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Biomedical subjects

A Filippini

Publications and source records attributed to A Filippini.

At least 19 recordsLinked to original sources
PubMed2001

Testicular FasL is expressed by sperm cells.

The testis is the main source of Fas ligand (FasL) mRNA in rodents; it is generally believed that this molecule, expressed on bordering somatic Sertoli cells, bestows an immune-privileged status in the testis by eliminating infiltrating inflammatory Fas-bearing leukocytes. Our results demonstrate that the attribution of testicular expression of FasL to Sertoli cells is erroneous and that FasL transcription instead occurs in meiotic and postmeiotic germ cells, whereas the protein is only displayed on mature spermatozoa. These findings point to a significant role of the Fas system in the biology of mammalian reproduction.

Animals↗
PubMed2000

TNF-alpha and IFN-gamma regulate expression and function of the Fas system in the seminiferous epithelium.

Sertoli cells have long been considered to be involved in the regulation of the immune response in the testis. More recently, the Fas system has been implicated in the maintenance of the immune privilege in the testis as well as in the regulation of germ cell apoptosis. However, the control of Fas and Fas ligand (FasL) expression in the testis remains unknown. In the present study, we demonstrate that cultured mouse Sertoli cells constitutively express a low level of membrane-bound Fas protein, but not a soluble form of Fas. Sertoli cells stimulated with TNF-alpha and IFN-gamma markedly increase the expression of both soluble and membrane-bound Fas in a dose-dependent manner. The up-regulated membrane-bound Fas protein is functionally active because it induces a significant level of Sertoli cell death in the presence of Neuro-2a FasL+ effector cells. Interestingly, the soluble form of Fas, which is induced by the same cytokines but has an antiapoptotic effect, is also functional. In fact, conditioned media from TNF-alpha-stimulated Sertoli cell cultures inhibit Neuro-2a FasL+-induced cell death. Taken together, our data suggest a possible regulatory role of TNF-alpha and IFN-gamma on Fas-mediated apoptosis in the testis through disruption of the balance between different forms of Fas.

Adjuvants, Immunologic↗
PubMed2000

Muscarinic receptors modulate intracellular calcium level in chick sensory neurons.

In the present work we have studied the variation of intracellular calcium levels induced by muscarinic agonists in chick dorsal root ganglia neurons. Muscarinic agonists such as muscarine and oxotremorine cause an increase of intracellular calcium levels in fura-2AM-loaded DRG neurons of E18 chick embryos. This increase was abolished following treatment with 1 microM atropine but not by 1 microM mecamylamine, indicating that the observed intracellular calcium increase, was dependent on muscarinic receptor activation. Stimulation in absence of external calcium or pre-incubation of the DRG cultures with thapsigargin or Mn(2+) demonstrated that [Ca(2+)](i) increase is mainly due to its release from intracellular stores. The use of selective antagonists of muscarinic receptor subtypes also indicated that M(1) and to a lesser extent M(3) receptor subtypes are responsible for the observed intracellular calcium mobilization. Finally pre-treatment of DRG cultures with pertussis toxin showed that the variation of [Ca(2+)](i) levels was dependent on PTX-insensitive G-protein. Moreover muscarinic agonists induce in DRG also the increase of IPs level, suggesting that the variations of intracellular calcium levels may be due at least in part to the activation of the phosphoinositide transduction pathway. In conclusion the reported observations demonstrate the activity of muscarinic receptors in sensory neurons, suggesting a functional role for acetylcholine in sensory transduction.

Animals↗
PubMed2000

Platelet-derived growth factor-BB stimulates hypertrophy of peritubular smooth muscle cells from rat testis in primary cultures.

The tunica propria of seminiferous tubules contains a particular type of smooth muscle cell (myoid cells) arranged in a contractile epithelioid layer that is responsible for sperm and tubular fluid flow. Unlike other types of smooth muscle (SM) cells, highly purified populations of peritubular smooth muscle cells (PSMC) survive and maintain their contractile phenotype in primary cultures in controlled conditions. We used this culture model to investigate the response of the SM contractile phenotype to prolonged exposure to platelet-derived growth factor (PDGF), one of the main factors involved in vascular SM pathologies. We observed that 4-day continuous exposure of PSMC to PDGF-BB at nanomolar concentrations in plain medium enhances contractile phenotype traits and induces cell hypertrophy without inducing proliferation. In Northern and Western blotting experiments, SM-alpha-actin transcript and protein were found to be markedly increased in the PDGF-BB-treated samples, which is in line with the formation of conspicuous SM-alpha-actin-containing stress fibers. Moreover, binding sites for endothelin-1 were increased, and the calcium response to the contractile agonist, determined in single fura-2-loaded cells, was enhanced. In response to PDGF-BB, the cells underwent immediate, transient contraction, as seen in a scanning electron microscope, followed by a gradual increase in size, as evaluated by cytofluorometry, and enhancement of protein synthesis. The observed pattern of response to PDGF-BB was not accompanied by cell proliferation, as assessed by [3H]thymidine incorporation and direct cell counts. Unlike other SM cell types, in which proliferation and loss of contractile traits are induced by PDGF, chronic treatment of PSMC with this growth factor results in hypertrophy rather than hyperplasia.

Actins↗
PubMed2000

Nerve-sparing surgery in rectal cancer: feasibility and functional results.

Traditional rectal cancer surgery has been burdened with a high rate of sexual and urinary dysfunctions due to intraoperative injury or the cutting of the sympathetic and/or parasympathetic nerves. The experience acquired in the last ten years with total mesorectal excisions has permitted the use of the "nerve-sparing" technique. The present study regards 239 patients from two surgical centres, most of whom underwent sphincter-saving radical surgery between 1994 and 1998 with the above mentioned technique for resectable colon cancer. Details regarding the technique were recorded in the last 58 patients, in order to examine the severity of the surgical damage. The subgroup with the longest follow-up, which included 36 patients, was diagnostically evaluated by a surgeon, psychologist, urologist and neurologist to analyze the risk of sexual and urinary dysfunctions. A complete nerve-sparing was performed in 86.3% of the cases. The parasympathetic nerve trunks were those most often damaged because of perineural tumor spreading. Partial to complete sexual impotence was observed in 44% of the patients and surprisingly, preoperative dysfunctions were detected by means of the multidisciplinary approach in one third of these patients. Therefore, only 30.5% of the patients presented with strictly postoperative sexual impotency, above all, those who had undergone high-dose preoperative chemoradiation for T3 or T4 middle to low rectal cancer. A prospective study was initiated to evaluate the genitourinary dysfunctions after rectal cancer surgery in all of the clinical phases by means of a multidisciplinary approach aimed at functional recovery and improved quality of life.

Humans↗
PubMed1999

Activation of Jun N-terminal kinase/stress-activated protein kinase pathway by tumor necrosis factor alpha leads to intercellular adhesion molecule-1 expression.

Tumor necrosis factor alpha (TNF-alpha) is a cytokine implicated in the pathogenesis of numerous chronic and acute inflammatory conditions. We have previously shown that mouse Sertoli cells respond to TNF-alpha by increasing interleukin-6 production and intercellular adhesion molecule-1 (ICAM-1) expression (1). In this cell type TNF-alpha activates the mitogen-activated protein kinase (MAPK) pathways p42/p44 MAPK, JNK/SAPK, and p38, the last of which is responsible for interleukin-6 production (1). To determine which MAPK signaling pathway is required for TNF-alpha induction of ICAM-1 expression, we have utilized the protein kinase inhibitor dimethylaminopurine, demonstrating that treatment of Sertoli cells with such compound significantly reduced ICAM-1 expression and JNK/SAPK activation. Moreover, dimethylaminopurine treatment increased the expression of MAPK phosphatase-2, providing a possible mechanism of action of this compound. By using agonist antibodies to p55 and to p75 TNF-alpha receptors and both human and mouse TNF-alpha, we demonstrate that both TNF receptors are expressed and that only the p55 receptor is involved in ICAM-1 expression. The p55 receptor activates all of the three pathways, whereas p75 failed to activate any of the MAPKs. Altogether our results demonstrate that TNF-alpha up-regulates ICAM-1 expression through the activation of the JNK/SAPK transduction pathway mediated by the p55 receptor.

Adenine↗
PubMed1999

Cyclic expression of endothelin-converting enzyme-1 mediates the functional regulation of seminiferous tubule contraction.

The potent smooth muscle agonist endothelin-1 (ET-1) is involved in the local control of seminiferous tubule contractility, which results in the forward propulsion of tubular fluid and spermatozoa, through its action on peritubular myoid cells. ET-1, known to be produced in the seminiferous epithelium by Sertoli cells, is derived from the inactive intermediate big endothelin-1 (big ET-1) through a specific cleavage operated by the endothelin-converting enzyme (ECE), a membrane-bound metalloprotease with ectoenzymatic activity. The data presented suggest that the timing of seminiferous tubule contractility is controlled locally by the cyclic interplay between different cell types. We have studied the expression of ECE by Sertoli cells and used myoid cell cultures and seminiferous tubule explants to monitor the biological activity of the enzymatic reaction product. Northern blot analysis showed that ECE-1 (and not ECE-2) is specifically expressed in Sertoli cells; competitive enzyme immunoassay of ET production showed that Sertoli cell monolayers are capable of cleaving big ET-1, an activity inhibited by the ECE inhibitor phosphoramidon. Microfluorimetric analysis of intracellular calcium mobilization in single cells showed that myoid cells do not respond to big endothelin, nor to Sertoli cell plain medium, but to the medium conditioned by Sertoli cells in the presence of big ET-1, resulting in cell contraction and desensitization to further ET-1 stimulation; in situ hybridization analysis shows regional differences in ECE expression, suggesting that pulsatile production of endothelin by Sertoli cells (at specific "stages" of the seminiferous epithelium) may regulate the cyclicity of tubular contraction; when viewed in a scanning electron microscope, segments of seminiferous tubules containing the specific stages characterized by high expression of ECE were observed to contract in response to big ET-1, whereas stages with low ECE expression remained virtually unaffected. These data indicate that endothelin-mediated spatiotemporal control of rhythmic tubular contractility might be operated by Sertoli cells through the cyclic expression of ECE-1, which is, in turn, dependent upon the timing of spermatogenesis.

Animals↗
PubMed1999

Is hyperdiploidy of immature ejaculated germ cells predictive of testis malignancy? A comparative study in healthy normozoospermic, infertile, and testis tumor suffering subjects.

The possibility of diagnosing neoplastic testis pathologies by studying immature germ cells released from the seminiferous epithelium and present in the semen has been reported. It has been suggested that carcinoma in situ (CIS) of the testis and testis tumor may be identified by studying specific surface antigenic determinants or ploidy of chromosome 1 of malignant germ cells recovered from the semen. A noninvasive diagnostic approach of this type would be of great interest if we consider that CIS is supposed to precede the development of testicular germ cell tumors and that the frequency of that preneoplastic condition is increased in specific andrologic pathologies. To evaluate the reliability of this diagnostic approach, we have quantified the presence of immature hyperdiploid germ cells in the seminal fluid of normal healthy subjects, of infertile oligozoospermic patients affected by maldescended testis or left vancocele, and of patients suffering from CIS or testis tumor. Cell ploidy was evaluated on seminal cell fractions highly enriched in immature germ cells, by means of in situ hybridization with a DNA-probe specific for chromosome 1. Our observations indicate that chromosome 1 hyperdiploidy is not necessarily a predictive parameter of testis tumoral pathologies. The percentage of hyperdiploid immature seminal germ cells is in fact increased in nontumoral pathologies associated with infertility, such as cryptorchidism and left varicocele.

Adult↗
PubMed1998

Tumor necrosis factor-alpha induces interleukin-6 production and integrin ligand expression by distinct transduction pathways.

Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic cytokine that elicits a large number of biological effects. However, the intracellular signaling mechanisms that are responsible for the TNF-alpha effects remain largely unknown. We have previously demonstrated that cultured mouse Sertoli cells, after TNF-alpha treatment, increase the surface expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and interleukin-6 (IL-6) production (Riccioli, A., Filippini, A., De Cesaris, P., Barbacci, E., Stefanini, M., Starace, G., and Ziparo, E. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 5808-5812). Here, we show that, in cultured Sertoli cells, TNF-alpha activates the mitogen-activated protein kinase pathway (p38, c-Jun N-terminal protein kinase/stress-activated protein kinase, and the p42/p44 mitogen-activated protein kinases) as revealed by an increased phosphorylation of p38, activating transcription factor-2, c-Jun, and Elk-1. Furthermore, our data indicate that the biological effects induced by TNF-alpha in Sertoli cells (enhancement of ICAM-1, VCAM-1, and IL-6 expression) depend on the activation of different signaling pathways. SB203580, a highly specific p38 inhibitor, does not affect ICAM-1 and VCAM-1 expression, but strongly inhibits IL-6 production. Moreover, interferon-gamma, which up-regulates adhesion molecule expression and reduces IL-6 production, does not induce phosphorylation of p38. Our data strongly support the hypothesis that, in response to TNF-alpha, activation of p38 leads to IL-6 production, whereas ICAM-1 and VCAM-1 expression could be induced by activation of the c-Jun N-terminal protein kinase/stress-activated protein kinase pathway.

Activating Transcription Factor 2↗
PubMed1998

Contractile response of peritubular myoid cells to prostaglandin F2alpha.

Prostaglandin (PG) F2alpha, a well known agonist of smooth muscle, is produced in the male gonad. We have investigated whether PG F2alpha stimulates seminiferous tubule contractility through direct action on peritubular myoid cells. Myoid cells from prepubertal rats were highly purified through Percoll density gradient and cultured in vitro. Stimulation with PG F2alpha was observed to induce: (i) rapid and dose-dependent production of inositol phosphates; (ii) mobilization of Ca2+ from intracellular stores and (iii) cell contraction. Moreover, at a concentration of 10 microM the agonist was found to induce immediate contractile response of peritubular tissue in freshly explanted tubular fragments from both young and adult rats; the explants were examined in whole-mount preparations and the peritubular myoid cell layer was identified by selective staining for alkaline phosphatase activity. Our observations demonstrate that myoid cells are a direct target for PG F2alpha and suggest a role of the eicosanoid in the intragonadal control of seminiferous tubule contractility.

Aging↗
PubMed1998

Testicular germ cells of HIV-seropositive asymptomatic men are infected by the virus.

In situ PCR hybridization studies in the testis of infected asymptomatic subjects detected the presence of HIV-1 proviral DNA in the nuclei of germ cells at all stages of differentiation suggesting that HIV-seropositive men produce infected spermatozoa that are released in the genital tract. In all subjects studied spermatogenesis was normal, the presence of provirus was not associated with germ cell damage and a very mild local immune response was observed. The HIV hybridization pattern observed in germ cells supports the hypothesis of a clonal infection. It is suggested the possibility of a direct infection of the germ cells by cell-free virus and that the testis might represent a site of early viral localization, well tolerated because of the immune privilege of this organ.

HIV Seropositivity↗
PubMed1998

Presence and cellular distribution of HIV in the testes of seropositive subjects: an evaluation by in situ PCR hybridization.

Cellular distribution of HIV-1 proviral DNA has been studied, by in situ PCR hybridization, in the testes of infected men who died at various stages of the disease. In seropositive asymptomatic subjects, HIV-1 proviral DNA was present in the nuclei of germ cells at all stages of their differentiation. The presence of provirus did not induce germ cell damage, was associated with normal spermatogenesis, and was not accompanied by morphologic signs of immune response. The observed HIV hybridization pattern of germ cells suggests clonal infection. Mechanisms responsible for HIV penetration in testicular germ cells remain to be clarified; however, the possibility of a direct infection of the germ cells by cell-free virus is suggested. In the testes of AIDS-deceased men, histologic features of hypoplasia with arrested spermatogenesis were evident, and few infected spermatogonia and spermatocytes were observed. The whole of these data demonstrates that the testis is a site of early viral localization that fails to elicit an immunological response, and that HIV-seropositive men produce infected spermatozoa that are released in the genital tract.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Dual control of seminiferous tubule contractility mediated by ETA and ETB endothelin receptor subtypes.

Testicular myoid cells surrounding the seminiferous tubule are contractile cells responsible for peritubular contractility and for the propulsion of tubular fluid and spermatozoa. We have investigated the contractile response of rat myoid cells to endothelins (ETs) in cell and organ culture and analyzed the cell signaling involved. ET-2, ET-3, and IRL 1620, a highly selective agonist of ETB receptor, elicit [Ca2+]i increases, though with dissimilar potencies and kinetics. Competition binding assays using [125I]ET-1, [125I]ET-3 and [125I]IRL 1620 show that myoid cells express both ETA and ETB receptors with high affinity for ET-1 and ET-1/ ET-3, respectively. All endothelin isoforms activate phosphoinositide (PI) turnover, but only stimulation of the ETA receptor mediates both PI turnover and mobilization of [Ca2+]i. Although stimulation of the ETB receptor with IRL 1620 fails to produce a significant effect on inositol phosphate (IP) production, it induces mobilization of a thapsigargin-sensitive intracellular Ca2+ pool in the absence of any measurable IP production. We also studied the effect of U-73122 [1-(6-[17-beta-3-methoxyestra-1,3,5 (10)-trien-17-yl] amino/hexyl)-1H-pirrole-2,5-dione] and its inactive analog, U-73343, on Ca2+ mobilization and IP production after selective stimulation of ET receptors. U-73122 (1 microM) completely inhibited the effect of ETA-mediated ET-1 stimulation of IP production, whereas U-73343 was inactive. However, in the presence of U-73122, the selective stimulation of ETB receptors induced the mobilization of a thapsigargin-sensitive and inositol phosphate-independent intracellular Ca2+ pool. The ETB receptor-dependent mobilization of [Ca2+]i resulted mainly from Ca2+ release from intracellular Ca2+ stores. This paper illustrates contraction of myoid cells in the seminiferous tubule in response to selective activation of either ET receptor. Scanning electron microscopy of the peritubular tissue demonstrates that the contractile response to ET was inhibited by a combination of BQ-123 and BQ-788, but not by either antagonist alone. Moreover, the observation that selective stimulation of ETB receptor with IRL 1620 also resulted in cell contraction strongly suggests that stimulation of either ETA or ETB receptors alone may be sufficient to elicit seminiferous tubule contractility. Two types of receptors account for the actions of endothelin on contractile activity of seminiferous tubule: 1) an ETA receptor that is positively coupled to phospholipase C (PLC) and Ca2+ mobilization; and 2) an ETB receptor that induces the mobilization of a thapsigargin-sensitive intracellular Ca2+ pool in a manner independent of the formation of inositol phosphates. ET may play a complex role in regulating the flux of spermatozoa along the seminiferous tubule through its contractile effect on peritubular myoid cells.

Animals↗
PubMed1996

Identification and characterization of an ecto-ATPase activity in rat Sertoli cells.

A membrane associated extracellular ATPase (ecto) has been identified on rat Sertoli cells. Sertoli cell ecto-ATPase demonstrated a Km for ATP of 114 muM and a V(max) of 1.79 mumol/min/2 x 10(5) cells and was activated by either Mg2+ or Ca2+. This ecto-ATPase hydrolyzes other nucleoside triphosphates, but is inactive with ADP. The effects of some possible inhibitors of ectonucleotidases on the breakdown of extracellular ATP by Sertoli cells were also investigated.

Adenosine Triphosphatases↗
PubMed1996

Direct visualization of rat peritubular myoid cell contraction in response to endothelin.

Seminiferous tubule contractility is fundamental for sperm progression towards the rete testis; hence its regulation represents a key point in male fertility. Endothelin-1 (ET-1), a potent stimulator of smooth muscle contractility, has recently been shown to be produced in the testis, as well as to bind to specific receptors on myoid cells and thereby activate intracellular signaling. The present paper illustrates contraction of isolated myoid cells in response to ET-1 both in phase-contrast and scanning electron microscopy. Moreover, a simple method is described that allows visualization of a dramatic endothelin-induced rearrangement of myoid cell actin filaments in whole-mount preparations of seminiferous tubules. The response, which can be observed within seconds from stimulation, was paralleled by cell shape changes that were well apparent in scanning electron microscopy. The present report provides the first direct evidence that endothelin is an agonist of myoid cell contractility. Moreover, the experimental approach described could represent a promising tool for the screening of potential agonists of peritubular contractility and for the analysis of their mechanisms of action. In this regard, preliminary evidence is presented on myoid cell contractile response to [Arg8]-vasopressin.

Actin Cytoskeleton↗
PubMed1995

Inflammatory mediators increase surface expression of integrin ligands, adhesion to lymphocytes, and secretion of interleukin 6 in mouse Sertoli cells.

The expression of the cell adhesion molecules ICAM-1, ICAM-2, and VCAM-1 and the secretion of the cytokine interleukin 6 have been measured in mouse Sertoli cells cultured in vitro. Cytometric analysis revealed that, in basal conditions, low levels of ICAM-1 and VCAM-1 were present on the surface of the cells, whereas treatment with interleukin 1, tumor necrosis factor alpha, lipopolysaccharide, or interferon gamma induced, with different kinetics, increases in their expression. ICAM-2 was not detectable in basal conditions, nor was it inducible. Electron microscopic analysis and binding experiments using 51Cr-labeled lymphocytes demonstrated that increased expression of ICAM-1 and VCAM-1 on the surface of Sertoli cells, induced by inflammatory mediators, determines an augmented adhesion between the two cell types. The same stimuli, with the exception of interferon gamma, produced a rapid and remarkable increment of interleukin 6 production by Sertoli cells. These results suggest the presence of both direct and paracrine mechanisms of interaction between Sertoli and immune-competent cells, possibly involved in the control of immune reactions in the testis. Such mechanisms are of interest for the understanding of autoimmune pathologies of the testis and, if confirmed in humans, they could be involved in the sexual transmission of human immunodeficiency virus infection.

Animals↗