Numb Expression Contributes to the Maintenance of an Undifferentiated State in Human Epidermis

The epidermis is a stratified epithelium with a stem cell subpopulation in the basal layer that constantly replicates and periodically detaches from the base, undergoing a differentiation process that involves various developmen- tal signals and regulatory pathways. During the last 10 years, a number of studies tried to elucidate the intricate scenario that maintains the epithelial shield during the entire life span. In our study, we investigated the role of Numb in the skin compartment and, in particular, its involvement in stem cell maintenance. Numb expression in the skin compartment was assessed by immunofluorescence and immunohistochemistry analysis. We evaluated Numb expression in primary epithelial cells at various differentiative stages. Moreover, we overexpressed Numb in the isolated population enriched for undifferentiated progenitors to establish its involvement in in vitro differ- entiation. We demonstrated that Numb in high-proliferating epithelial undifferentiated progenitors contributes to the maintenance of an undifferentiated state. This regulation involves the E3 ligases Itch binding. Moreover, the analysis of a cohort of cutaneous carcinomas showed that Numb is highly expressed in squamous cell carcinoma (SCC), where we observed a direct correlation between the expression of Numb and Ki-67. Our data indicate for the first time that Numb is involved in the maintenance of the undifferentiated proliferating stem cell pool in the epithelial basal layer and its expression could become a new marker in skin cancer

Rhinovirus infection induces cytotoxicity and delays wound healing in bronchial epithelial cells

BACKGROUND: Human rhinoviruses (RV), the most common triggers of acute asthma exacerbations, are considered not cytotoxic to the bronchial epithelium. Recent observations, however, have questioned this knowledge. The aim of this study was to evaluate the ability of RV to induce epithelial cytotoxicity and affect epithelial repair in-vitro. METHODS: Monolayers of BEAS-2B bronchial epithelial cells, seeded at different densities were exposed to RV serotypes 1b, 5, 7, 9, 14, 16. Cytotoxicity was assessed chromatometrically. Epithelial monolayers were mechanically wounded, exposed or not to RV and the repopulation of the damaged area was assessed by image analysis. Finally epithelial cell proliferation was assessed by quantitation of proliferating cell nuclear antigen (PCNA) by flow cytometry. RESULTS: RV1b, RV5, RV7, RV14 and RV16 were able to induce considerable epithelial cytotoxicity, more pronounced in less dense cultures, in a cell-density and dose-dependent manner. RV9 was not cytotoxic. Furthermore, RV infection diminished the self-repair capacity of bronchial epithelial cells and reduced cell proliferation. CONCLUSION: RV-induced epithelial cytotoxicity may become considerable in already compromised epithelium, such as in the case of asthma. The RV-induced impairment on epithelial proliferation and self-repair capacity may contribute to the development of airway remodeling

Segregation of Virulent Influenza A(H1N1) Variants in the Lower Respiratory Tract of Critically Ill Patients during the 2010–2011 Seasonal Epidemic

BACKGROUND: Since its appearance in 2009, the pandemic influenza A(H1N1) virus circulated worldwide causing several severe infections. METHODS: Respiratory samples from patients with 2009 influenza A(H1N1) and acute respiratory distress attending 24 intensive care units (ICUs) as well as from patients with lower respiratory tract infections not requiring ICU admission and community upper respiratory tract infections in the Lombardy region (10 million inhabitants) of Italy during the 2010-2011 winter-spring season, were analyzed. RESULTS: In patients with severe ILI, the viral load was higher in bronchoalveolar lavage (BAL) with respect to nasal swab (NS), (p<0.001) suggesting a higher virus replication in the lower respiratory tract. Four distinct virus clusters (referred to as cluster A to D) circulated simultaneously. Most (72.7%, n = 48) of the 66 patients infected with viruses belonging to cluster A had a severe (n = 26) or moderate ILI (n = 22). Amino acid mutations (V26I, I116M, A186T, D187Y, D222G/N, M257I, S263F, I286L/M, and N473D) were observed only in patients with severe ILI. D222G/N variants were detected exclusively in BAL samples. CONCLUSIONS: Multiple virus clusters co-circulated during the 2010-2011 winter-spring season. Severe or moderate ILI were associated with specific 2009 influenza A(H1N1) variants, which replicated preferentially in the lower respiratory tract

Clinical anatomic, immunomorphologic and molecular anatomic data suggest interplay of thyroidal molecules, autoantibodies and Hsp60 in Hashimoto’s disease

Hsp60 is, typically, a mitochondrial protein, but it also occurs in the cytosol, vesicles, and plasma membrane, and in the intercellular space and biological fluids, e.g., blood. Changes in the levels and distribution of Hsp60 are linked to several pathologies, including cancer and chronic inflammatory and autoimmune disorders. What is the histopathological pattern of Hsp60 in the thyroid of Hashimoto’s patients? Are there indications of a pathogenic role of Hsp60 that may make Hashimoto’s thyroiditis a chaperonopathy? Experiments reported here provide information regarding those questions. We found by various immunomorphological techniques increased levels of Hsp60 in the thyroid from HT patients, localized to thyrocytes of small and degenerated follicles and to oncocytes (Hurtle cells). Immunofluorescence showed the chaperonin both inside the cells and also in the plasma membrane, especially in oncocytes. We also found that Hsp60 levels in the blood of HT patients were increased compared to controls and correlated with those of autoantibodies against two distinctive thyroidal proteins, thyroglobulin (TG) and thyroid peroxidase (TPO) (r=0.379, p=0.0103; r=0.484, p=0.0008; respectively). Molecular analysis of these two proteins in comparison with Hsp60 demonstrated various regions of high structural similarity shared by them, which could very well be immunologically crossreactive epitopes. Thus, it is likely that the three proteins potentiate each other as immunogens to elicit autoantibodies and, as antigens, to cause antigen-antibody reactions at those sites in which Hsp60 is exposed, for example the surface of oncocytes. This would lead to inflammation and oncocyte lysis with destruction of thyroidal tissue. The cytometric bead assay revealed that recombinant Hsp60 did not induce increment of cytokine production by peripheral blood mononuclear cells from HT patients. Consequently, we propose that Hsp60 is implicated in the pathogenesis of Hashimoto’s thyroiditis as autoantigen, via a participation of autoantibodies that also recognize TG and TPO, whereas participation of inflammatory cytokines induced by the chaperonin is unlikely. Supported by IEMEST (FC and AJLM)

Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury

Several studies demonstrated that treatment with mesenchymal stem cells (MSCs) reduces cisplatin mortality in mice. Microvesicles (MVs) released from MSCs were previously shown to favor renal repair in non lethal toxic and ischemic acute renal injury (AKI). In the present study we investigated the effects of MSC-derived MVs in SCID mice survival in lethal cisplatin-induced AKI. Moreover, we evaluated in vitro the effect of MVs on cisplatin-induced apoptosis of human renal tubular epithelial cells and the molecular mechanisms involved. Two different regimens of MV injection were used. The single administration of MVs ameliorated renal function and morphology, and improved survival but did not prevent chronic tubular injury and persistent increase in BUN and creatinine. Multiple injections of MVs further decreased mortality and at day 21 surviving mice showed normal histology and renal function. The mechanism of protection was mainly ascribed to an anti-apoptotic effect of MVs. In vitro studies demonstrated that MVs up-regulated in cisplatin-treated human tubular epithelial cells anti-apoptotic genes, such as Bcl-xL, Bcl2 and BIRC8 and down-regulated genes that have a central role in the execution-phase of cell apoptosis such as Casp1, Casp8 and LTA. In conclusion, MVs released from MSCs were found to exert a pro-survival effect on renal cells in vitro and in vivo, suggesting that MVs may contribute to renal protection conferred by MSCs

Rhinovirus-induced basic fibroblast growth factor release mediates airway remodeling features

BACKGROUND: Human rhinoviruses, major precipitants of asthma exacerbations, induce lower airway inflammation and mediate angiogenesis. The purpose of this study was to assess the possibility that rhinoviruses may also contribute to the fibrotic component of airway remodeling. METHODS: Levels of basic fibroblast growth factor (bFGF) mRNA and protein were measured following rhinovirus infection of bronchial epithelial cells. The profibrotic effect of epithelial products was assessed by DNA synthesis and matrix metalloproteinase activity assays. Moreover, epithelial cells were exposed to supernatants from cultured peripheral blood mononuclear cells, obtained from healthy donors or atopic asthmatic subjects and subsequently infected by rhinovirus and bFGF release was estimated. bFGF was also measured in respiratory secretions from atopic asthmatic patients before and during rhinovirus-induced asthma exacerbations. RESULTS: Rhinovirus epithelial infection stimulated mRNA expression and release of bFGF, the latter being positively correlated with cell death under conditions promoting rhinovirus-induced cytotoxicity. Supernatants from infected cultures induced lung fibroblast proliferation, which was inhibited by anti-bFGF antibody, and demonstrated increased matrix metalloproteinase activity. Rhinovirus-mediated bFGF release was significantly higher in an in vitro simulation of atopic asthmatic environment and, importantly, during rhinovirus-associated asthma exacerbations. CONCLUSIONS: Rhinovirus infection induces bFGF release by airway epithelium, and stimulates stroma cell proliferation contributing to airway remodeling in asthma. Repeated rhinovirus infections may promote asthma persistence, particularly in the context of atopy; prevention of such infections may influence the natural history of asthma