatory response, suggesting modulation of the proangiogenic response. Endogenous inhibitors of angiogenesis induced during acute inflammation are poorly characterized. Here, we looked for endothelial cell-derived modulators of angiogenesis that may account for delayed neovascularization during inflammation. Gene profiling of endothelial cells showed that the inflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interferon-gamma (IFN-gamma) selectively promote expression of the antiangiogenic molecules, IFN-inducible protein-10, monokine induced by IFN-gamma, tryptophanyl-tRNA synthetase, and tissue inhibitor of metalmetalloproteinase-1, and inhibit expression of the proangiogenic molecules, platelet-endothelial cell adhesion molecule-1, vascular endothelial growth factor receptor-2, stromal cell-derived factor-1 (SDF-1), collagen type IV, endothelial cell growth factor-1, and carcinoembryonic antigen-related cell adhesion molecule-1. Reduced endothelial cell expression of SDF-1 protein by TNF-alpha and IFN-gamma disrupts extracellular matrix-dependent endothelial cell tube formation, an in vitro morphogenic process that recapitulates critical steps in angiogenesis. Replacement of SDF-1 onto the endothelial cell surface reconstitutes this morphogenic process. In vivo, TNF-alpha and IFN-gamma inhibit growth factor-induced angiogenesis and SDF-1 expression in endothelial cells. These results demonstrate that SDF-1/CXC chemokine receptor-4 constitutes a TNF-alpha- and IFN-gamma-regulated signaling system that plays a critical role in mediating angiogenesis inhibition by these inflammatory cytokines.     

Novel MC1R variants in Ligurian melanoma patients and controls

Several variant forms of the melanocortin-1 receptor gene (MC1R) have been associated with red hair, fair skin and an increased risk for melanoma. Their involvement in melanoma susceptibility is apparently linked both to skin sensitivity and to non-pigmentary pathways. We investigated the frequency of the MC1R variants in the Italian region of Liguria, where the occurrence and penetrance of melanoma are low and primary susceptibility is characterized by prevalence of the CDKN2A c.301G>T [p.G101W] founder mutation. Additionally, we attempted to establish the frequency of the red hair/fair skin phenotype in our region. As predicted by anecdotal evidence, the frequency of red hair/phototype I was very low (0.7%). Screening of 17 red-haired individuals and their red-haired relatives, 207 controls and 214 melanoma patients unselected for hair color but all of Ligurian descent, led to the detection of 8 novel substitutions (c.133T>C [p.F45L], c.248C>T [p.S83L], c.332C>T [p. A111V], c.479G>A [p.R160Q], c.637C>T [p.R213W], c.793G>A [p. V265I], c.923C>T [p. T308M], c.943T>C [p.C315R]), 1 novel deletion (c.520_523delGTC [p.V174del]) and 3 novel synonymous variants (c.366G>C [p. V122V], c.684G>A [p. Q228Q], c.726C>T [p.T241T]). Preliminary genotype/phenotype correlation seems to indicate that other genes involved in the regulation of human pigmentation may mask the recessive action of high-penetrance MC1R alleles, thus determining the low frequency of at-risk phototypes and of incidence and/or penetrance of melanoma in Liguria.     

Identification of a gene disrupted by a microdeletion in a patient with X-linked retinitis pigmentosa (XLRP)

The gene for the most frequent from of X-linked retinitis pigmentosa (XLRP), RP3, has been assigned by genetic and physical mapping to a segment of less than 1000 kbp, which is flanked by the marker DXS1110 and the ornithine transcarbamylase (OTC) gene. In search of microdeletions, we have screened the DNA of 30 unrelated patients with XLRP by employing a representative set of YAC-derived DNA fragments that were generated by restriction enzyme digestion and PCR amplification. In one of these patients, a 6.4 kbp microdeletion was detected which was not present in the DNA of 444 male controls. A cosmid contig spanning the deletion was constructed and used to isolate cDNAs from retina-specific libraries. Exons corresponding to these expressed sequences as well as other putative exons were identified by sequencing more than 30 kbp of the critical region. So far, no point mutations in these putative exon sequences have been identified.