Modulatory effect of pioglitazone on sperm parameters and oxidative stress, apoptotic and inflammatory biomarkers in testes of streptozotocin-induced diabetic rats

Background and Aims: Diabetes mellitus causes testicular damage by increasing oxidative stress and inflammation. In the present study, modulation of oxidative stress by pioglitazone, a synthetic ligand of peroxisome proliferator-activated receptor-Gamma, was examined in testis of streptozotocin-induced diabetic rats

Materials and Methods: Diabetes was induced by a single dose of streptozotocin [65 mg/kg, i.p.] injection in male Wistar rats. 32 adult male rats were divided into four groups [n=8]: control, diabetic, diabetic pioglitazone [1 mg/kg/day] and diabetic pioglitazone [10 mg/kg/day]. Rats were treated with pioglitazone for 5 weeks. Serum testosterone levels were estimated. Reproductive damage was evaluated by sperm parameters [viability, motility, morphology and count]. Oxidative stress markers were evaluated in testicular homogenate. Pro-inflammatory cytokines [tumor necrosis factor-Alpha and interleukin-1Beta] levels, expressions of inflammatory [inducible nitric oxide synthase and nuclear factor-Kappa B] and apoptotic markers [caspase-3] in testicular tissue were performed by enzyme-linked immunosorbent assay and western blot

Results: Pioglitazone treatment significantly increased sperm parameters [p<0.05]. No significant decrease in serum level of testosterone was observed in the pioglitazone treated mice [p>0.05]. Aside from reducing the elevated tissue nitric oxide and malondialdehyde levels, pioglitazone increased the reduced superoxide dismutase, catalas and total antioxidant capacity in testes compared to diabetic rats [p<0.05]. Pioglitazone reduced testicular inflammation by decreasing the expressions of inducible nitric oxide synthase, nuclear factor-Kappa B and pro-inflammatory cytokines levels and inhibited cell death by decreasing the expressions of caspase-3 [p<0.05]

Conclusions: Pioglitazone attenuated testicular damage in diabetic rats by decreasing oxidative stress, testicular inflammation and testicular damage

Comparison the effects of two monocyte isolation methods, plastic adherence and magnetic activated cell sorting methods, on phagocytic activity of generated dendritic cells

It is believed that monocyte isolation methods and maturation factors affect the phenotypic and functional characteristics of resultant dendritic cells [DC]. In the present study, we compared two monocyte isolation methods, including plastic adherence-dendritic cells [Adh-DC] and magnetic activated cell sorting- dendritic cells [MACS-DC], and their effects on phagocytic activity of differentiated immature DCs [immDCs]. In this experimental study, immDCs were generated from plastic adherence and MACS isolated monocytes in the presence of granulocyte-macrophage colony-stimulating factor [GM-CSF] and interleukin 4 [IL-4] in five days. The phagocytic activity of immDCs was analyzed by fluorescein isothiocyanate [FITC]-conjugated latex bead using flow cytometry. One way ANOVA test was used for statistical analysis of differences among experimental groups, including Adh-DC and MACS-DC groups. We found that phagocytic activity of Adh-DC was higher than MACS-DC, whereas the mean fluorescence intensity [MFI] of phagocytic cells was higher in MACS-DC [p<0.05]. We concluded that it would be important to consider phagocytosis parameters of generated DCs before making any decision about monocyte isolation methods to have fully functional DCs

Effect of cytosolic extract of alternaria alaternata fungus on monocyte-derived dendritic cell phagocytosis ability and T-lymphocyte proliferation in the presence of myelin basic protein

Multiple sclerosis [MS] is an autoimmune disease with impairment in function of CNS, meanwhile macrophages and dendritic cells [DC] can cause inflammation and damage to the myelin of nerve cells by releasing Reactive oxygen species [ROS] and other harmful substances when these cells get matured. We investigated the effect of Alternaria alternata [A. alternata] extract on phagocytic T cell stimulation activity of DC pulsed with Myelin Basic Protein [MBP] as a laboratory model of MS. Plastic adherent monocytes were cultured with granulocyte-macrophage colony stimulating factor [GM-CSF], interleukin-4 for converting these cells to MoDc [Monocyte-Derived Dendritic Cell], pulsed with MBP, matured in the presence of monocyte-conditioned medium [MCM] in control group and MCM+Alternaria. alternata extract in treatment groups. Phagocytic activity of DC was evaluated and T cell responses were investigated by MTT test. Phagocytic activity in treatment groups decreased significantly in compare with control group. Meanwhile, DC couldn't stimulate T cell proliferation. A. alternata extract decreased phagocytic activity of MoDc-pulsed with MBP and had no effect on T cell proliferation may provide a new strategy on immunotraphy of multiple sclerosis

Phenotypic and functional comparison between flask adherent and magnetic activated cell sorted monocytes derived dendritic cells

Generation of an effective dendritic cell [DC] based cancer vaccine depends on appropriate differentiation of monocytes in vitro. To compare the effects of monocyte separation methods, flask adherence [Flask-DC] and magnetic activated cell sorting [MACS-DC], on phenotypic and functional characteristics of resultant DCs. DCs from healthy volunteers were generated from plastic adherence and MACS isolated monocytes in the presence of GM-CSF and IL-4 as well as TNF-alpha and monocyte conditioned medium [MCM] in 7 day cultures. Mature DCs were then subjected to phenotypic analysis using anti-CD14, anti-CD83 and HLA-DR monoclonal antibodies. Functional and cytokine release assays were carried out using [[3]H] thymidine uptake test and commercially available ELISA kits for the determination of IL-12, IL-10, IFN-alpha and IL-4, respectively. We found that MACS-DCs were more homogenous and the yield and viability were fairly higher than Flask-DCs. MACS-DCs expressed higher levels of CD83 and HLA-DR as well as CD14 compared to the Flask-DCs. Induction of T cell proliferative responses were higher in Flask-DCs and also they elicited higher levels of IL- 12: IL-10 and IFN-alpha: IL-4 ratios in cytokine generation assays. MACS method was superior for mass production of viable homogenous and fully mature DCs but their cytokine profile had the potential to polarize the immune system toward Th2 type immune response

Effects of fibroblasts conditioned media on differentiation of programmable cells of monocytic origin to insulin-producing cells

The characteristic of stem cells in self renewal and differentiation to different types of cells has stimulated the interests for using stem cells as a starting material for generating insulin secreting cells. We've evaluated the differentiation potential of Programmable cells of monocytic origin [PCMOs] into insulin producing cells effected from the growth factors and fibroblasts conditioned media [FCM]. Peripheral blood monocytes of rat were cultured for 6 days in RPMI with 15% FBS, beta- mercaptoethanol, MCSF and interleukin-3. Then, these cells were incubated in differentiation media with HGF, EGF, Nicotinamide, 15% fibroblasts conditioned media and glucose for 15days. Morphological differences of cells were studied by invert microscope. In several stages, the amounts of insulin in supernatant of cells were measured by radioimmunoassay kit. Also productions of insulin from differentiated cells were studied with DTZ special staining. In response to MCSF and IL-3, monocytes dedifferentiated. These programmable cells of monocytic origin [PCMOs] were capable of differentiating into insulin producing cells in differentiation media. The morphology of differentiated cells was similar to Beta cells and the amount of insulin in supernatant of differentiated cells was much higher than PCMOs [P<0.05]. HGF, EGF, Nicotinamide and fibroblasts conditioned media are differentiation factors of PCMOs into insulin producing cells. According to the results insulin producing cells can be differentiated from programmable cells of monocytic origin in presence of fibroblasts conditioned media

Inducing maturation of monocyte-derived dendritic cells on human epithelial cell feeder layer

Nowadays, dendritic cells [DCs] have a special place in cancer treatment strategies and they have been used for tumor immunotherapy as they can induce immune response against tumor cells. Researchers have been trying to generate efficient dendritic cells in vitro; therefore, this research was done to generate them for use in research and tumor immunotherapy. This study took place at Urmia University in 2010-2011 years. In this study plastic adherent monocytes were incubated with granulocyte-macrophage colony stimulating factor [GM-CSF] and interleukin-4 [IL-4] for five days. Finally, fully matured and stable DCs were generated by 48 hours of incubation in a monocyte conditioned medium [MCM] containing tumor necrosis factor-alpha [TNF-alpha] and epithelial cells. Phenotypic and functional analysis were carried out by using anti-CD 14, anti-CD80, anti-CD86, and anti-CD83 monoclonal antibodies, and by determining their phagocytic activity, mixed lymphocyte reaction [MLR] and cytokine production, respectively. Dendritic cells were produced with high levels of surface molecule, i.e. of CD80, CD83, CD86, HLA-DR, expression and low levels of CD14 expression. Dendritic cells showed efficient phagocytosis and ability to stimulate T-lymphocytes. Moreover, dendritic cells could secrete high levels of interleukin-12 [IL-12] cytokine which was depictive of their full maturation. Measurement of the produced cytokines showed the generation of type-1 dendritic cells [DC1]. Our study showed that skin epithelial cells could induce maturation of monocyte-derived dendritic cells [DCs]. This feeder layer led to the production of efficient dendritic cells with the ability to be used for tumor immunotherapy

Functional recovery of sciatic nerve through inside-out vein graft in rats / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>Present study aimed at further comprehensive functional, histomorphometrical and immunohistochemical assessment of peripheral nerve regeneration using rat sciatic nerve transection model.</p><p><b>METHODS</b>The 10-mm rat sciatic nerve gap was created in rats. In control group nerve stumps were sutured to adjacent muscle and in treatment group the gap was bridged using an inside-out vein graft. In sham-operated group the nerve was manipulated and left intact. All animals underwent walking track analysis test 4, 8, and 12 weeks after surgery. Subsequently, muscle mass measurement was performed to assess reenervation, histological examination to observe the sciatic nerve regeneration morphologically and immunohistochemistry to detect Schwann cells using anti S-100. Results were analyzed using a factorial ANOVA with two between-subjects factors. Bonferroni test for pairwise comparisons was used to examine the effect of treatments.</p><p><b>RESULTS</b>Functional analysis of myelinated nerve fibers showed that nerve function improved significantly in the time course in treatment group. However, quantitative morphometrical analysis of myelinated nerve fibers showed that there was no significant difference between 8 and 12 weeks in treatment group. Muscle weight ratio was bigger and weight loss of the gastrocnemius muscle was ameliorated by inside-out vein grafting. The position of positive immunohistochemical reactions further implied that regenerated axons and Schwann cell-like cells existed after vein grafting was performed, and was accompanied by the process of myelination and structural recovery of regenerated nerves.</p><p><b>CONCLUSION</b>Functional analysis of peripheral nerve repair is far more reliable than quantitative morphometrical analysis.</p>

Adipose-derived stromal vascular fraction improves tendon healing in rabbits / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To evaluate the potential effects of uncultured adipose-derived stromal vascular fraction on tendon healing.</p><p><b>METHODS</b>Twenty five adult male New Zealand white rabbits weighing 2.5-3.0 kg were used. Five rabbits were used as donors of adipose tissue and the rest were divided into control and treatment groups. The injury model was completed by unilateral tenotomy through the middle one third of deep digital flexor tendon. Immediately after suture repair, either fresh stromal vascular fraction from enzymatic digestion of adipose tissue or placebo was intratendinously injected at tendon stumps in treatment and control groups, respectively. Immobilization with cast was continued for two weeks after surgery. Animals were sacrificed at eight weeks after surgery and tendons underwent histological, immunohistochemical, and mechanical evaluations. Statistical analyses of quantitative and qualitative data were assessed using one-way analysis of variance and Mann-Whitney U-test, respectively.</p><p><b>RESULTS</b>Histological evaluations demonstrated superior fibrillar linearity and continuity, and decreased vascularity in treatment group indicated improved organization and remodeling of neotendons. Immunohistochemistry de- monstrated a significant increase in collagen I expression in treatment group. Ultimate load and energy absorption capacity were both significantly increased in cell-treated repairs compared with controls.</p><p><b>CONCLUSION</b>The present study shows that intratendinous injection of uncultured adipose-derived stromal vascular fraction results in improved structural and mechanical properties of tendon repairs and it could be an effective modality for treating tendon injury.</p>

Generation of mature monocyte-derived dendritic cells in the presence of heparin and monocyte conditioned medium: phenotypic and functional comparison

Dendritic cells [DC] induce tumor or pathogen-specific T cell responses in humans. Several laboratories have developed culture systems, including maturation factors for human DC from peripheral blood monocytes. We comprehensively compared standard maturation stimulus, an autologous monocyte-conditioned medium [MCM], with heparin for their ability to promote uniformly mature DC that elicit T cell responses. A short [4-day] priming of plastic adherent monocytes with granulocyte-macrophage colony stimulating factor [GM-CSF] and IL-4 with or without heparin was followed by 48-hour incubation in MCM to generate fully mature and stable DC. Phenotypic and functional analyses were carried out using anti-CD14 and anti-CD83 monoclonal antibodies, and mixed lymphocyte reaction, respectively. We found that fully matured DC with a large amount of cytoplasm and copious dendritic projections were visible at the end of culturing period in the presence of MCM, heparin and MCM plus heparin. Thus, DC generated with these maturation factors are non-adherent and have typical satellite morphology. Flow cytometric analysis using anti-CD14 [monocyte marker] and anti-CD83 [mature DC marker] revealed that expression of CD14 decreased in MCM plus heparin-treated DC, and the expression of CD83 was increased when heparin and MCM used as a maturation factor. Functionally, MCM and MCM plus heparin-treated DC showed stronger mixed leukocyte reaction than heparin alone. These results support the use of the MCM with heparin as maturation factor that could result in functionally mature monocyte-derived DC in comparison to either MCM or heparin alone