Identification of miRNAs from stem cell derived microparticles in umbilical cord blood.

Umbilical cord blood CD34+ (UCB-CD34+) stem cells are clinically used in hematopoietic cell transplantation. However, there are limitations in the use of umbilical cord blood transplants because of the small number of cells and delayed engraftment. To gain a better understanding of functional components of UCB, we have detected and characterized CD34+ microparticles (CD34+MPs) from cord blood units. We collected cord blood units and assessed the numbers of CD34+MPs before and after red blood cell and plasma depletion by SEPAX processing using flow cytometry analysis. In parallel we identified MPs by electron microscopy. CD34+MPs and cells were isolated by MACs sorting. MicroRNAs (miR-106, miR-221, miR-517, miR-519, and miR-221) exhibited a characteristic microRNA profile that was further validated in isolated CD34+MPs. We found that in cord blood, there are CD34+MPs that carry microRNAs.

In vitro and in vivo study on the effect of antifungal agents on hematopoietic cells in mice.

Liposomal amphotericin B, voriconazole, and caspofungin are currently used for systemic and severe fungal infections. Patients with malignant diseases are treated with granulocyte-colony stimulating factor (G-CSF) for the recovery of granulocytes after chemotherapy or hematopoietic cell (HC) transplantation. Since they have a high incidence of fungal infections, they inevitably receive antifungal drugs for treatment and prophylaxis. Despite their proven less toxicity for various cell types comparatively with amphotericin B and the decrease in the number of leukocytes that has been reported as a possible complication in clinical studies, the effect of liposomal amphotericin B, voriconazole, and caspofungin on HCs has not been clarified. The present study aimed to examine the in vitro and in vivo effect of these three modern antifungals on HCs. Colony-forming unit (CFU) assays of murine bone marrow cells were performed in methylcellulose medium with or without cytokines and in the presence or absence of various concentrations of liposomal amphotericin B, voriconazole, and caspofungin. In the in vivo experiments, the absolute number of granulocytes was determined during leukocyte recovery in sublethally irradiated mice receiving each antifungal agent separately, with or without G-CSF. In vitro, all three antifungal drugs were nontoxic and, interestingly, they significantly increased the number of CFU-granulocyte-macrophage colonies in the presence of cytokines, at all concentrations tested. This was contrary to the concentration-dependent toxicity and the significant decrease caused by conventional amphotericin B. In vivo, the number of granulocytes was significantly higher with caspofungin plus G-CSF treatment, higher and to a lesser extent higher, but not statistically significantly, with voriconazole plus G-CSF and liposomal amphotericin B plus G-CSF treatments, respectively, as compared with G-CSF alone. These data indicate a potential synergistic effect of these antifungals with the cytokines, in vitro and in vivo, with subsequent positive effect on hematopoiesis.

Safety and efficacy of suicide gene therapy with adenosine deaminase 5-fluorocytosine silmutaneously in in vitro cultures of melanoma and retinal cell lines.

Local treatment as a treatment modality is gaining increased general acceptance over time. Novel drugs and methodologies of local administration are being investigated in an effort to achieve disease local control. Suicide gene therapy is a method that has been investigated as a local treatment with simultaneously distant disease control. In our current experiment we purchased HTB-70 (melanoma cell line, derived from metastatic axillary node) and CRL-2302 (human retinal epithelium) were from ATCC LGC Standards and Ancotil(®), 2.5 g/250 ml (1 g/00ml) (5-Flucytosine) MEDA; Pharmaceuticals Ltd. UK. Adenosine Cytosine Deaminase (Ad.CD) was also used in order to convert the pro-drug 5-Flucytosine to the active 5-Fluoracil. Three different concentrations of 5-Flucytosine (5-FC) were administered (0.2ml, 0.8ml and 1.2ml). At indicated time-points (4h, 8h and 24h) cell viability and apoptosis were measured. Our concept was to investigate whether suicide gene therapy with Ad. CD-5-FC could be used with safety and efficiency as a future local treatment for melanoma located in the eye cavity. Indeed, our results indicated that in every 5-FC administration had mild cytotoxicity for the retinal cells, while increased apoptosis was observed for the melanoma cell line.

Protective effect of mesenchymal stem cell-conditioned medium on hepatic cell apoptosis after acute liver injury.

The aim of this study was to investigate the role of Mesenchymal Stem Cell (MSC) conditioned medium (CM(MSC)) on apoptosis of cultured mouse primary hepatocytes after in vivo carbon tetrachloride (CCl4)-induced acute liver injury. The acute liver injury was induced by injecting CCl4 intraperitoneally in C57/BL6 mice. Hepatocytes were isolated by liver perfusion, cultured in a defined medium to maintain their differentiation and characterized by reverse transcriptase polymerase chain reaction (RT-PCR) using the hepatic cell specific genes albumin, hepatocyte nuclear factor 4 (HNF4) and cytokeratin 18 (CK18). CM(MSC) was generated from cultured bone marrow-derived MSCs (BM-MSCs). BM-MSCs were positive for CD73, CD90, CD44 by flow cytometry and able to differentiate into chondrocytes, adipocytes and osteocytes. Apoptosis was evaluated by both annexin V. CM(MSC) were examined by flow cytometry to detect MSC-derived annexin V- and CD54/CD44-positive microparticles (MPs). In the CCl4-CM(MSC) treated hepatocytes, interleukin-6 (IL-6) was increased on the first day of culture compared to control and CCl4 and was followed by upregulation of fibroblast-like-protein (FGL1) expression after 48 hrs. This was associated with a significant decrease of annexin V positive CCl4-CM(MSC) treated hepatocytes at day 3 post plating. Recombinant IL-6 was induced FGL1 expression in hepatocytes derived from CCl4-treated mice suggesting that CM(MSC), which is enriched also in microparticles, attenuates CCl4-induced early apoptosis in hepatocytes through activation of FGL1.

Potential prognostic value of intracellular cytokine detection by flow cytometry in pulmonary sarcoidosis.

In pulmonary sarcoidosis, differential cytokine production in the lungs could be related to variable prognosis of patients at different stages of disease. Twenty patients with pulmonary sarcoidosis (10 at radiographic stage I and 10 at stages II-IV), as well as 10 age-matched healthy volunteers participated in the study. A 4-colour flow cytometric technique was used to measure interferon-γ (IFN-γ), interleukin (IL)-2, tumour necrosis factor-α (TNF-α), IL-4, and IL-13 production in phorbol myristate acetate (PMA)/ionomycin-stimulated CD4+ and CD8+ T cells from bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) of patients, and PB of control subjects. CD4+ T cells from patients showed higher expression of IFN-γ in BALF than in PB. Significant correlations were observed between the percentages of BALF CD4+ and CD8+ T cells expressing intracellular IFN-γ, IL-2, and TNF-α. Stage I patients had lower percentages of IFN-γ-producing CD4+ and CD8+ T cells, as well as TNF-α-producing CD8+ T cells, in BALF (but not in PB) than stage II-IV patients. A decreased TH1 and TC1 response was demonstrated in BALF of patients at stage I of disease, which could explain their anticipated better prognosis.

The proteasome inhibitor bortezomib drastically affects inflammation and bone disease in adjuvant-induced arthritis in rats.

OBJECTIVE: To explore the effect of bortezomib in splenocytes and fibroblast-like synoviocytes (FLS) and its in vivo potency in a rat model of adjuvant-induced arthritis (AIA), which resembles human rheumatoid arthritis (RA). METHODS: AIA was induced with Freund's complete adjuvant. Splenocyte and FLS proliferation and apoptosis were measured by radioactivity incorporation and flow cytometry, respectively. The invasiveness of FLS from rats with AIA was tested in a Transwell system. The pattern of cytokine secretion was evaluated by cytometric bead array in splenocyte supernatants. Bortezomib was administered prophylactically or therapeutically, and arthritis was assessed clinically and histologically. Immunohistochemistry was performed for markers of inflammation and angiogenesis in joints. Hematologic and biochemical parameters were tested in peripheral blood (PB). Representative animals were examined by computed tomography (CT) scanning before and after bortezomib administration. The expression of Toll-like receptor 2 (TLR-2), TLR-3, and TLR-4 in PB and FLS was measured by real-time polymerase chain reaction, and alterations in specific cell populations in PB and spleen were determined by flow cytometry. RESULTS: In vitro, bortezomib exhibited significant inhibitory and proapoptotic activity in splenocytes and FLS from rats with AIA, altered the inflammatory cytokine pattern, and reduced the invasiveness of FLS from rats with AIA. In vivo, bortezomib significantly ameliorated disease severity. Remission was associated with improved histology and decreased expression of CD3, CD79a, CD11b, cyclooxygenase 1, and factor VIII in target tissues as well as down-regulation of TLR expression in PB and cultured FLS. CT scanning demonstrated a bone healing effect after treatment. CONCLUSION: Our findings suggest that bortezomib affects AIA in a pleiotropic manner and that this drug may be effective in RA.