Urine is a novel source of autologous mesenchymal stem cells for patients with epidermolysis bullosa.

BACKGROUND: Regenerative medicine is strictly dependent on stem cells as a source for a high diversity of somatic cells. However, the isolation of such from individuals suffering from severe genetic skin blistering diseases like epidermolysis bullosa (EB) is often associated with further organ damage. METHODS: Stem cells were isolated from 112 urine samples from 21 different healthy donors, as well as from 33 urine samples from 25 donors with EB. The cultivation of these cells was optimized by testing different media formulations and pre-coating of culture vessels with collagen. The identity of cells was confirmed by testing marker expression, differentiation potential and immune-modulatory properties. RESULTS: We provide here an optimized protocol for the reproducible isolation of mesenchymal stem cells from urine, even from small volumes as obtained from patients with EB. Furthermore, we offer a basic characterization of those urine-derived stem cells (USCs) from healthy donors, as well as from patients with EB, and demonstrate their potential to differentiate into chondrocytes, osteoblasts and adipocytes, as well as their immune-modulatory properties. CONCLUSIONS: Thus, USCs provide a novel and non-invasive source of stem cells, which might be applied for gene-therapeutic approaches to improve medical conditions of patients with EB.

MicroRNAs differentially present in the plasma of HIV elite controllers reduce HIV infection in vitro.

Elite controllers maintain HIV-1 viral loads below the limit of detection. The mechanisms responsible for this phenomenon are poorly understood. As microRNAs (miRNAs) are regulators of gene expression and some of them modulate HIV infection, we have studied the miRNA profile in plasma from HIV elite controllers and chronically infected individuals and compared against healthy donors. Several miRNAs correlate with CD4+ T cell count or with the known time of infection. No significant differences were observed between elite controllers and healthy donors; however, 16 miRNAs were different in the plasma of chronic infected versus healthy donors. In addition, levels of hsa-miR-29b-3p, hsa-miR-33a-5p and hsa-miR-146a-5p were higher in plasma from elite controllers than chronic infected and hsa-miR-29b-3p and hsa-miR-33a-5p overexpression significantly reduced the viral production in MT2 and primary T CD4+ cells. Therefore, levels of circulating miRNAs might be of diagnostic and/or prognostic value for HIV infection, and hsa-miR-29b-3p and miR-33a-5p may contribute to the design of new anti-HIV drugs.

HIV-1 induces telomerase activity in monocyte-derived macrophages, possibly safeguarding one of its reservoirs.

Monocyte-derived macrophages (MDM) are widely distributed in all tissues and organs, including the central nervous system, where they represent the main part of HIV-infected cells. In contrast to activated CD4(+) T lymphocytes, MDM are resistant to cytopathic effects and survive HIV infection for a long period of time. The molecular mechanisms of how HIV is able to persist in macrophages are not fully elucidated yet. In this context, we have studied the effect of in vitro HIV-1 infection on telomerase activity (TA), telomere length, and DNA damage. Infection resulted in a significant induction of TA. This increase was directly proportional to the efficacy of HIV infection and was found in both nuclear and cytoplasmic extracts, while neither UV light-inactivated HIV nor exogenous addition of the viral protein Tat or gp120 affected TA. Furthermore, TA was not modified during monocyte-macrophage differentiation, MDM activation, or infection with vaccinia virus. HIV infection did not affect telomere length. However, HIV-infected MDM showed less DNA damage after oxidative stress than noninfected MDM, and this resistance was also increased by overexpressing telomerase alone. Taken together, our results suggest that HIV induces TA in MDM and that this induction might contribute to cellular protection against oxidative stress, which could be considered a viral strategy to make macrophages better suited as longer-lived, more resistant viral reservoirs. In the light of the clinical development of telomerase inhibitors as anticancer therapeutics, inhibition of TA in HIV-infected macrophages might also represent a novel therapeutic target against viral reservoirs.

Longitudinal analysis of the 5'UTR, E2-PePHD and NS5A-PKRBD genomic regions of hepatitis C virus genotype 1a in association with the response to peginterferon and ribavirin therapy in HIV-coinfected patients.

BACKGROUND: The rate of non-response to pegylated interferon plus ribavirin (peg-IFN+RBV) in HCV/HIV coinfected patients is higher than in HCV-monoinfected patients. In this sense, the contribution of HCV genetic variability is unknown. The 5' untranslated (5'UTR), the nonstructural 5A (NS5A) and the second envelope (E2) HCV genomic regions have been implicated to peg-IFN therapy response. The proteins appear to block interferon (IFN)-induced RNA-dependent protein kinase (PKR) and the 5'UTR may influence the viral lymphotropism. METHODS: We examined comparatively the pretreatment HCV variability between HIV coinfected and HCV monoinfected patients as well as assessed longitudinally the impact of peg-IFN+RBV on HCV variability when HIV is co-present. For this purpose, 15 HIV coinfected and 20 HCV monoinfected patients were compared. They were peg-IFN+RBV non-responders and infected with HCV 1a. RESULTS: Irrespectively of the HIV-coexistence, at baseline the amino acid variation in the NS5A-related domains was significantly higher than in the E2-PePHD (p<0.001). The number of amino acid variations (mean±SD) at the NS5A-ISDR domain was higher among HCV/HIV patients than HCV-monoinfected ones (1.80±0.77 vs. 0.95±1.05; p=0.009) but such difference was slightly lower when comparing NS5A-PKRBD sequences (2.47±1.13 vs. 1.60±1.57; p=0.06). No differences were found at the E2-PePHD (0±0 vs. 0.2±0.4). At intra-HIV coinfected patient level, only minor (HCV genetic analysis) or no (HCV substitution rate and quasispecies heterogeneity) changes were observed during therapy (basal, 24h, 4weeks, and 12weeks). CONCLUSIONS: Among HCV-1a/HIV coinfected and HCV-monoinfected peg-IFN+RBV non-responder patients, the HCV variability at the 5'UTR, E2-PePHD and NS5A-PKRBD/ISDR domains was mostly comparable exhibiting a low number of variations. Four well-defined amino acid substitutions in NS5A-ISDR domain appeared most frequently when HIV coexists. The interferon-based therapy did not exert any effect in the variation, selection or diversity in the above mentioned HCV regions that could influence clinical responsiveness to IFN therapy.

Telomerase activity in peripheral blood mononuclear cells from HIV and HIV-HCV coinfected patients.

Replicative senescence of peripheral blood mononuclear cells (PBMC) plays an important role in the pathophysiology of chronic viral infections. Although there are controversial reports concerning telomerase activity in HIV monoinfected subjects, no data on HIV-HCV coinfected individuals is available. In this cross-sectional study telomerase activity was quantified in non-stimulated and mitogen-stimulated PBMC lysates from HIV-1 monoinfected and HIV-HCV coinfected individuals using real-time PCR. Up-regulation of telomerase activity after mitogen stimulation was impaired in PBMC of HIV monoinfected and HIV-HCV coinfected patients. The lack of an appropriate induction of this enzymatic activity after stimulus could partly account for immunosuppressive conditions observed in such patients.