Cell cycle reactivation of cochlear progenitor cells in neonatal FUCCI mice by a GSK3 small molecule inhibitor.

Due to the lack of regenerative capacity of the mammalian auditory epithelium, sensory hair cell loss results in permanent hearing deficit. Nevertheless, a population of tissue resident stem/progenitor cells has been recently described. Identification of methods to trigger their activity could lead to exploitation of their potential therapeutically. Here we validate the use of transgenic mice reporting cell cycle progression (FUCCI), and stemness (Lgr5-GFP), as a valuable tool to identify regulators of cell cycle re-entry of supporting cells within the auditory epithelium. The small molecule compound CHIR99021 was used to inhibit GSK3 activity. This led to a significant increase in the fraction of proliferating sphere-forming cells, labeled by the FUCCI markers and in the percentage of Lgr5-GFP + cells, as well as a selective increase in the fraction of S-G2-M cells in the Lgr5 + population. Using whole mount cultures of the organ of Corti we detected a statistically significant increment in the fraction of proliferating Sox2 supporting cells after CHIR99021 treatment, but only rarely appearance of novel MyoVIIa +/Edu + hair cells. In conclusion, these tools provide a robust mean to identify novel regulators of auditory organ regeneration and to clarify the contribution of stem cell activity.

The W520X mutation in the TSHR gene brings on subclinical hypothyroidism through an haploinsufficiency mechanism.

BACKGROUND: TSHR is a G-protein-coupled seven transmembrane domain receptor that activates the two major signal transduction pathways: the Gαs/adenylate cyclase and the Gαq/11/phospholipase C pathways. Inactivating mutations in the TSHR gene have been demonstrated to be responsible for subclinical hypothyroidism, a disorder characterized by elevated serum TSH concentrations despite normal thyroid hormones levels. AIM: We identified in a child a nonsense mutation (W520X) in the third transmembrane domain of the TSHR that causes the lack of the C-terminus portion of the receptor. The functional significance of this variation was assessed in vitro. MATERIAL/SUBJECT AND METHODS: The W520X mutation was introduced into the pSVL vector containing the wild-type sequence of TSHR gene. Wild-type and mutated vectors were expressed in Chinese Hamster Ovary (CHO) cells, and cAMP, inositol phosphate (IP), immunofluorescence and FACS analyses were performed. RESULTS: Transfection with pSVL-TSHR vector induced basal cAMP and IP production in the absence of TSH stimulation, indicating a constitutive activity for the TSHR. An impairment of receptor function was demonstrated by the observation that cells expressing the mutant TSHR exhibited a lower second messenger production with respect to the wild-type, despite a normal expression of the receptor at the cell surface. CONCLUSIONS: The mechanism through which the W520X mutation exerts its effect is more likely haploinsufficiency rather than a dominant-negative effect. This could explain the phenotype of our patient, who has a hormonal pattern in the range of a mild subclinical hypothyroidism, without an overt disease phenotype.

HPV infection and intraepithelial lesions: comparison between HIV positive and negative women.

INTRODUCTION: Human Papillomavirus infections have been shown to be crucial for the development of cervical intraepithelial neoplasia and subsequent cervical cancer. The aim of this study is to describe the prevalence of different genotypes of HPV, in a population of HIV-positive women, compared to the negative ones, and their oncogenic risk. PATIENTS AND METHOD: A case-control study comparing HPV genotype distribution between 93 HIV-seropositive and 186 HIV-seronegative women, matched for age and severity of cervical lesions, who attending colposcopic service of our departments for periodical Pap smear and HPV DNA full genotyping by SPF-10 LiPA assay. RESULTS: No significant difference was found in genotype distribution between HIV positive and HIV negative women. Only the prevalence of HPV56 was higher in HIV positive women (p=0,046). The rates of HPV 6, 11, 16 and 18 were similar in both groups. The likelihood of the detection of three or more HPV genotypes was significantly associated with CIN (OR=2.0; 95% CI=1.1-3.8; p= 0.026) but only marginally to HIV-positive serostatus (OR=1.68; 95% CI=0.89-3.16; p= 0.1). High grade cervical lesions are associated with high risk viruses like HPV 16 and 18 and with multiple cervical HPV infections. CONCLUSIONS: The tendency to treat HIV disease with high active antiretroviral therapy may reduce the impact of immunosuppression and make the course of such HPV infections more similar to that among women who are not HIVinfected. As in immunocompetent women, high oncogenic risk viral type and multiple infections are associated with a histologically proven cervical intraepithelial lesions.

Progenitor cells isolated from the human heart: a potential cell source for regenerative therapy.

BACKGROUND: In recent years, resident cardiac progenitor cells have been identified in, and isolated from the rodent heart. These cells show the potential to form cardiomyocytes, smooth muscle cells, and endothelial cells in vitro and in vivo and could potentially be used as a source for cardiac repair. However, previously described cardiac progenitor cell populations show immature development and need co-culture with neonatal rat cardiomyocytes in order to differentiate in vitro. Here we describe the localisation, isolation, characterisation, and differentiation of cardiomyocyte progenitor cells (CMPCs) isolated from the human heart. METHODS: hCMPCs were identified in human hearts based on Sca-1 expression. These cells were isolated, and FACS, RT-PCR and immunocytochemistry were used to determine their baseline characteristics. Cardiomyogenic differentiation was induced by stimulation with 5-azacytidine. RESULTS: hCMPCs were localised within the atria, atrioventricular region, and epicardial layer of the foetal and adult human heart. In vitro, hCMPCs could be induced to differentiate into cardiomyocytes and formed spontaneously beating aggregates, without the need for co-culture with neonatal cardiomyocytes. CONCLUSION: The human heart harbours a pool of resident cardiomyocyte progenitor cells, which can be expanded and differentiated in vitro. These cells may provide a suitable source for cardiac regeneration cell therapy. (Neth Heart J 2008;16:163-9.).

Phospholipase D1 is an effector of Rheb in the mTOR pathway.

The mammalian target of rapamycin (mTOR) assembles a signaling network essential for the regulation of cell growth, which has emerged as a major target of anticancer therapies. The tuberous sclerosis complex 1 and 2 (TSC1/2) proteins and their target, the small GTPase Rheb, constitute a key regulatory pathway upstream of mTOR. Phospholipase D (PLD) and its product phosphatidic acid are also upstream regulators of the mitogenic mTOR signaling. However, how the TSC/Rheb and PLD pathways interact or integrate in the rapamycin-sensitive signaling network has not been examined before. Here, we find that PLD1, but not PLD2, is required for Rheb activation of the mTOR pathway, as demonstrated by the effects of RNAi. The overexpression of Rheb activates PLD1 in cells in the absence of mitogenic stimulation, and the knockdown of Rheb impairs serum stimulation of PLD activation. Furthermore, the overexpression of TSC2 suppresses PLD1 activation, whereas the knockdown or deletion of TSC2 leads to elevated basal activity of PLD. Consistent with a TSC-Rheb-PLD signaling cascade, AMPK and PI3K, both established regulators of TSC2, appear to lie upstream of PLD as revealed by the effects of pharmacological inhibitors, and serum activation of PLD is also dependent on amino acid sufficiency. Finally, Rheb binds and activates PLD1 in vitro in a GTP-dependent manner, strongly suggesting that PLD1 is a bona fide effector for Rheb. Hence, our findings reveal an unexpected interaction between two cascades in the mTOR signaling pathways and open up additional possibilities for targeting this important growth-regulating network for the development of anticancer drugs.

Stem cell sources for cardiac regeneration.

Cell-based cardiac repair has the ambitious aim to replace the malfunctioning cardiac muscle developed after myocardial infarction, with new contractile cardiomyocytes and vessels. Different stem cell populations have been intensively studied in the last decade as a potential source of new cardiomyocytes to ameliorate the injured myocardium, compensate for the loss of ventricular mass and contractility and eventually restore cardiac function. An array of cell types has been explored in this respect, including skeletal muscle, bone marrow derived stem cells, embryonic stem cells (ESC) and more recently cardiac progenitor cells. The best-studied cell types are mouse and human ESC cells, which have undisputedly been demonstrated to differentiate into cardiomyocyte and vascular lineages and have been of great help to understand the differentiation process of pluripotent cells. However, due to their immunogenicity, risk of tumor development and the ethical challenge arising from their embryonic origin, they do not provide a suitable cell source for a regenerative therapy approach. A better option, overcoming ethical and allogenicity problems, seems to be provided by bone marrow derived cells and by the recently identified cardiac precursors. This report will overview current knowledge on these different cell types and their application in cardiac regeneration and address issues like implementation of delivery methods, including tissue engineering approaches that need to be developed alongside.

[Perinatal transmission of HIV]. / Infezione perinatale da HIV.

Paediatric HIV infection is still the most important pandemic, despite the substantial reductions of mother to child transmission achieved in North America and Europe. The total number of people living with the human immunodeficiency virus (HIV) rose in 2004 to reach its highest level ever: an estimated 39.4 million people are living with the virus. This number has been rising in every region, compared with two years ago, with the steepest increases occurring in East Asia, in Eastern Europe and central Asia. Sub-Saharan Africa remains by far the worst-affected region, with 25,4 million people living with HIV at the end of 2004. The AIDS epidemic is affecting women and girls in increasing number in Africa; them make up almost 57% of all people infected with HIV, but became a striking 76% in Sub-Saharan area. This review will focus on the current knowledge available regarding the timing of HIV transmission and the subsequent implications for its prevention. Mother to child transmission can take place during pregnancy, labour, delivery and post-partum, through breastfeeding. Different factors may influence HIV transmission during each of these time periods, and hence interventions to reduce transmission during each of these periods may also require different preventive strategies. The risk of mother to child transmission of HIV infection can be substantially reduced from 15-20% without interventions to less than 2% with the use of antiretroviral therapy during pregnancy, during labour and in the neonatal period, with an elective caesarean section delivery and refraining from breastfeeding. Factors associated with an increased risk of perinatal HIV transmission include advanced maternal blood, prolonged duration of ruptured membranes, and increased quantity of HIV in maternal blood at delivery.

Regulation of the small GTPase Rheb by amino acids.

The mTOR/S6K/4E-BP1 pathway integrates extracellular signals derived from growth factors, and intracellular signals, determined by the availability of nutrients like amino acids and glucose. Activation of this pathway requires inhibition of the tumor suppressor complex TSC1/2. TSC2 is a GTPase-activating protein for the small GTPase Ras homologue enriched in brain (Rheb), GTP loading of which activates mTOR by a yet unidentified mechanism. The level at which this pathway senses the availability of amino acids is unknown but is suggested to be at the level of TSC2. Here, we show that amino-acid depletion completely blocks insulin- and TPA-induced Rheb activation. This indicates that amino-acid sensing occurs upstream of Rheb. Despite this, amino-acid depletion can still inhibit mTOR/S6 kinase signaling in TSC2-/- fibroblasts. Since under these conditions Rheb-GTP levels remain high, a second level of amino-acid sensing exists, affecting mTOR activity in a Rheb-independent fashion.