Comparison of colony formation of human spermatogonial stem cells (SSCs) with and without collagen.

OBJECTIVE: To investigate the effects of collagen and growth factors on in vitro proliferation of human spermatogonial stem cells obtained from patients with non-obstructive azoospermia. METHODS: The experimental cross-sectional study was conducted from February 2013 to April 2015 after obtaining approval from the ethics committee of Ahvaz Jundishapur University of Medical Sciences, Iran. Testicular sperm extractions of non-obstructive azoospermic patients were obtained from the Clinical Urology and Embryology, In Vitro Fertilization Department of Imam Khomeini Hospital. Spermatogonial stem cells and Sertoli cells, obtained from human testis biopsies by a two-step enzymatic digestion method, were purified using fluorescence- activated cell-sorting and daturastramonium-lectin, and were cultured separately. To investigate a more direct influential factor on colony formation, one control and two experimental groups were formed. Group 1 acted as the control in which spermatogonial stem cells were co-cultured with Sertoli cells alone. In group 2 they were co-cultured with Sertoli cells and growth factors such as leukaemia inhibitory factor, epidermal growth factor and glial cell-derived neurotrophic factor, and in group 3 with Sertoli cells along with growth factors in the presence of collagen-coated dishes. Number and diameter of the colonies were evaluated after 7 weeks. RESULTS: Specimens obtained related to 21 patients. Number and diameter of the colonies in group 3 (18±2.6 and 276.6±45.5) were significantly more than both groups 1 (3.5±1 and D1:81.6±12) and group 2(11±2.2 and 165.2±32.5) (p<0.05 each). Also, the number and diameter of colony in group 2 were significantly better than the control group (p<0.05).Expression profile of the VASA, promyelocytic leukaemia zinc-finger (PLZF), Octamer-binding transcription factor 4 (OCT4) and integrin a6 (INTGa6) were detected in all groups. Based on cytochemical findings, OCT4 was expressed in the colonies of all three groups. CONCLUSIONS: According to positive effects of collagen and growth factors on the colonisation of spermatogonial stem cells, it seems that using the cells may lead to better colonisation of this type of stem cells.

Targeting integrin α6 stimulates curative-type bone metastasis lesions in a xenograft model.

Laminin-binding integrin receptors are key mediators of epithelial cell migration and tumor metastasis. Recent studies have demonstrated a role for the α6 integrin (ITGA6/CD49f) in maintaining stem cell compartments within normal bone marrow and in residency of tumors metastatic to bone. In this study, we tested a function-blocking antibody specific for ITGA6, called J8H, to determine if preexisting cancer lesions in bone could be slowed and/or animal survival improved. Human prostate tumors were established by intracardiac injection into male SCID mice and treatment with J8H antibody was initiated after 1 week. Tumor progression was monitored by micro-computed tomography (CT) imaging of skeletal lesions. Animals that received weekly injections of the anti-ITGA6 antibody showed radiographic progression in only 40% of osseous tumors (femur or tibia), compared with control animals, where 80% of the lesions (femur or tibia) showed progression at 5 weeks. Kaplan-Meier survival analysis demonstrated a significant survival advantage for J8H-treated animals. Unexpectedly, CT image analysis revealed an increased proportion of bone lesions displaying a sclerotic rim of new bone formation, encapsulating the arrested lytic lesions in animals that received the anti-ITGA6 antibody treatment. Histopathology of the sclerotic lesions demonstrated well-circumscribed tumor within bone, surrounded by fibrosis. These data suggest that systemic targeting of the ITGA6-dependent function of established tumors in bone may offer a noncytotoxic approach to arrest the osteolytic progression of metastatic prostate cancer, thereby providing a new therapeutic strategy for advanced disease.

The effect of butyric acid on adhesion molecule expression by human gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Short-chain fatty acids, such as butyric acid, are detected in periodontal pockets and are thought to be involved in the initiation and progression of periodontal disease. In the present study, we examined the effects of butyric acid on adhesion molecule expression by human gingival epithelial cells. MATERIAL AND METHODS: The human gingival carcinoma cell line, Ca9-22, was cultured in media that contained different concentrations of butyric acid. RESULTS: Cell numbers were significantly decreased in a dose-dependent manner by butyric acid at concentrations of > or = 0.2 mM. The expression of intercellular adhesion molecule-1 mRNA was significantly increased 6 h after stimulation. By contrast, the expression levels of integrins alpha 6 and beta 4 were decreased. Similar results were obtained by flow cytometry. CONCLUSION: The results of the present study indicate that butyric acid alters the expression of adhesion molecules by Ca9-22 cells. The elucidation of the mechanism of action of butyric acid on the periodontium may help to clarify several aspects of the onset and progression of periodontal disease.

Blockade of alpha6 integrin inhibits IL-1beta- but not TNF-alpha-induced neutrophil transmigration in vivo.

In vitro and in vivo evidence supports a functional role for the integrin alpha6beta1 in neutrophil migration through the perivascular basement membrane, a response that in vivo appears to be associated with platelet/endothelial cell adhesion molecule-1 (PECAM-1)-mediated up-regulation of alpha6beta1 on the cell surface of transmigrating leukocytes. As the involvement of PECAM-1 in leukocyte migration is cytokine-specific, the aim of the present study was to investigate whether alpha6beta1 exhibited a similar profile of stimulus specificity in this context. The cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) were used to elicit neutrophil migration in two murine models of inflammation, migration through cremasteric venules, as observed by intravital microscopy, and migration into the peritoneal cavity. The role of alpha6beta1 was investigated using an alpha6 integrin-blocking monoclonal antibody GoH3. In both models, GoH3 significantly inhibited neutrophil transmigration induced by IL-1beta but not TNF-alpha. This cytokine-specific role of alpha6 integrin was associated with enhanced cell-surface expression of alpha6beta1 on transmigrated neutrophils (as compared with blood cells) in response to IL-1beta but not TNF-alpha. Using lipopolysaccharide as an inflammatory stimulus in the cremaster muscle model, the study also provides evidence for the involvement of alpha6 integrin in leukocyte transmigration as mediated by endogenously generated IL-1beta. Collectively, the findings demonstrate that alpha6beta1 blockade inhibits neutrophil migration induced by exogenous and endogenous IL-1beta but not TNF-alpha, observations that are associated with increased expression of the integrin on transmigrated leukocytes.

Mechanisms of poly-N-acetyl glucosamine polymer-mediated hemostasis: platelet interactions.

BACKGROUND: Investigations were performed to determine whether poly-N-acetyl glucosamine (p-GlcNAc) induces hemostasis by the activation of platelets. METHODS: Platelets were isolated from human blood, fixed in the presence poly-N-acetyl glucosamine fibers, and visualized with scanning electron microscopy. Platelet activation surface markers were measured by fluorescence multiphoton microscopy. Platelet aggregation in the presence of p-GlcNAc fibers and integrin receptor blockers was measured. RESULTS: Scanning electron microscopy indicated that contact of platelets with poly-N-acetyl glucosamine fibers resulted in platelet activation. Fluorescent microscopy showed that contact of platelets with the marine polymer increased intracellular levels of free calcium and resulted in surface exposure of platelet phosphatidylserine, P selectin, and the alphaIIbbeta3 integrin. Antibody inhibitors of the platelet alphaIIbbeta3 integrin inhibited p-GlcNAc to stimulate fibrin polymerization. CONCLUSION: Poly-N-acetyl glucosamine fiber material promotes hemostasis by the activation of platelets.

Effect of fucoidan on fibroblast growth factor-2-induced angiogenesis in vitro.

Fucoidans are sulfated polysaccharides extracted from brown marine algae. A purified fucoidan fraction exhibits the same venous antithrombotic activity as heparin in rabbits, but with a lower anticoagulant effect. Because of its heparin-like structure, we postulated that fucoidan might modulate heparin-binding angiogenic growth factor activity. We thus studied its effect, at antithrombotic concentrations, on fibroblast growth factor (FGF)-2-induced proliferation and differentiation of human umbilical vein endothelial cells. The fucoidan effect on endothelial cell differentiation was evaluated by studying the expression of surface proteins (i.e. integrin, adhesion molecule) known to be modulated by FGF-2 and involved in angiogenesis, and by quantifying closed areas delimited by vascular tubes formed on reconstituted basement membrane. Fucoidan had no modulatory effect on the mitogenic activity of FGF-2, but significantly increased tubular structure density induced by FGF-2. Fucoidan alone increased alpha(6) integrin subunit expression with only partially organized tubular structure. In the presence of FGF-2, fucoidan enhanced alpha(6), beta(1) and PECAM-1 and inhibited alpha(v)beta(3) integrin expression. Heparin had no effect in these systems. The most striking effect of fucoidan was observed on alpha(6) expression and tube formation was abolished by monoclonal anti-alpha(6) antibodies. Fucoidan plus FGF-2 effect on alpha(6) expression was markedly decreased by monoclonal anti-FGF-2 antibodies, indicating that fucoidan acts mainly via FGF-2. These results show that, at antithrombotic concentrations, contrary to heparin, fucoidan can enhance vascular tube formation induced by FGF-2 with a modulation of the expression of surface proteins (mainly alpha(6)) involved in angiogenesis.