Umbilical Cord Mesenchymal Stromal Cells for Cartilage Regeneration Applications.

Chondropathies are increasing worldwide, but effective treatments are currently lacking. Mesenchymal stromal cell (MSCs) transplantation represents a promising approach to counteract the degenerative and inflammatory environment characterizing those pathologies, such as osteoarthritis (OA) and rheumatoid arthritis (RA). Umbilical cord- (UC-) MSCs gained increasing interest due to their multilineage differentiation potential, immunomodulatory, and anti-inflammatory properties as well as higher proliferation rates, abundant supply along with no risks for the donor compared to adult MSCs. In addition, UC-MSCs are physiologically adapted to survive in an ischemic and nutrient-poor environment as well as to produce an extracellular matrix (ECM) similar to that of the cartilage. All these characteristics make UC-MSCs a pivotal source for a stem cell-based treatment of chondropathies. In this review, the regenerative potential of UC-MSCs for the treatment of cartilage diseases will be discussed focusing on in vitro, in vivo, and clinical studies.

Role of non-coding RNAs in age-related vascular cognitive impairment: An overview on diagnostic/prognostic value in Vascular Dementia and Vascular Parkinsonism.

Age is the pivotal risk factor for different common medical conditions such as cardiovascular diseases, cancer and dementia. Among age-related disorders, cardiovascular and cerebrovascular diseases, represent the leading causes of premature mortality strictly related to vascular ageing, a pathological condition characterized by endothelial dysfunction, atherosclerosis, hypertension, heart disease and stroke. These features negatively impact on the brain, owing to altered cerebral blood flow, neurovascular coupling and impaired endothelial permeability leading to cerebrovascular diseases (CVDs) as Vascular Dementia (VD) and Parkinsonism (VP). It is an increasing opinion that neurodegenerative disorders and cerebrovascular diseases are associated from a pathogenetic point of view, and in this review, we discuss how cerebrovascular dysfunctions, due to epigenetic alterations, are linked with neuronal degeneration/dysfunction that lead to cognitive impairment. The relation between neurodegenerative and cerebrovascular diseases are reviewed with a focus on role of ncRNAs in age-related vascular diseases impairing the endothelium in the blood-brain barrier with consequent dysfunction of cerebral blood flow. In this review we dissert about different regulatory mechanisms of gene expression implemented by ncRNAs in the pathogenesis of age-related neurovascular impairment, aiming to highlight the potential use of ncRNAs as biomarkers for diagnostic/prognostic purposes as well as novel therapeutic targets.

Successful Treatment of Kaposi Sarcoma-Associated Herpesvirus Inflammatory Cytokine Syndrome After Kidney-Liver Transplant: Correlations With the Human Herpesvirus 8 miRNome and Specific T Cell Response.

After transplant, patient infection with human herpesvirus 8 (HHV-8) and Kaposi sarcoma-associated herpesvirus (KSHV) is known to cause aggressive tumors and severe nonneoplastic complications. These latter syndromes are driven by HHV-8/KSHV lytic reactivations and related hyperinflammatory host responses typically characterized by high viral loads, elevated levels of cytokines and other inflammation biomarkers, cytopenia, organ failure, high fever, and worsening conditions (with no evidence of B cell neoplasias). These disorders are associated with a high mortality rate, often due to lack of prompt diagnosis, effective therapeutic approaches, and adequate follow-up. These features resemble most of those defining the so-called KSHV-associated inflammatory cytokine syndrome (KICS), which was recently recognized in patients positive for human immunodeficiency virus (HIV). In this report, we describe-for the first time-a case of a KICS-like nonneoplastic recurrent complication occurring after transplant in an HIV-negative patient that was successfully treated by a combination of anti-CD20 monoclonal therapy, antivirals, and modification of the immunosuppressive regimen. In addition to clinical and laboratory findings collected during 3-year follow-up, we report novel experimental data on HHV-8-specific T cell dynamics and circulating microRNA profile, showing correlations with clinical course and other laboratory markers (including viral load, C-reactive protein, and cytokine levels), providing useful information about abnormal cellular and cytokine dynamics underlying HHV-8-associated inflammatory disorders in posttransplant patients.

In vivo multiclonal transfer of bla(KPC-3) from Klebsiella pneumoniae to Escherichia coli in surgery patients.

During active surveillance at the Mediterranean Institute for Transplantation and Advanced Specialized Therapies (ISMETT, Palermo, Italy) with the CARBA screening medium, five pairs of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae and Escherichia coli strains were isolated in each of five colonized patients. In each patient, lateral gene transfer was demonstrated by comparing K. pneumoniae and E. coli strains, both possessing KPC-3, Tn4401a and pKpQIL-IT elements. The isolates were found to be multiclonal by multilocus sequence typing (sequence type (ST) 512 related to ST258, and ST307 belonging to a clonal complex different from the habitual sequence clone ST258 isolated in Italy) and pulsed-field gel electrophoresis. The results of our study highlight the easy transfer of KPC among Enterobacteriaceae colonizing the human intestine, and the active and careful surveillance required to identify and prevent the spread of these multidrug-resistant microorganisms.

Pandemic influenza A/H1N1 virus in a swine farm house in Sicily, Italy.

This report describes a pandemic A/H1N1 (H1N1 pdm) virus outbreak occurred in December, 2009 in a swine farm used as research facility (Istituto Mediterraneo Trapianti e Terapie ad Alta Specializzazione) for preclinical studies, located in Sicily, Italy. All the 13 pigs of the farm, showed cough, fever, inappetence and weakness. At the same time, an unvaccinated worker of the stabling showed influenza-like symptoms. RNAv extracted from two swabs collected from infected pigs resulted positive by Real Time RT-PCR for Influenza A virus. Furthermore, after growth on embryonated eggs, viral isolates were identified by Real Time RT-PCR specific for H1N1 pdm virus and characterized antigenically. Sequencing of the whole genome was also performed. All sera taken from animals and from the worker were tested by a competitive influenza A ELISA and by the haemoagglutination inhibition test. Serological findings confirmed the circulation of influenza virus H1N1 pdm in pigs and the presence of specific antibodies against H1N1 pdm in human serum. The results of this study seem to support a H1N1 pdm transmission from man to animals showing the importance of serological and virological investigation to control the pig farms and the importance of close cooperation between the different authorities like veterinarian and human public.

Primary and reactivated HHV8 infection and disease after liver transplantation: a prospective study.

Human herpesvirus 8 (HHV8) is pathogenic in humans, especially in cases of immunosuppression. We evaluated the risk of HHV8 transmission from liver donors, and its clinical impact in southern Italy, where its seroprevalence in the general population is reported to be as high as 18.3%. We tested 179 liver transplant recipients and their donors for HHV8 antibodies at the time of transplantation, and implemented in all recipients a 12-month posttransplant surveillance program for HHV8 infection. Of the 179 liver transplant recipients enrolled, 10.6% were HHV8 seropositive before transplantation, whereas the organ donor's seroprevalence was 4.4%. Eight seronegative patients received a liver from a seropositive donor, and four of them developed primary HHV8 infection. Two of these patients had lethal nonmalignant illness with systemic involvement and multiorgan failure. Among the 19 HHV8 seropositive recipients, two had viral reactivation after liver transplantation. In addition, an HHV8 seronegative recipient of a seronegative donor developed primary HHV8 infection and multicentric Castleman's disease. In conclusion, primary HHV8 infection transmitted from a seropositive donor to a seronegative liver transplant recipient can cause a severe nonmalignant illness associated with high mortality. Donor screening for HHV8 should be considered in geographic areas with a high prevalence of such infection.

Severe outcome of influenza A/H1N1/09v infection associated with 222G/N polymorphisms in the haemagglutinin: a multicentre study.

In a multicentre study, influenza A/H1N1/09v 222G/N variants were more frequently detected in patients admitted to the intensive-care unit for invasive mechanical ventilation or extracorporeal membrane oxygenation (10/23; 43.5%) than in patients hospitalized in other units (2/27; 7.4%) and community patients (0/81; 0.0%) (p <0.01). A significantly higher virus load (p 0.02) in the lower vs the upper respiratory tract was observed. Predominance of 222G/N variants in the lower respiratory tract (40% of total virus population) vs the upper respiratory tract (10%) was shown by clonal analysis of haemagglutinin sequences in paired nasal swab and bronchoalveolar lavage samples. The time from illness onset to sampling was significantly longer in patients with severe infection vs community patients (p <0.001). It was concluded that the 222G/N variants showed increased virulence; mutant variants were probably selected in individual patients; and the longer duration of illness might have favoured the emergence of adaptive mutations through multiple replication cycles.

[HIV-1 and renal cells: pathogenesis of HIV-associated nephropathy]. / HIV-1 e cellule renali: patogenesi della nefropatia da HIV.

Kidney is one of the organs, as haematopoietic tissue and central nervous system, representing a reservoir of HIV-1, where the virus can exert a direct pathogenic activity. HIV-associated nephropathy (HIVAN) is the prominent illness among the numerous renal injuries occurring in HIV-1 infection. It is characterized by heavy proteinuria and rapid progression to end stage renal disease. Histopathologically, HIVAN is a collapsing form of focal segmental glomerulosclerosis with podocyte hyperplasia and dedifferentiation, associated with severe tubulopathy which is characterized by tubular apoptosis, microcysts, and interstitial fibrosis. Several clinical and experimental data point to a direct role of HIV-1 in kidney damage. In renal biopsies of HIVAN cases viral transcripts have been found in glomerular and tubular epithelial cells. Transgenic mice expressing replication-defective HIV proviral constructs develop a renal disease similar to HIVAN both from the histopathological and clinical point of view. In vitro studies using cultures of human renal cells have shown that HIV-1 can induce in glomerular and tubular cells distinct pathogenic effects, which mimic the pathological features of HIVAN in vivo. A large body of evidence suggests that an abnormal response of podocytes to HIV-1 infection and/or HIV-1 proteins is the key event in HIVAN pathogenesis. Since the highly-active antiretroviral therapy has demonstrated scant beneficial effects on the development of HIVAN, the elucidation of the pathogenic mechanisms activated by HIV-1 in the renal cells might allow designing specific therapeutic strategies.

Primary and immortalised human pancreatic islet endothelial cells: phenotypic and immunological characterisation.

AIMS/HYPOTHESIS: Studies on the biology of the microvascular endothelial cells (MECs) that surround and penetrate the pancreatic islets are hampered by difficulties in isolating and culturing large numbers of pure cells. We aimed to morphologically and functionally characterise primary MECs purified and cultured from human islets, and to establish a simian virus 40 (SV40)-immortalised cell line from these primary cultures. MATERIALS AND METHODS: Human islet MECs were extracted and purified using anti-CD105 coated immunomagnetic beads, and endothelial markers and surface molecules analysed by flow cytometric analysis. An immortalised cell line was then established by using a chimeric adeno5/SV40 virus. RESULTS: Islet MECs expressed classic and specific endothelial markers, a high basal level of intercellular adhesion molecule-1, and low levels of E-selectin and TNF (previously known as TNF-alpha) inducible vascular cell adhesion molecule-1. IFNG (previously known as IFN-gamma) induced expression of HLA class II molecules. The immortalised islet MECs expanded rapidly, exhibited increased DNA synthesis, and were passaged approximately 30 times, without signs of senescence. They retained the endothelial characteristics of the parental cells, and behaved as the primary cells in terms of TNF stimulation of expression of adhesion molecules and support of leucocyte adhesion and transmigration. CONCLUSIONS/INTERPRETATION: The immortalised islet MECs that we have established could effectively represent a substitute for primary counterparts for in vitro studies on the role of the microvasculature in pathophysiological processes involved in type 1 and type 2 diabetes.

Nephrin redistribution on podocytes is a potential mechanism for proteinuria in patients with primary acquired nephrotic syndrome.

We investigated the distribution of nephrin by immunofluorescence microscopy in renal biopsies of patients with nephrotic syndrome: 13 with membranous glomerulonephritis (GN), 10 with minimal change GN, and seven with focal segmental glomerulosclerosis. As control, six patients with IgA GN without nephrotic syndrome and 10 normal controls were studied. We found an extensive loss of staining for nephrin and a shift from a podocyte-staining pattern to a granular pattern in patients with nephrotic syndrome, irrespective of the primary disease. In membranous GN, nephrin was co-localized with IgG immune deposits. In the attempt to explain these results, we investigated in vitro whether stimuli acting on the cell cytoskeleton, known to be involved in the pathogenesis of GN, may induce redistribution of nephrin on the surface of human cultured podocytes. Aggregated but not disaggregated human IgG(4), plasmalemmal insertion of membrane attack complex of complement, tumor necrosis factor-alpha, and puromycin, induced the shedding of nephrin with a loss of surface expression. This phenomenon was abrogated by cytochalasin and sodium azide. These results suggest that the activation of cell cytoskeleton may modify surface expression of nephrin allowing a dislocation from plasma membrane to an extracellular site.

Role of platelet-activating factor in functional alterations induced by xenoreactive antibodies in porcine endothelial cells.

BACKGROUND: Platelet-activating factor (PAF) is a phospholipid mediator of inflammation which has been implicated in rejection. The interaction of anti-alpha-galactosyl natural antibodies (anti-alpha gal Abs) with endothelial cells is the initial step for the development of xenograft rejection. In our study, we stimulated porcine aortic endothelial cells (PAEC) with anti-alpha gal IgG to investigate the synthesis of PAF from PAEC and its biological consequences. METHODS AND RESULTS: PAF was extracted and chromatographically purified from cultured PAEC stimulated with baboon anti-alpha gal Abs. The Abs induced a dose-dependent synthesis of PAF peaking after 30 min of incubation, and decreasing thereafter. Concomitant cell shape change, motility, and cytoskeleton redistribution were observed. These events were prevented by addition of a panel of PAF-receptor antagonists. An SV40 T-large antigen-immortalized PAEC line was engineered to express PAF acetyl-hydrolase (PAF-AH) cDNA, the major PAF-inactivating enzyme. These transfected cells exposed to anti-alpha gal Abs showed reduced cell contraction and motility compared with empty vector-transfected cells. Moreover, in PAEC stimulated with anti-alpha gal Abs, the synthesis of PAF promoted the adhesion of a monocytic cell line as shown by the inhibitory effect of PAF-receptor antagonists and of PAF-AH expression. Finally, studies on cell monolayer demonstrated an enhanced permeability 48 hr after exposure to anti-alpha gal Abs, and this increase was prevented by PAF-inactivation and by PAF-receptor blockade. CONCLUSIONS: These results demonstrate that on stimulation with anti-alpha gal Abs, PAEC synthetize PAF which can contribute to several vascular events involved in xenograft rejection.

Motility induced by human immunodeficiency virus-1 Tat on Kaposi's sarcoma cells requires platelet-activating factor synthesis.

In the present study, we evaluated whether motility of Kaposi's sarcoma (KS) spindle cells induced by HIV-1 Tat protein is dependent on the synthesis of platelet-activating factor (PAF). The results obtained indicate that Tat induced a dose-dependent synthesis of PAF from KS cells at a concentration as low as 0.1 ng/ml. PAF production started rapidly after Tat stimulation, peaking at 30 minutes and declining thereafter. Tat-induced cell migration was also a rapid event starting at 30 minutes. The motility was abrogated by addition of a panel of chemically unrelated PAF receptor antagonists (WEB 2170, CV 3988, CV 6209, and BN 52021), suggesting that the synthesized PAF mediates the motogenic effect of Tat. This effect was also present on cells plated on a type-I collagen-, fibronectin-, or basement membrane extract-coated surface. Expression of PAF receptor-specific mRNA was detected in KS cells. In addition, examination of the cytoskeletal organization showed that Tat-mediated KS cell redistribution of actin filaments and shape change was also inhibited by a PAF receptor antagonist. Moreover, PAF receptor blockade prevented the up-regulation of beta1 integrin and the down-regulation of alphavbeta3 observed after stimulation of KS cells with Tat. In conclusion, the results of the present study indicate that Tat-induced PAF synthesis plays a critical role in triggering the events involved in motility of KS cells.

The synthesis of platelet-activating factor modulates chemotaxis of monocytes induced by HIV-1 Tat.

HIV-1 Tat protein has been shown to induce chemotaxis and recruitment of monocytes. In the present study, we evaluated whether HIV-1 Tat protein was able to induce the synthesis of platelet-activating factor (PAF), which is a potent mediator of cell motility, and whether the synthesis of PAF was instrumental in triggering Tat-induced monocyte chemotaxis. The results obtained indicate that Tat, but not gp120 and gp41, induced a time-dependent synthesis of PAF from monocytes at concentration as low as 0.1 ng/ml. As inferred by the inhibitory effect of anti-Flt-1 antibody and by the desensitization of monocytes following preincubation with vascular endothelial growth factor, the synthesis of PAF by monocytes stimulated with Tat was induced by activation of vascular endothelial growth factor receptor 1. Moreover, the Tat-induced chemotaxis of monocytes was abrogated both by WEB 2170 and by CV 3988, two chemically unrelated PAF receptor antagonists, suggesting that the synthesized PAF modulates the chemotactic response of monocytes to Tat. In conclusion, the results of the present study indicate that Tat-induced PAF synthesis plays a critical role in triggering the events involved in the migratory response of monocytes.

Potentiation of the malignant phenotype of the undifferentiated ARO thyroid cell line by insertion of the bcl-2 gene.

We have reported that bcl-2 is expressed in normal human thyroid epithelium and that its expression is down-regulated in undifferentiated thyroid tumors. Production of IL-6 was concomitantly down-regulated in these forms. Based on these observations, we analyzed whether insertion of bcl-2 would reverse the highly malignant phenotype of a thyroid cell line (ARO) derived from an undifferentiated carcinoma. This cell line fails to produce Bcl-2 and IL-6. By infection with a bcl-2 retroviral vector, ARO cells expressing bcl-2 (ARObcl-2) were obtained. Compared with parental cells, expression of bcl-2 was associated with enhancement of growth potential (DNA synthesis, in vitro proliferation rate, anchorage-independent growth in semi-solid media). Chemotaxis and invasive potential in Boyden chambers were also increased. bcl-2-expressing cells showed a reduced response to apoptotic stimuli (low-serum conditions or anti-neoplastic drugs). Large branched colonies were formed in Matrigel from ARObcl-2 cells but not from parental cells. Finally, ARObcl-2 cells showed a decreased latency of tumor appearance when injected into immunodeficient mice. Potentiation of the malignant phenotype of ARO cells by bcl-2 was not ascribed to altered expression of (i) cytokine/growth factors (IL-4, IL-6, IL-8, IL-10, IL-12, TGF-alpha, TGF-beta), (ii) thyroid-specific transcripts (TG, TPO, TSH-R, PIGF, PAX-8) or (iii) genes influencing tumor aggressiveness [VEGF, HMGI (Y), HMGI-C]. Our data indicate that bcl-2 potentiates the malignant phenotype of ARO cells not only by limiting the response to apoptotic stimuli but also by enhancing proliferation and tumor aggressiveness.

HIV-1 kills renal tubular epithelial cells in vitro by triggering an apoptotic pathway involving caspase activation and Fas upregulation.

HIV-infected patients suffer several renal syndromes, which can progress rapidly from renal insufficiency to end-stage renal disease. Histologically, HIV-induced nephropathy is characterized by prominent tubulopathy with apoptosis of tubular cells. Clinical and experimental evidence suggests that renal injury may be directly related to virus infection. Although HIV-1 is a polytropic and not solely lymphotropic pathogen, the susceptibility of renal cells to HIV-1 remains to be determined. This paper demonstrates in vitro the permissiveness of proximal tubular epithelial cells (PTEC) to HIV-1 and describes the effects of PTEC infection to explain the pathogenesis of tubular damage in vivo. The results indicate that PTEC express HIV-specific receptor and coreceptors and sustain virus replication. We observed that HIV-1 infection causes the death of tubular cells by triggering an apoptotic pathway involving caspase activation. Fas upregulation but not Fas ligand expression was found in the infected PTEC. However, after HIV-1 infection, tubular cells became susceptible to apoptosis induced through Fas stimulation. Caspase inhibition prevented the death of the infected PTEC in spite of persistent viral replication. These findings may explain the prominent histopathology of HIV-associated nephropathy and demonstrate that the apoptosis of nonlymphoid cells can be directly induced by HIV-1.

Antibody response to fibronectin-binding adhesin FnbpA in patients with Staphylococcus aureus infections.

We have analyzed antibody reactivity to a fibronectin-binding microbial surface component that recognizes adhesive matrix molecules (MSCRAMM) in blood plasma collected from patients with staphylococcal infections. All patients had elevated levels of anti-MSCRAMM antibodies compared to those of young children who, presumably, had not been exposed to staphylococcal infections. The anti-MSCRAMM antibodies preferentially reacted with the ligand-binding repeat domain of the adhesin. However, these antibodies did not inhibit fibronectin binding. Essentially, all patients had antibodies which specifically recognized the fibronectin-MSCRAMM complex but not the isolated components. Epitopes recognized by these anti-ligand-induced binding sites antibodies were found in each repeat unit of the MSCRAMM. These results demonstrate that staphylococci have bound fibronectin some time during infection and that each repeat unit in the MSCRAMM can engage in ligand binding. Furthermore, our previously proposed model, suggesting that an unordered structure in the MSCRAMM undergoes a conformational change upon ligand binding (K. House-Pompeo, Y. Xu, D. Joh, P. Speziale, and M. Höök, J. Biol. Chem. 271:1379-1384, 1996), is presumably operational in patients during infections.

Reduced expression of interleukin 6 in undifferentiated thyroid carcinoma: in vitro and in vivo studies.

Cytokines appear to play an important role in the development and progression of epithelial tumors. Cultured normal human thyroid follicular cells constitutively release high levels of interleukin-6 (IL-6) and IL-8, together with low to moderate levels of transforming growth factor-alpha (TGF-alpha) and TGF-beta. IL-6 appears to play multiple functions in thyroid physiology and disease. Because certain data indicate an inverse relationship between IL-6 production and epithelial tumor aggressiveness, we used both tissue culture methods and histochemical techniques to search for possible alterations of cytokine expression in thyroid carcinomas. As compared to cultures from normal tissue and well-differentiated carcinoma, production of IL-6 was strongly down-regulated in cultures derived from undifferentiated carcinoma. In contrast, levels of IL-8, TGF-alpha, and TGF-beta produced by neoplastic TFC were similar to those produced by normal cells. Actually, production of TGF-alpha was slightly enhanced in cultures from well-differentiated carcinoma. Immunoassay results were confirmed by reverse transcriptase-PCR analysis. Immunohistochemistry of human thyroid carcinomas (n = 99) and normal thyroid tissue (n = 85) showed that immunoreactive IL-6 was strongly diminished in undifferentiated forms (n = 34) and slightly reduced in well-differentiated carcinoma (n = 65). In agreement with the in vitro results, TGF-alpha expression was significantly increased in neoplastic thyrocytes, as compared to their normal counterpart. The results indicate that, as in the mammary and salivary glands, down-regulation of IL-6 expression may represent a marker of undifferentiated thyroid carcinoma.