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Biomedical subjects

G Romeo

Publications and source records attributed to G Romeo.

At least 109 records · Page 6Linked to original sources
PubMed1997

High glucose level unmasks a genetic predisposition to enhanced extracellular matrix production in mesangial cells from the Milan normotensive strain.

A growing body of evidence indicates that the individual genetic background plays a role in the pathogenesis of diabetic glomerular disease by either favoring or protecting against injury produced by hyperglycemia. Two genetically related rat strains, the Milan normotensive strain (MNS) and the Milan hypertensive strain (MHS) display different susceptibilities to develop glomerulosclerosis with age. Glomerular sclerosing lesions occur in the MNS rats, which remain normotensive throughout their entire life-span, but not in the MHS rats, despite the presence of arterial hypertension. Previous studies have reported that extracellular matrix production and cell proliferation increased with donor-aging in mesangial cells isolated from MNS rats, but not in those from MHS rats, thus suggesting the existence of an inherited defect in the regulation of cell and matrix turnover, which translates into an abnormal response to growth-promoting stimuli favoring the development of glomerulosclerosis. In the study presented here, it was hypothesized that, in addition to donor-aging, other independent risk factors for the development of glomerular disease, such as metabolic injury by hyperglycemia, would be able to trigger and/or precipitate the occurrence of these changes in mesangial cells from the susceptible normotensive strain, but not in those from the protected hypertensive strain. To test this hypothesis, mesangial cells obtained from these rat strains (before the onset of either glomerulosclerosis or hypertension) were used to assess the effects of prolonged (4 wk) exposure to high (30 mmol/L) versus normal (5.5 mmol/L) glucose concentrations on extracellular matrix and cytokine production and cell proliferation. The accumulation and/or gene expression of the matrix components fibronectin, laminin, and collagen IV, and of the cytokines insulin-like growth factor-I (IGF-I) and transforming growth factor-beta (TGF-beta) did not change under normal glucose and increased progressively in response to high glucose in both MNS and MHS cells. These increases, with the exception of the increment in TGF-beta gene expression, were significantly more pronounced in MNS cells than in MHS cells. In contrast, the proliferative response to serum was not affected by high glucose, but increased in MNS cells, and decreased, although not significantly, in MHS cells during the 4-wk period, thus mimicking the changes previously observed in these rat strains as a function of age. These results indicate that high glucose unmasks a genetic tendency to produce increasing amounts of extracellular matrix, not yet evident under normal glucose conditions, and suggest that a genetically determined propensity of mesangial cells to hyperrespond to chronic hyperglycemia may be implicated in the pathogenesis of diabetic glomerular disease.

Animals↗
PubMed1997

Upregulation of mesangial growth factor and extracellular matrix synthesis by advanced glycation end products via a receptor-mediated mechanism.

Enhanced advanced glycosylation end product (AGE) formation has been shown to participate in the pathogenesis of diabetes-induced glomerular injury by mediating the increased extracellular matrix (ECM) deposition and altered cell growth and turnover leading to mesangial expansion. These effects could be exerted via an AGE-receptor-mediated upregulation of growth factors, such as the IGFs and transforming growth factor-beta (TGF-beta). We tested this hypothesis in human and rat mesangial cells grown on nonglycated or native bovine serum albumin (BSA), glycated BSA with AGE formation (BSA-AGE), or glycated BSA in which AGE formation was prevented by the use of aminoguanidine (BSA-AM), in the presence or absence of an antibody, alpha-p60, directed against the p60/OST protein named AGE-receptor 1 (AGE-R1), or normal control (pre-immune) serum. The mRNA and/or protein levels of IGF-I, IGF-II, IGF receptors, IGF binding proteins (IGFBPs), TGF-beta1 and the ECM components fibronectin, laminin, and collagen IV were measured, together with cell proliferation. Both human and rat mesangial cells grown on BSA-AGE showed increased IGF-I and total and bioactive TGF-beta medium levels and enhanced IGF-I, IGF-II, and TGF-beta1 gene expression, compared with cells grown on BSA, whereas total IGFBP and IGFBP-3 medium content, IGF receptor density and affinity, and IGF-I receptor transcripts were unchanged. Moreover, cells grown on BSA-AGE showed increased ECM protein and mRNA levels versus cells cultured on BSA, whereas cell proliferation was unchanged in human mesangial cells and slightly reduced in rat mesangial cells. Growing cells on BSA-AM did not affect any of the measured parameters. Co-incubation of BSA-AGE with anti-AGE-R1, but not with pre-immune serum, prevented AGE-induced increases in IGF-I, TGF-beta1, and ECM production or gene expression; anti-AGE-R1 also reduced growth factor and matrix synthesis in cells grown on BSA. These results demonstrate that mesangial IGF and TGF-beta1 synthesis is upregulated by AGE-modified proteins through an AGE-receptor-mediated mechanism. The parallelism with increased ECM production raises the speculation that the enhanced synthesis of these growth factors resulting from advanced nonenzymatic glycation participates in the pathogenesis of hyperglycemia-induced mesangial expansion.

Animals↗
PubMed1997

Early, but not advanced, glomerulopathy is reversed by pancreatic islet transplants in experimental diabetic rats: correlation with glomerular extracellular matrix mRNA levels.

In this study, we investigated 1) whether long-term restoration of euglycemia by means of pancreatic islet transplants is capable of preventing and/or reversing renal functional and structural alterations in an experimental model of insulin-deficient diabetes, and 2) whether changes in extracellular matrix (ECM) and cell turnover at the glomerular level and biochemical abnormalities associated with hyperglycemia correlate with the renal outcome after transplantation. Male Lewis rats, rendered diabetic by intravenous injection of streptozotocin, underwent homologous islet transplantation via the portal vein at 2 weeks (study A), at 4 months (study B), and at 8 months (study C) after the induction of diabetes and killed 12 months after transplantation in study A and 4 months after transplantation in studies B and C. Age-matched nondiabetic and untreated diabetic rats were used as control animals and were studied at 4, 8, and 12 months. In the untreated diabetic animals, metabolic derangement was associated with increased erythrocyte polyol and fructose levels, tail-tendon content of advanced glycation end products (AGEs), total proteinuria, albuminuria, kidney weight, and mean glomerular volume as well as with marked glomerular and extraglomerular lesions. Glomerular gene expression for the ECM components fibronectin and collagen IV and for TGF-beta was also increased, whereas glomerular cell proliferation was unaffected by diabetes. In study A, changes in renal function and structure observed in diabetic rats at 12 months were completely prevented by successful islet transplants. In study B, all functional and structural abnormalities detected in diabetic rats at 4 months of disease duration were virtually reversed by 4 months of euglycemia in transplanted animals, whereas they progressed further in untreated diabetic rats. In study C, the course of functional and structural changes observed in untreated diabetic rats was not reversed by islet transplantation. Likewise, tissue AGE accumulation and particularly upregulation of glomerular ECM and transforming growth factor (TGF)-beta gene expression, which are believed to play a role in the pathogenesis of altered renal function and structure in diabetes, were normalized in transplanted rats from study A and study B, but not in those from study C. These experiments show that restoration of euglycemia by islet transplants is capable of preventing experimental diabetic glomerulopathy and reversing early changes in renal function and structure induced by diabetes. In a later phase of the disease, when glomerular matrix gene expression becomes independent of hyperglycemia, possibly because of the persistent increase in tissue AGE accumulation, metabolic control is not capable of reversing renal abnormalities.

Animals↗
PubMed1997

Identification of Mediterranean beta-thalassemia mutations by reverse dot-blot in Italians and Egyptians.

beta-Thalassemia is a significant public health problem in Egypt where over 1000 of the annual 1.5 million newborns are expected to be affected with this disorder. A preventive program of the disease should be multifaceted with its technical component based on carrier screening and prenatal diagnosis through mutation detection. In addition, it should have an information and educational component with the aim of increasing public awareness of the disease. Proper selection of the technique(s) to be utilized in such a program is highly important. The appropriate technique to be used in screening should be reliable, simple and cost effective. It should also circumvent the problem of marked heterogeneity of the disease in Egypt. The reverse dot-blot technique has been used in the present study for the characterization of mutations in 138 Italian and 108 Egyptian thalassemia chromosomes, confirming its reliability as a screening method. The technique is now in routine use for thalassemia diagnosis in the Microcitemia Center of the Galliera Hospital in Genoa, Italy. Based on these results, we recommend the reverse dot-blot method as the technique of choice in the preventive program of this disease in Egypt.

Alleles↗
PubMed1997

Retinoic acid and IFN inhibition of cell proliferation is associated with apoptosis in squamous carcinoma cell lines: role of IRF-1 and TGase II-dependent pathways.

Both retinoids and IFNs are known to inhibit proliferation of many normal and transformed cells and to have an in vivo antitumor effect against a variety of cancers, including squamous cell carcinoma. Because the combination of IFNs and all-trans retinoic acid (RA) could improve their antitumor effectiveness (depending on the histological origin and state of differentiation of the cells), we compared the activity of RA and/or IFN-alpha 2b with regard to the mechanism of growth inhibition of ME180 and SiHa cell lines, derived from squamous cervix carcinoma at different stages of differentiation. We reported previously that, in the ME180 cell line, the combined treatment significantly increased the growth inhibitory effect of the single agents. Here, we show that the SiHa cell line appears more sensitive to IFN-alpha 2b than the ME180 cell line, and resistant to RA, which does not significantly inhibit SiHa cell growth. Induction of apoptotic cell death clearly occurs and correlates with the inhibition of cell proliferation in both cell lines. It is interesting that the induction of the transcription factor IFN regulatory factor 1 correlates with the subsequent induction of apoptosis, whereas TGase I and II expression does not. In particular, TGase I and II appear differentially expressed in the ME180 and SiHa cell lines; i.e., TGase I is expressed in ME180 and specifically inhibited by RA, whereas TGase II is expressed in SiHa. It is interesting that both IFN-alpha and RA are able to increase TGase II expression and activity in this cell line.

Apoptosis↗
PubMed1996

Exclusion of linkage between RET and neuronal intestinal dysplasia type B.

Neuronal Intestinal Dysplasia type B (NID B) is a complex alteration of the enteric nervous system belonging to the group of intestinal dysganglionoses which may involve rectum, colon, and small intestine. Second only to Hirschsprung disease (HSCR), NID B is one of the most frequent causes of chronic constipation and pseudo-obstructive intestinal dysmotility. Since NID B is often associated with HSCR and point mutations in the RET proto-oncogene have been identified in HSCR patients, we analyzed two NID B pedigrees to investigate if RET mutations might cause also the NID B phenotype. Linkage analysis demonstrated that the NID B locus is not linked to RET in the pedigrees analyzed. Further genetic analyses will possibly improve the understanding of the cause and facilitate diagnostic procedures in NID B.

Drosophila Proteins↗
PubMed1996

Strategies for the Identification of Intron-Exon Boundaries and Point Mutations: The Example of the RET Proto-Oncogene

For any given gene, the identification of exon boundaries and the corresponding flanking intron sequences is the essential prerequisite for asking interesting questions related to its structural organization, spanning from the study of its evolution to the molecular mechanisms underlying possible alternative splicing. In addition, this type of knowledge allows us to carry out mutation screenings for whole exons when the gene in question is known or suspected to be responsible for a human disease phenotype. Several different strategies already used to establish the genomic organization of genes are presented in detail, along with appropriate examples, and they are compared to each other with respect to their requirements, efficiency, and applicability. Particular emphasis is given to the detailed description of the strategy used to study the intron-exon junctions and to establish the intron flanking sequences of the RET proto-oncogene, whose mutations cause Hirschsprung disease (or congenital megacolon) as well as different types of thyroid cancer.

Journal Article↗
PubMed1996

Regulatory role of eicosanoids in extracellular matrix overproduction induced by long-term exposure to high glucose in cultured rat mesangial cells.

Accumulation of extracellular matrix in the mesangium and altered renal eicosanoid synthesis are two prominent features of diabetic glomerular disease. We investigated the relationship between eicosanoid and extracellular matrix production in rat mesangial cells cultured under high glucose vs normal glucose conditions. Long-term exposure of rat mesangial cells to high glucose, but not to iso-osmolar mannitol, significantly increased extracellular matrix accumulation and gene expression and transforming growth factor-beta (TGF-beta) mRNA levels, and decreased prostaglandin (PG) E2 synthesis without affecting production of either thromboxane (TX) B2 or PGF2 alpha, with respect to cells incubated in normal glucose. Addition of exogenous PGE2 resulted in a dose-dependent reduction of matrix protein and mRNA levels and TGF-beta gene expression in cells cultured in either normal or high glucose conditions, whereas exposure to exogenous PGF2 alpha produced a significant increment in matrix production and matrix and TGF-beta gene expression in cells grown in normal glucose, but only a slight increase in those cultured in high glucose. Stimulation of endogenous endoperoxide metabolism towards PGE2 and PGF2 alpha synthesis with FCE-22,178, a drug originally developed as TXA2 synthase inhibitor, resulted in a dose-dependent decrease in matrix accumulation and matrix and TGF-beta gene expression which was suppressed by coincubation with the cyclo-oxygenase inhibitor fenoprofen blocking the FCE-22,178-enhanced PG production. In both cell lines, the rate of synthesis of TXA2 was very low and the selective blockade of its synthesis (by two other TXA2 synthase inhibitors, OKY-046 and Ridogrel) or action (by the TXA2 receptor antagonist BM-13,177) did not alter matrix production or TGF-beta mRNA levels. These results suggest that the cyclo-oxygenase pathway is involved in the regulation of matrix changes induced by high glucose in rat mesangial cells; the reduced production of PGE2 may enhance the synthesis or potentiate the effect of stimulators of ECM formation such as TGF-beta, whereas TXA2 does not appear to be involved. These data also indicate that glucose-enhanced mesangial matrix accumulation may be prevented by exogenous PGE2 or by drugs capable of increasing endogenous PGE2 synthesis.

Animals↗
PubMed1996

Increased activity of the insulin-like growth factor system in mesangial cells cultured in high glucose conditions. Relation to glucose-enhanced extracellular matrix production.

Recent evidence suggests that several growth factors participate in diabetic glomerular disease by mediating increased extracellular matrix accumulation and altered cell growth and turnover leading to mesangial expansion. Transforming growth factor (TGF)-beta has been demonstrated to be upregulated both in vivo and in vitro, whereas studies on the activity of the renal insulin-like growth factor (IGF) system in experimental diabetes have provided conflicting results. We investigated the effects of prolonged exposure (4 weeks) of cultured human and rat mesangial cells to high (30 mmol/l) glucose vs iso-osmolar mannitol or normal (5.5 mmol/l) glucose levels on: 1) the autocrine/paracrine activity of the IGF system (as assessed by measuring IGF-I and II, IGF-I and II receptors, and IGF binding proteins); and, in parallel, on 2) TGF-beta 1 gene expression; 3) matrix production; and 4) cell proliferation. High glucose levels progressively increased the medium content of IGF-I and the mRNA levels for IGF-I and IGF-II, increased IGF-I and IGF-II binding and IGF-I receptor gene expression, and reduced IGF binding protein production. TGF-beta 1 transcripts and matrix accumulation and gene expression were increased in parallel, whereas cell proliferation was reduced. Iso-osmolar mannitol did not affect any of the above parameters. These experiments demonstrated that high glucose levels induce enhanced mesangial IGF activity, together with enhanced TGF-beta 1 gene expression, increased matrix production, and reduced cell proliferation. It is possible that IGFs participate in mediating diabetes-induced changes in matrix turnover leading to mesangial expansion, by acting in a paracrine/autocrine fashion within the glomerulus.

Animals↗
PubMed1996

RET mutations in human disease.

The RET proto-oncogene is at the origin of one of the most interesting models of human disease caused by mutations in a receptor tyrosine kinase gene. Somatic rearrangements of RET are involved in the aetiology of a variable proportion of papillary thyroid carcinomas (PTC), the most common type of thyroid tumour whose prevalence is increasing in areas heavily exposed to radioactive fallout after the Chernobyl accident of 1986. Moreover, germline RET mutations are associated with the three variants of the inherited cancer syndrome known as multiple endocrine neoplasia type 2 (MEN2A, MEN2B and FMTC). Finally, RET mutations or heterozygous deletions of the whole gene cause the autosomal dominant form of Hirschsprung disease (HSCR), a congenital disorder of the enteric nervous system (ENS).

Animals↗
PubMed1996

Localization of a gene responsible for autosomal recessive demyelinating neuropathy with focally folded myelin sheaths to chromosome 11q23 by homozygosity mapping and haplotype sharing.

Hereditary motor and sensory neuropathy (HMSN) with focally folded myelin sheaths, or Charcot-Marie-Tooth type 4B (CMT4B), is a distinct clinical entity belonging to the heterogeneous group of autosomal recessive demyelinating neuropathies. We first described a large pedigree with CMT4B, which showed a high consanguinity level and an autosomal recessive pattern of inheritance. Through conventional linkage analysis, we excluded linkage of the locus segregating in this pedigree to any of the known genes responsible for other HMSNs. Using homozygosity mapping and haplotype sharing analysis, we were able to localize the disease gene in a 4 cM interval on chromosome 11q23, between the D11S1332 and D11S917 loci. On the basis of the clinical characteristics of the disease, we propose that this locus corresponds to the CMT4B gene.

Charcot-Marie-Tooth Disease↗
PubMed1996

Volume regulatory taurine release in human tracheal 9HTEo- and multidrug resistant 9HTEo-/Dx cells.

The intracellular taurine release evoked by hypotonic shock is accomplished by volume-activated Cl- channels whose activity has been related to the expression of the multidrug resistance protein (MDR-1). We studied taurine transport in 9HTEo- cells and in the derived cell line 9HTEo-/Dx expressing MDR-1. [3H]taurine release from preloaded cells increased upon reduction of extracellular osmolality. This process was not inhibited by preincubation with phorbol 12-myristate 13-acetate but was reduced by inhibitors of volume-sensitive Cl- channels such as 1,9-dideoxiforskolin, La3+, and arachidonate. Verapamil, a substrate of MDR-1, increased the osmotically evoked taurine efflux. Replacement of extracellular Cl- with I- or gluconate or of extracellular Na+ with Li+ significantly reduced the taurine efflux, whereas substitution of N-methyl-D-glucamine for Na+ increased it. Application of ATP and 2-chloroadenosine stimulated the efflux in isotonic medium. No differences were seen between 9HTEo- and 9HTEo-/Dx cells with respect to hypotonically induced taurine efflux and the response to phorbol ester, channel blockers, ion replacement, and purinergic agents. Our results reveal novel properties of the osmotically induced taurine release and demonstrate its independence from MDR-1 gene expression.

Biological Transport↗
PubMed1996

Villous adenocarcinoma of the duodenum invading the ampulla of Vater case report and review of literature.

We report a case of villous adenocarcinoma of duodenum arising from the ampulla of Vater with a review of the literature. Although preoperative endoscopic biopsies were performed, no malignancy was identified. Because of the pathological uncertainty we decided to perform a pylorus-preserving pancreatoduodenectomy. Microscopic examination demonstrated glandular dysplasia with aspects of villous adenoma and well differentiated adenocarcinoma. We conclude that both in malignant cases and in cases with uncertain diagnosis a pylorus-preserving pancreatoduodenectomy is the best surgical treatment because it results in better 5 year survival.

Adenocarcinoma↗
PubMed1996

Physical map and cosmid contig encompassing a new interstitial deletion of the X-linked lymphoproliferative syndrome region.

The X-linked lymphoproliferative syndrome (XLP) is an inherited immuno-deficiency to Epstein-Barr virus infection that has been mapped to chromosome Xq25. Molecular analysis of XLP patients from ten different families identified a small interstitial constitutional deletion in 1 patient (XLP-D). This deletion, initially defined by a single marker, DF83, known to map to interval Xq24-q26.1, is nested within a previously reported and much larger deletion in another XLP patient (XLP-739). A cosmid minilibrary was constructed from a single mega-YAC and used to establish a contig encompassing the whole XLP-D deletion and a portion of the XLP-739 deletion. Based on this contig, the size of the XLP-D deletion can be estimated at 130 kb. The identification of this minimal deletion, within which at least a portion of the XLP gene is likely to reside, should greatly facilitate efforts in isolating the gene.

Adolescent↗
PubMed1996

Prevalence and parental origin of de novo RET mutations in Hirschsprung's disease.

In contrast with the reported almost exclusive paternal origin of de novo mutations in MEN 2A, FMTC and MEN 2B, de novo mutations in Hirschsprung patients arise both on paternal and maternal chromosomes. This distinctive feature of RET mutations associated with Hirschsprung's disease and of the RET mutations associated with thyroid cancer indicates a basic biological difference between the mutational events leading to the different phenotypes.

DNA Mutational Analysis↗