Biomedical subjects

Fabrizio Padula

Publications and source records attributed to Fabrizio Padula.

9 recordsLinked to original sources

PubMed2006

c-Flip(L) is expressed in undifferentiated mouse male germ cells.

Apoptosis represents a fundamental process during fetal/post-natal testis development. Therefore pro- and anti-apoptotic proteins are essential to regulate testis physiology. c-Flip(L) is a known inhibitor of caspase 8/10 activity; in this study its perinatal expression in mouse male germ cells was investigated. In testis sections and seminiferous tubule whole mount c-Flip(L) was found to be expressed in undifferentiated spermatogonia and to co-localize with germ stem cells markers. In vivo investigations in the vitamin-A deficient mouse, lacking differentiated germ cells, confirmed c-Flip(L) expression in undifferentiated spermatogonia. Further analyses showed Fas expression but no significant caspase 8/10 activity when c-Flip(L) was highly expressed. Altogether these data suggest that c-Flip may control the survival rate of undifferentiated spermatogonia.

Animals↗

PubMed2006

Sequential valproic acid/all-trans retinoic acid treatment reprograms differentiation in refractory and high-risk acute myeloid leukemia.

Epigenetic alterations of chromatin due to aberrant histone deacetylase (HDAC) activity and transcriptional silencing of all-trans retinoic acid (ATRA) pathway are events linked to the pathogenesis of acute myeloid leukemia (AML) that can be targeted by specific treatments. A pilot study was carried out in eight refractory or high-risk AML patients not eligible for intensive therapy to assess the biological and therapeutic activities of the HDAC inhibitor valproic acid (VPA) used to remodel chromatin, followed by the addition of ATRA, to activate gene transcription and differentiation in leukemic cells. Hyperacetylation of histones H3 and H4 was detectable at therapeutic VPA serum levels (>or=50 microg/mL) in blood mononuclear cells from seven of eight patients. This correlated with myelomonocytic differentiation of leukemic cells as revealed by morphologic, cytochemical, immunophenotypic, and gene expression analyses. Differentiation of the leukemic clone was proven by fluorescence in situ hybridization analysis showing the cytogenetic lesion +8 or 7q- in differentiating cells. Hematologic improvement, according to established criteria for myelodysplastic syndromes, was observed in two cases. Stable disease and disease progression were observed in five and one cases, respectively. In conclusion, VPA-ATRA treatment is well tolerated and induces phenotypic changes of AML blasts through chromatin remodeling. Further studies are needed to evaluate whether VPA-ATRA treatment by reprogramming differentiation of the leukemic clone might improve the response to chemotherapy in leukemia patients.

Antineoplastic Agents↗

PubMed2006

Platelet-derived growth factor-BB-induced hypertrophy of peritubular smooth muscle cells is mediated by activation of p38 MAP-kinase and of Rho-kinase.

Peritubular smooth muscle cells (PSMC) from rat testis in primary serum-free cultures unexpectedly undergo contraction and subsequent cell hypertrophy in response to the growth factor PDGF-BB, remaining stationary. The present study investigates the transduction pathways involved in the observed paradoxical upregulation of the differentiated phenotype and induction of hypertrophy in PSMC. PI3K, ERK, JNK, and p38 kinases, known to mediate PDGF-BB signaling in the canonic dedifferentiative and proliferative response of smooth muscle cells (SMC) were rapidly activated by PDGF-BB but only p38 remained activated after 2-day stimulation. Immunofluorescence and immunoblotting experiments showed that in 4-day treatment: (i) continuous inhibition of PI3K, of ERK, of JNK, failed to inhibit either cell enlargement and formation of prominent alpha-SM actin containing stress fibers or the typical increase in alpha-SM actin; (ii) when stimulated in the presence of the p38 inhibitor SB203580 both responses were significantly inhibited and cytofluorimetric analysis of cell size showed a remarkable reduction of the hypertrophic response. PDGF-BB was also found to activate the small GTPase RhoA and inhibition of Rho-dependent kinase ROCK by Y27632 counteracted the effects of PDGF-BB similarly to SB203580. Both the transcription factor ATF2 and the nucleosomal kinase MSK1, downstream targets of p38, were activated by PDGF-BB, but p38 inhibitor SB203580 inhibited only the phosphorylation of MSK1 which appeared unaffected by ROCK inhibitor Y27632. In concluding, p38 and the Rho-ROCK system were found to play prominent, probably independent roles in the upregulation of PSMC differentiated phenotype and induction of hypertrophy by PDGF-BB.

Actins↗

PubMed2005

Beta-chemokine receptors 5 and 3 are expressed on the head region of human spermatozoon.

Induction of human sperm chemotaxis is an established phenomenon, though signaling systems physiologically involved have not been identified. Recently, it has been demonstrated that RANTES is present in the follicular fluid and that this molecule is a chemoactractant for human spermatozoa. However, the presence of beta-chemokine receptors on human spermatozoa has never been reported. By cytometric, Western blotting and immunofluorescence analysis, we demonstrate the presence of CCR5 and CCR3 on ejaculated spermatozoa from healthy subjects. CCR5 was detected in the periacrosomal region of the sperm surface, whereas CCR3 was also present in the postacrosomal cap. Individual variability was observed on CCR5 and CCR3 positive sperm percentages. Presence of Delta32+/-) mutation was demonstrated in two subjects expressing CCR5 in half of the ejaculated spermatozoa. Our findings represent the missing information in favor of the possibility that beta-chemokines and their receptors are involved in sperm chemotaxis. Identification of molecular mechanisms of sperm chemotaxis may allow us to identify predictive parameters of sperm fertilizing ability in hypofertile or infertile subjects. Finally, both CCR5 and CCR3 expressed on the sperm cell surface may be involved in HIV-1 adhesion to spermatozoa, thus allowing these cells to perform as virion cellular carriers during sexual transmission of HIV-1 infection.

Blotting, Western↗

PubMed2005

HIV-1 chemokine co-receptor CCR5 is expressed on the surface of human spermatozoa.

Viruses adhering to the sperm surface are described in the semen of HIV-1-infected individuals, although viral adhesion mechanisms have yet to be fully understood. We demonstrate, by cytometric analysis and immunofluorescence microscopy, the presence of beta-chemokine receptor 5 (CCR5) on the periacrosomal region of ejaculated spermatozoa. CCR5 expressed on the sperm cell surface may allow sperm to act as virion cellular carriers during the sexual transmission of HIV-1 infection.

Flow Cytometry↗

PubMed2005

Histone deacetylase inhibitor valproic acid enhances the cytokine-induced expansion of human hematopoietic stem cells.

Ex vivo amplification of human hematopoietic stem cells (HSC) without loss of their self-renewing potential represents an important target for transplantation, gene and cellular therapies. Valproic acid is a safe and widely used neurologic agent that acts as a potent inhibitor of histone deacetylase activities. Here, we show that valproic acid addition to liquid cultures of human CD34+ cells isolated from cord blood, mobilized peripheral blood, and bone marrow strongly enhances the ex vivo expansion potential of different cytokine cocktails as shown by morphologic, cytochemical, immunophenotypical, clonogenic, and gene expression analyses. Notably, valproic acid highly preserves the CD34 positivity after 1 week (range, 40-89%) or 3 weeks (range, 21-52%) amplification cultures with two (Flt3L + thrombopoietin) or four cytokines (Flt3L + thrombopoietin + stem cell factor + interleukin 3). Moreover, valproic acid treatment increases histone H4 acetylation levels at specific regulatory sites on HOXB4, a transcription factor gene with a key role in the regulation of HSC self-renewal and AC133, a recognized marker gene for stem cell populations. Overall, our results relate the changes induced by valproic acid on chromatin accessibility with the enhancement of the cytokine effect on the maintenance and expansion of a primitive hematopoietic stem cell population. These findings underscore the potentiality of novel epigenetic approaches to modify HSC fate in vitro.

AC133 Antigen↗

PubMed2005

Germ cell apoptosis control during spermatogenesis.

The aim of the present study was to investigate the expression and role of c-Flip long isoform (c-FlipL), a known anti-apoptotic protein. No data are currently available on c-FlipL in male gonad before puberty; therefore, this study was carried out in prepuberal mouse testis. We investigated pachytene spermatocytes and spermatogonia by immunostaining of testis sections and found a strong and specific expression of c-FlipL in pachytene spermatocytes, while spermatogonia expressed very low levels of c-FlipL. This finding inversely correlated with the caspases activity, which was higher in spermatogonia as compared to pachytene spermatocytes. Other experiments carried out in an organ-culture model revealed that Fas-induced apoptosis was higher in spermatogonia as compared to pachytene spermatocytes. These data suggest that c-FlipL may play a role as an anti-apoptotic molecule in the prepuberal mouse testis and open new perspectives in the comprehension of the mechanisms controlling germ cells apoptosis.

Aging↗

PubMed2004

RGDS peptide induces caspase 8 and caspase 9 activation in human endothelial cells.

Peptides containing the Arg-Gly-Asp (RGD) motif inhibit cell adhesion and exhibit a variety of other biologic effects including anticoagulant and antimetastatic activities. The aim of the present study was to examine the anchorage-independent effects of an RGD-containing peptide, Arg-Gly-Asp-Ser (RGDS), on human umbilical vein endothelial cells (HUVECs). Assays were performed on HUVECs seeded onto collagen IV; under these experimental conditions RGDS did not exert antiadhesive effects but significantly reduced FGF-2-dependent chemotaxis after 4 hours of treatment and reduced proliferation after 24 hours of treatment. Experiments carried out with caspase-specific inhibitors indicated that the observed antichemotactic effects required caspase 8 and caspase 9 activation. RGDS activated both caspase 8 and caspase 9 after 4 hours of treatment and caspase 3 after 24 hours of treatment, and markedly enhanced HUVEC apoptosis by transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)/Hoechst staining and fluorescence-activated cell sorting (FACS) analysis. Finally, confocal microscopy showed that RGDS localizes in the cytoplasm of live HUVECs within 4 hours and in vitro experiments showed that RGDS directly interacts with recombinant caspases 8 and 9 in a specific way. In summary, these results indicate that RGDS directly binds and activates caspases 8 and 9, inhibits chemotaxis, and induces apoptosis of HUVECs with a mechanism independent from its antiadhesive effect.

Caspase 8↗

PubMed2002

Complete remission through blast cell differentiation in PLZF/RARalpha-positive acute promyelocytic leukemia: in vitro and in vivo studies.

Acute leukemia with the t(11;17) expressing the PLZF-RARalpha gene fusion is a rare variant of acute promyelocytic leukemia (APL) that has been associated with poor clinical response to all-trans retinoic acid (ATRA) treatment. However, some recent reports have put into question the absolute refractoriness of this leukemia to ATRA. We describe here a patient with PLZF/RARalpha APL who was treated at relapse with ATRA and low-dose hydroxyurea. Complete hematologic remission was obtained through differentiation of leukemic blasts, as proven by morphologic, immunophenophenotypic, and genetic studies carried out in sequential bone marrow samples. Moreover, in vitro studies indicated that blast differentiation was potentiated by the addition of the histone deacetylase inhibitor tricostatin A, but not of hydroxyurea, to ATRA. Our findings indicate that the maturation block may be overcome and terminal differentiation obtained in this leukemia subset and support the view that sensitivity/refractoriness of this form to ATRA should be revisited.

Antineoplastic Combined Chemotherapy Protocols↗