A mechanism of male germ cell apoptosis induced by bisphenol-A and nonylphenol involving ADAM17 and p38 MAPK activation.

Germ cell apoptosis regulation is pivotal in order to maintain proper daily sperm production. Several reports have shown that endocrine disruptors such as Bisphenol-A (BPA) and Nonylphenol (NP) induce germ cell apoptosis along with a decrease in sperm production. Given their ubiquitous distribution in plastic products used by humans it is important to clarify their mechanism of action. TACE/ADAM17 is a widely distributed extracellular metalloprotease and participates in the physiological apoptosis of germ cells during spermatogenesis. The aims of this work were: 1) to determine whether BPA and NP induce ADAM17 activation; and 2) to study whether ADAM17 and/or ADAM10 are involved in germ cell apoptosis induced by BPA and NP in the pubertal rat testis. A single dose of BPA or NP (50 mg/kg) induces germ cell apoptosis in 21-day-old male rats, which was prevented by a pharmacological inhibitor of ADAM17, but not by an inhibitor of ADAM10. In vitro, we showed that BPA and NP, at similar concentrations to those found in human samples, induce the shedding of exogenous and endogenous (TNF-α) ADAM17 substrates in primary rat Sertoli cell cultures and TM4 cell line. In addition, pharmacological inhibitors of metalloproteases and genetic silencing of ADAM17 prevent the shedding induced in vitro by BPA and NP. Finally, we showed that in vivo BPA and NP induced early activation (phosphorylation) of p38 MAPK and translocation of ADAM17 to the cell surface. Interestingly, the inhibition of p38 MAPK prevents germ cell apoptosis and translocation of ADAM17 to the cell surface. These results show for the first time that xenoestrogens can induce activation of ADAM17 at concentrations similar to those found in human samples, suggesting a mechanism by which they could imbalance para/juxtacrine cell-to-cell-communication and induce germ cell apoptosis.

TACE/ADAM17 is involved in germ cell apoptosis during rat spermatogenesis.

The pathways leading to male germ cell apoptosis in vivo are poorly understood, but are highly relevant for the comprehension of sperm production regulation by the testis. In this work, we show the evidence of a mechanism where germ cell apoptosis is induced through the inactivation and shedding of the extracellular domain of KIT (c-kit) by the protease TACE/a disintegrin and metalloprotease 17 (ADAM17) during the first wave of spermatogenesis in the rat. We show that germ cells undergoing apoptosis lacked the extracellular domain of the KIT receptor. TACE/ADAM17, a membrane-bound metalloprotease, was highly expressed in germ cells undergoing apoptosis as well. On the contrary, cell surface presence of ADAM10, a closely related metalloprotease isoform, was not associated with apoptotic germ cells. Pharmacological inhibition of TACE/ADAM17, but not ADAM10, significantly prevented germ cell apoptosis in the male pubertal rat. Induction of TACE/ADAM17 by the phorbol-ester phorbol 12-myristate 13-acetate (PMA) induced germ cell apoptosis, which was prevented when an inhibitor of TACE/ADAM17 was present in the assay. Ex-vivo rat testis culture showed that PMA induced the cleavage of the KIT extracellular domain. Isolation of apoptotic germ cells showed that even though protein levels of TACE/ADAM17 were higher in apoptotic germ cells than in nonapoptotic cells, the contrary was observed for ADAM10. These results suggest that TACE/ADAM17 is one of the elements triggering physiological germ cell apoptosis during the first wave of spermatogenesis.

Effects of PKF242-484 and PKF241-466, novel dual inhibitors of TNF-alpha converting enzyme and matrix metalloproteinases, in a model of intestinal reperfusion injury in mice.

Tumor necrosis factor (TNF)-alpha plays an important role in the mediation of reperfusion-induced tissue injury and lethality. Here, we assessed the effects of PKF242-484 and PKF241-466, two dual inhibitors of TNF-alpha converting enzyme (TACE) and matrix metalloproteinases (MMPs), in a model of ischemia and reperfusion injury in mice. Reperfused animals that received PKF242-484 or PKF241-466 treatment had a dose-dependent reduction of TNF-alpha concentrations in serum. Both drugs delayed and partially inhibited the reperfusion-associated lethality. Maximal inhibition occurred at 10 mg/kg. At this dose, both inhibitors reduced reperfusion-associated local and remote tissue injury, as assessed by changes in vascular permeability, neutrophil recruitment and hemorrhage. In addition, the compounds markedly reduced production of TNF-alpha, CXCL1 (keratinocyte-derived chemokine, KC) and CCL2 (monocyte chemoattractant protein-1, MCP-1) in intestine and lungs of animals which underwent reperfusion. FN-439, an inhibitor of MMPs which possesses no effect on TACE, decreased MMP-2 and MMP-3 activity, but failed to affect tissue injury, TNF-alpha production or lethality. Thus, combined TACE and MMP inhibitors might be effective co-adjuvants in treatments of injuries that follow reperfusion of an ischemic vascular territory. The effects of these drugs on TNF-alpha production appear to be more relevant than their effects on MMP inhibition.

Eficacia de la asociación rituximab-vindesina en el tratamiento de un caso de púrpura trombótico-trombopénico (PTT) adquirido idiopático refractario / Successful treatment of idiopathic acquired refractory thrombotic thrombocytopenic purpura with an association of rituximab - vindesine. Report of one case

We report a 23 years old female who presented a second episode of thrombotic thrombocytopenic purpura (TTP). She was treated with fresh frozen plasma infusions and 14 plasma exchange (PE) sessions without response. Therefore a second-line therapy was started, associating a weekly cycle administration of vindesine (Vds) 2 mg/m2 and rituximab (R) 375 mg/m2. Five cycles of this association plus one cycle of R exclusively, were administered. After the third course, biological signs of improvement were observed and complete normalization of blood cell counts and other specific parameters was seen after 8 weeks. From the beginning of her second relapse we detected a severe deficit (<5%) in von Willebrand-cleaving factor (ADAMTS13) associated to the presence of ADAMTS13 inhibitors. The combined treatment induced an improvement in ADAMTS13 values without detectable inhibitors. After 21 months of follow-up the patient was well, without signs of relapse but ADAMTS13 values were still under normal, which may be an unfavorable prognostic factor. PE is the treatment of choice for acquired idiopathic TTP, but for refractory cases or TTP cases with severe ADAMTS13 values/high inhibitor titers, PE associated to an immunosuppressive treatment should be considered.

[Successful treatment of idiopathic acquired refractory thrombotic thrombocytopenic purpura with an association of rituximab-vindesine. Report of one case]. / Eficacia de la asociación rituximab-vindesina en el tratamiento de un caso de púrpura trombótico-trombopénico (PTT) adquirido idiopático refractario.

We report a 23 years old female who presented a second episode of thrombotic thrombocytopenic purpura (TTP). She was treated with fresh frozen plasma infusions and 14 plasma exchange (PE) sessions without response. Therefore a second-line therapy was started, associating a weekly cycle administration of vindesine (Vds) 2 mg/m2 and rituximab (R) 375 mg/m2. Five cycles of this association plus one cycle of R exclusively, were administered. After the third course, biological signs of improvement were observed and complete normalization of blood cell counts and other specific parameters was seen after 8 weeks. From the beginning of her second relapse we detected a severe deficit (<5%) in von Willebrand-cleaving factor (ADAMTS13) associated to the presence of ADAMTS13 inhibitors. The combined treatment induced an improvement in ADAMTS13 values without detectable inhibitors. After 21 months of follow-up the patient was well, without signs of relapse but ADAMTS13 values were still under normal, which may be an unfavorable prognostic factor. PE is the treatment of choice for acquired idiopathic TTP, but for refractory cases or TTP cases with severe ADAMTS13 values/high inhibitor titers, PE associated to an immunosuppressive treatment should be considered.