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Biomedical subjects

M Fornaro

Publications and source records attributed to M Fornaro.

At least 19 recordsLinked to original sources
PubMed2004

Low-power laser biostimulation enhances nerve repair after end-to-side neurorrhaphy: a double-blind randomized study in the rat median nerve model.

Previous studies have shown that low-power laser biostimulation (lasertherapy) promotes posttraumatic nerve regeneration. The objective of the present study was to investigate the effects of postoperative lasertherapy on nerve regeneration after end-to-side neurorrhaphy, an innovative technique for peripheral nerve repair. After complete transection, the left median nerve was repaired by end-to-side neurorrhaphy on the ulnar "donor" nerve. The animals were then divided into four groups: one placebo group, and three laser-treated groups that received lasertherapy three times a week for 3 weeks starting from postoperative day 1. Three different types of laser emission were used: continuous (808 nm), pulsed (905 nm), and a combination of the two. Functional testing was carried out every 2 weeks after surgery by means of the grasping test. At the time of withdrawal 16 weeks postoperatively, muscle mass recovery was assessed by weighing the muscles innervated by the median nerve. Finally, the repaired nerves were withdrawn, embedded in resin and analyzed by light and electron microscopy. Results showed that laser biostimulation induces: (1) a statistically significant faster recovery of the lesioned function; (2) a statistically significant faster recovery of muscle mass; (3) a statistically significant faster myelination of the regenerated nerve fibers. From comparison of the three different types of laser emissions, it turned out that the best functional outcome was obtained by means of pulsed-continuous-combined laser biostimulation. Taken together, the results of the present study confirm previous experimental data on the effectiveness of lasertherapy for the promotion of peripheral nerve regeneration and suggest that early postoperative lasertherapy should be considered as a very promising physiotherapeutic tool for rehabilitation after end-to-side neurorrhaphy.

Animals↗
PubMed2003

HuC/D confocal imaging points to olfactory migratory cells as the first cell population that expresses a post-mitotic neuronal phenotype in the chick embryo.

In the present study, the expression of the HuC/D RNA-binding proteins, a marker of neurons that have left the mitotic cycle, in cells migrating from the olfactory neuroepithelium toward the telencephalon in the chick embryo was investigated by means of immunofluorescence and confocal laser microscopy. Results showed that this migratory cell population is early and massively labeled by the a-HuC/D antibody starting from the first olfactory pit stage. At this developmental stage, olfactory migratory cells appeared to be the only neuronal population that expressed the HuC/D antigens in the whole embryo. In following developmental stages, the great majority of migratory cells, the number of which increased progressively, continued to be heavily immunopositive for the a-HuC/D antibody while immunopositivity to this antibody begins to be detected in other regions of the nervous system. HuC/D immunopositivity persisted until stage 30 HH (about 6.5 days), the later developmental stage investigated in this study, when colocalization with GnRH was detected. Negativity to the anti-proliferating cell nuclear antigen (anti-PCNA) immunostaining, a marker of S-phase, showed that migratory olfactory cells have left the mitotic cycle. Altogether, these results suggest that we have identified the first population of post-mitotic neurons in the developing nervous system of the chick embryo.

Animals↗
PubMed2003

Schwann-cell proliferation in muscle-vein combined conduits for bridging rat sciatic nerve defects.

Among the various grafting procedures that have been studied as alternatives to traditional fresh nerve autografts for the repair of severed peripheral nerves, muscle-vein-combined graft conduits have recently been devised and successfully employed. In the present study, the early presence, origin, and proliferation activity of Schwann cells (SCs) along this particular type of biological graft conduit have been investigated, using antibodies directed against glial fibrillar acid protein (GFAP), a protein that is specifically expressed in glial cells, and proliferating cell nuclear antigen (PCNA), a protein that is expressed by cells during DNA synthesis. Results showed that the muscle-vein-combined graft was progressively invaded by a number of GFAP-immunopositive SCs, many of which were also found to be immunopositive for PCNA, thus demonstrating that their proliferation continues to occur inside the graft. Among the molecules that could be involved in the stimulation of Schwann-cell proliferation is neuregulin-1 (NRG-1) that mediates its effects by binding to the ErbB receptor tyrosine kinase family. In the present study, the authors report on the RT-PCR analysis for NRG-1 and ErbB3 mRNAs, showing an overall increase in the content of these transcripts inside the muscle-vein-combined graft. These results suggest that the muscle-vein-combined graft conduit constitutes an environment favorable to potentiate Schwann-cell proliferation during the early regeneration phases.

Animals↗
PubMed2001

Unscheduled DNA synthesis in rat adult myenteric neurons: an immunohistochemical study.

Unscheduled DNA synthesis refers to DNA synthesis not followed by cell division. Previous studies have suggested that this phenomenon may occur in neurons from peripheral myenteric ganglia in conditions of functional hyperstimulation. In order to verify these observations, we have carried on an immunohistochemical study on myenteric neurons from the hypertrophic intestinal loops upstream from a partial obstruction (an experimental condition that induces a relevant increase of the neuronal workload) after labelling with two different markers: the proliferating cell nuclear antigen (PCNA), that is specifically expressed in cell nuclei during the S-phase, and the protein gene product 9.5 (PGP 9.5), a specific marker of nerve cells. While no myenteric neuron immunopositive for the anti-PCNA antibody was found in the control intestine, in the hypertrophic myenteric ganglia some neurons were positive for PCNA. These results provide an unequivocal evidence on the existence of unscheduled DNA synthesis in myenteric neurons from the hypertrophic intestine.

Animals↗
PubMed2001

Adult stem cells and neurogenesis: historical roots and state of the art.

Over the last few years, an impressive number of papers have addressed the stem cell issue. However, as often occurs when a scientific subject undergoes a period of fast growth, some confusion is generated. To help reduce the existing uncertainty, this paper focuses on the concept of adult stem cells in relation to the classification of cell populations on the basis their proliferative behavior. Particular attention is dedicated to adult neural stem cells, an issue that has recently seen the most amazing advances. Finally, the concept of adult stem cells is differentiated from that of developmental stem cells in relation to the employment of stem cells for transplantation therapies.

Adult↗
PubMed2001

Cyclin D1 gene contains a cryptic promoter that is functional in human cancer cells.

A novel cyclin D1 (CCND1)-TROP2 fusion oncogene has been isolated from human cancer cells. Unexpectedly, the chimeric cDNA was found to express TROP2 in the absence of exogenous promoters. Mutagenesis of the TROP2 and CCND1 sequences and in vitro transcription/translation show that a cryptic promoter is present in the 3' coding region of CCND1. The CCND1 cryptic promoter is functional in luciferase assays, where it augments the basal expression levels by eightfold and efficiently cooperates with an SV-40 enhancer. The transcription start sites of the cryptic promoter map at bases 797 and 935 of CCND1, as determined by RNase protection assays. The cryptic promoter possesses canonical binding sites for ubiquitous transcription factors and W/S, X1, and CAAT/Y boxes that are characteristic of major histocompatibility complex class II gene promoters. Remarkably, the cryptic CCND1 promoter is active in human cancer cells and generates a truncated transcript that contains CCND1 instability sequences. Thus, this novel CCND1 transcription unit may play a role in the regulation of the expression of cyclin D1 and in tumor cell growth.

3T3 Cells↗
PubMed2001

Confocal imaging of Schwann-cell migration along muscle-vein combined grafts used to bridge nerve defects in the rat.

Schwann cells guide axonal regrowth during peripheral nerve repair. In a case of a nerve lesion with substance loss, a graft conduit is necessary to enable axons to reach the distal nerve stump. If a non-nervous autograft is used, the question arises as to the presence and origin of Schwann cells along the grafted tube. We addressed this issue using a tubulization technique based on the use of an autologous vein filled with fresh skeletal muscle for the repair of sciatic nerve defects in the rat. We showed that both ends of the graft were early and progressively colonized by a number of glial fibrillar acid protein-immunopositive and S-100 immunonegative cells, an immunocytochemical pattern typical of immature Schwann cells. These cells, which were located in the interstice between grafted skeletal muscle fibers, are mainly organized into long chains oriented along the main axis of the graft and progressively colonize all the graft. Schwann cells coming from the distal nerve end are suitable for being responsible for guiding regeneration of nerve fibers along the graft toward the correct periphery (tissue specificity).

Animals↗
PubMed2001

Regulation of MCP-3 and BRCA2 mRNA expression levels by beta(1) integrins.

The integrin cytoplasmic domain has been shown to modulate several cellular functions, including cell proliferation, adhesion, migration, and intracellular signaling. The beta(1) integrin subunits beta(1C) and beta(1A), which contain variant cytoplasmic domains, differentially affect cancer and normal cell functions. To identify target genes selectively regulated by these beta(1) cytoplasmic variants, stable cell transfectants expressing either beta(1A) or beta(1C) under the control of a doxycycline-inducible promoter were obtained using murine beta(1)-deficient GD25 cells. Screening of 1176 murine cDNAs using first-strand cDNA of mRNA isolated from either beta(1C)- or beta(1A)-expressing cells showed a striking differential expression of few genes. The differential expression of two genes, MCP-3 and BRCA2 (monocyte chemoattractant protein-3 and breast cancer susceptibility gene 2, respectively), whose products are involved, respectively, in chemotaxis and embryonic proliferation, was confirmed by Northern blot analysis. Increased MCP-3 and decreased BRCA2 mRNA levels in cells expressing beta(1C) compared to those in cells expressing beta(1A) were observed. Since beta(1C) and beta(1A) stable cell transfectants showed comparable adhesion to fibronectin, upregulation of MCP-3 and downregulation of BRCA2 mRNA levels did not appear to be due to a differential ability of the beta(1C) cells to adhere to the beta(1) ligand fibronectin. Overall, our data show that beta(1) integrin cytoplasmic domain variants control expression of downstream target genes in a differential manner without affecting cell adhesion.

Animals↗
PubMed2001

Epitope-specific antibodies to the beta(1C) integrin cytoplasmic domain variant.

The beta(1C) integrin is an alternatively spliced variant of the beta(1) subunit that contains a unique 48-amino-acid sequence in its cytoplasmic domain. We have shown previously that beta(1C) is a potent inhibitor of cell proliferation and that in vivo its expression is downregulated in prostate and breast carcinoma. In this study, we describe a panel of specific monoclonal antibodies that react with the beta(1C) cytodomain. We show by immunoblot analysis that the newly generated monoclonal antibodies specifically recognize the beta(1C) cytodomain expressed as glutathione S-transferase fusion protein. The specificity of the antibodies to beta(1C) was confirmed in competition studies by immunoblotting using beta(1C)-specific synthetic peptides. These monoclonal antibodies reacted, in enzyme-linked immunosorbent assays, with the beta(1C) 785-808 peptide but failed to bind the beta(1C) 778-794, beta(1C) 805-825, or beta(1A) 765-798 peptides. Thus, the epitope recognized by the antibodies is located within the Q(795)-F(804) beta(1C) cytoplasmic sequence; this region overlaps the previously described Q(795)-Q(802) domain necessary for beta(1C) to inhibit cell proliferation. To our knowledge, these are the first monoclonal antibodies specific for a beta(1) cytoplasmic isoform. The monoclonal antibodies described here will be useful tools for dissecting functional differences, among beta(1) integrin variants, as well as for the study of the role of beta(1C) in prostate and breast epithelial cell proliferation.

Amino Acid Sequence↗
PubMed2001

Evidence of very early neuronal migration from the olfactory placode of the chick embryo.

Cell migration from the olfactory neuroepithelium is a very peculiar phenomenon in the development of the nervous system. In this paper, we provide evidence that, in the chick embryo, migration of cells from the olfactory neuroepithelium begins earlier than previously reported, namely at the same time as the first olfactory placode differentiation, that occurs several hours before the superficial ectodermal invagination that gives rise to the olfactory pit. Moreover, we provide evidence that very early migrating cells express the HuC/D RNA-binding protein antigen, a specific neuronal marker. These observations refocus our knowledge on the very first developmental stages of olfactory neuroepithelium.

Animals↗
PubMed2001

Confocal imaging of HuC/D RNA-binding proteins in adult rat primary sensory neurons.

HuC/D RNA-binding proteins are antigens that are specifically expressed in nerve cells. In the present study, immunocytochemistry and laser confocal microscopy were used to study the cellular distribution of HuC/D RNA-binding proteins in adult rat primary sensory neurons. Colocalization with the protein gene product 9.5 (PGP 9.5) was used to identify sensory neurons. Confocal laser imaging showed that HuC/D antigens are expressed in the cytoplasm of all rat primary sensory neurons. Most neurons also showed anti-HuC/D immuno-positivity in the nucleus. The demonstration of the ubiquitous expression of HuC/D antigens in primary sensory neurons gives us a possible explanation for the severe pathological lesions observed in dorsal root ganglia in association with the paraneoplastic Hu Syndrome.

Animals↗
PubMed2001

Integrins and prostate cancer metastases.

Integrins have emerged as modulators of a variety of cellular functions. They have been implicated in cell migration, survival, normal and aberrant cellular growth, differentiation, gene expression, and modulation of intracellular signal transduction pathways. In this review article, the structural and functional characteristics of integrins, their expression and their potential role in prostate cancer metastases will be discussed.

Adenocarcinoma↗
PubMed2000

Down-regulation of beta(1C) integrin in breast carcinomas correlates with high proliferative fraction, high histological grade, and larger size.

beta(1C) integrin is an unspliced form of the integrin beta(1) subfamily, which has been shown to inhibit cell proliferation in vitro. Using an affinity-purified rabbit antibody, we have investigated 283 previously untreated breast carcinomas, with the aim of ascertaining the actual prevalence of beta(1C) expression in these tumors and of defining its pathological correlates. Immunoblotting and reverse transcriptase-polymerase chain reaction experiments have also been performed in selected cases, to confirm the immunocytochemical findings. Overall, beta(1C) immunoreactivity was down-regulated (ie, expressed in < 50% of the neoplastic cells) in 114 cases (40.3%). Down-regulation of beta(1C) expression in breast carcinomas correlated significantly with the tumor grade, the proliferative fraction (as evaluated by Ki-67 immunostaining with the MIB-1 monoclonal antibody), the estrogen and progesterone receptor status, and the tumor size (pT classification) and marginally with the node status. In a multivariate analysis with all available measures fitted simultaneously, tumor grade (P = 0.004), Ki-67 immunolabeling (P = 0.01), and pT categories (P = 0.04) were significantly associated with beta(1C) immunoreactivity. Although the short follow-up time (2-3 years) of the current series of patients does not allow the performance of survival analyses, the correlation of beta(1C) expression with tumor size, grade, and proliferative fraction and its alleged role as an upstream regulator of p27(kip1) make this integrin variant a likely novel prognostic parameter for invasive carcinomas of the breast.

Breast↗
PubMed2000

Regulation of mRNA and protein levels of beta1 integrin variants in human prostate carcinoma.

Alterations of integrin expression levels in cancer cells correlate with changes in invasiveness, tumor progression, and metastatic potential. The beta1C integrin, an alternatively spliced form of the human beta1 integrin, has been shown to inhibit prostate cell proliferation. Furthermore, beta1C protein levels were found to be abundant in normal prostate glandular epithelium and down-regulated in prostatic adenocarcinoma. To gain further insights into the molecular mechanisms underlying abnormal cancer cell proliferation, we have studied beta1C and beta1 integrin expression at both mRNA and protein levels by Northern and immunoblotting analysis using freshly isolated neoplastic and normal human prostate tissue specimens. Steady-state mRNA levels were evaluated in 38 specimens: 33 prostatic adenocarcinomas exhibiting different Gleason's grade and five normal tissue specimens that did not show any histological manifestation of benign prostatic hypertrophy. Our results demonstrate that beta1C mRNA is expressed in normal prostate and is significantly down-regulated in neoplastic prostate specimens. In addition, using a probe that hybridizes with all beta1 variants, mRNA levels of beta1 are found reduced in neoplastic versus normal prostate tissues. We demonstrate that beta1C mRNA down-regulation does not correlate with either tumor grade or differentiation according to Gleason's grade and TNM system evaluation, and that beta1C mRNA levels are not affected by hormonal therapy. In parallel, beta1C protein levels were analyzed. As expected, beta1C is found to be expressed in normal prostate and dramatically reduced in neoplastic prostate tissues; in contrast, using an antibody to beta1 that recognizes all beta1 variants, the levels of beta1 are comparable in normal and neoplastic prostate, thus indicating a selective down-regulation of the beta1C protein in prostate carcinoma. These results demonstrate for the first time that beta1C and beta1 mRNA expression is down-regulated in prostate carcinoma, whereas only beta1C protein levels are reduced. Our data highlight a selective pressure to reduce the expression levels of beta1C, a very efficient inhibitor of cell proliferation, in prostate malignant transformation.

Adenocarcinoma↗
PubMed2000

Differential role of beta(1C) and beta(1A) integrin cytoplasmic variants in modulating focal adhesion kinase, protein kinase B/AKT, and Ras/Mitogen-activated protein kinase pathways.

The integrin cytoplasmic domain modulates cell proliferation, adhesion, migration, and intracellular signaling. The beta(1) integrin subunits, beta(1C) and beta(1A), that contain variant cytoplasmic domains differentially affect cell proliferation; beta(1C) inhibits proliferation, whereas beta(1A) promotes it. We investigated the ability of beta(1C) and beta(1A) to modulate integrin-mediated signaling events that affect cell proliferation and survival in Chinese hamster ovary stable cell lines expressing either human beta(1C) or human beta(1A). The different cytodomains of either beta(1C) or beta(1A) did not affect either association with the endogenous alpha(2), alpha(V), and alpha(5) subunits or cell adhesion to fibronectin or TS2/16, a mAb to human beta(1). Upon engagement of endogenous and exogenous integrins by fibronectin, cells expressing beta(1C) showed significantly inhibited extracellular signal-regulated kinase (ERK) 2 activation compared with beta(1A) stable cell lines. In contrast, focal adhesion kinase phosphorylation and Protein Kinase B/AKT activity were not affected. Selective engagement of the exogenously expressed beta(1C) by TS2/16 led to stimulation of Protein Kinase B/AKT phosphorylation but not of ERK2 activation; in contrast, beta(1A) engagement induced activation of both proteins. We show that Ras activation was strongly reduced in beta(1C) stable cell lines in response to fibronectin adhesion and that expression of constitutively active Ras, Ras 61 (L), rescued beta(1C)-mediated down-regulation of ERK2 activation. Inhibition of cell proliferation in beta(1C) stable cell lines was attributable to an inhibitory effect of beta(1C) on the Ras/MAP kinase pathway because expression of activated MAPK kinase rescued beta(1C) antiproliferative effect. These findings show that the beta(1C) variant, by means of a unique signaling mechanism, selectively inhibits the MAP kinase pathway by preventing Ras activation without affecting either survival signals stimulated by integrins or cellular interactions with the extracellular matrix. These findings highlight a role for beta(1)-specific cytodomain sequences in maintaining an intracellular balance of proliferation and survival signals.

Animals↗
PubMed1999

Prostatic carcinoma cell migration via alpha(v)beta3 integrin is modulated by a focal adhesion kinase pathway.

The highly invasive human prostate cancer PC3 cell line was found to express the alpha(v)beta3 integrin; in contrast, the noninvasive LNCaP prostate cancer cell line did not express alpha(v)beta3. PC3 cells adhered to and migrated on vitronectin (VN), an alpha(v)beta3 ligand expressed in mature bone where prostate cancer cells preferentially metastasize. In contrast, LNCaP cells did not adhere to or migrate on VN. Analysis of primary human prostate cancer cells isolated from 16 surgical specimens, showed that these cells expressed alpha(v)beta3, whereas normal prostate epithelial cells did not. In addition, only primary prostate cancer cells adhered to and migrated on VN. The role of alpha(v)beta3 in mediating prostate epithelial cell migration was confirmed using LNCaP cell transfectants expressing beta3 (beta3-LNCaP). Exogenous expression of alpha(v)beta3 induced LNCaP cells to adhere to and migrate on VN. In response to alpha(v)beta3 engagement, increased tyrosine phosphorylation of focal adhesion kinase (FAK), a signaling molecule activated by integrins and able to modulate cell migration, was detected. Transfection of FAK-related nonkinase, known to compete with FAK for its correct localization and phosphorylation, caused inhibition of beta3-LNCaP cell migration, specifically on VN. These data indicate that de novo expression of alpha(v)beta3 integrin in prostate cancer cells generates a migratory phenotype that is modulated by a FAK signaling pathway. This study points to alpha(v)beta3 as potential target in prostate cancer cell invasion and metastasis.

Cell Adhesion Molecules↗
PubMed1999

p27(kip1) acts as a downstream effector of and is coexpressed with the beta1C integrin in prostatic adenocarcinoma.

Integrins are a large family of transmembrane receptors that, in addition to mediating cell adhesion, modulate cell proliferation. The beta1C integrin is an alternatively spliced variant of the beta1 subfamily that contains a unique 48-amino acid sequence in its cytoplasmic domain. We have shown previously that in vitro beta1C inhibits cell proliferation and that in vivo beta1C is expressed in nonproliferative, differentiated epithelium and is selectively downregulated in prostatic adenocarcinoma. Here we show, by immunohistochemistry and immunoblotting analysis, that beta1C is coexpressed in human prostate epithelial cells with the cell-cycle inhibitor p27(kip1), the loss of which correlates with poor prognosis in prostate cancer. In the 37 specimens analyzed, beta1C and p27(kip1) are concurrently expressed in 93% of benign and 84%-91% of tumor prostate cells. Forced expression of beta1C in vitro is accompanied by an increase in p27(kip1) levels, by inhibition of cyclin A-dependent kinase activity, and by increased association of p27(kip1) with cyclin A. beta1C inhibitory effect on cell proliferation is completely prevented by p27(kip1) antisense, but not mismatch oligonucleotides. beta1C expression does not affect either cyclin A or E levels, or cyclin E-associated kinase activity, nor the mitogen-activated protein (MAP) kinase pathway. These findings show a unique mechanism of cell growth inhibition by integrins and point to beta1C as an upstream regulator of p27(kip1) expression and, therefore, a potential target for tumor suppression in prostate cancer.

Adenocarcinoma↗