Çetin Kocaefe

Functional Structure of Adipocytes Differentiated from Human Umbilical Cord Stroma‐Derived Stem Cells

It has been previously demonstrated that human umbilical cord stroma‐derived stem cells (HUCSCs) are competent to differentiate into adipocytes. However, controversies have arisen as to whether HUCSCs can become mature adipocytes or not, and to what extent these cells can be induced in adipogenic pathway. Here, we extensively analyzed their adipogenic potency with a structural and functional approach by determining lipid formation dynamics in concordance to adipocyte‐specific markers. During a 35‐day period, HUCSCs respond to adipogenic induction, at which point 88% of cells exhibited multilocular lipid granules (LGs) having a mean diameter of 3 μm in round‐shaped, F‐actin‐poor cells. Although the 1st week of induction did not generally display typical lipidogenic phenotypes, the degree of adipogenesis was dissected and confirmed by mRNA expressions of peroxisome proliferator‐activated receptor γ, C/EBP‐β, sterol regulatory element‐binding transcription factor 1, adipophilin, stearoyl‐CoA desaturase, glycerol 3‐phosphate dehydrogenase 1, LIPE, adiponectin, and leptin. All markers tested were found elevated in various amounts (3–70‐fold) around day 7 and reached a plateau after day 14 or 21 (5–335‐fold). Perilipin as a surface protein around the LGs was confined exclusively to the enlarging LGs. Conclusively, we propose that after the termination of proliferation, HUCSCs possess the biochemical and cellular machinery to successfully differentiate into maturing adipocytes under adipogenic conditions, and this feature will ultimately allow these fetus‐derived stem cells to be used for various therapeutic or esthetic purposes.

Histone Deacetylase Inhibition Activity and Molecular Docking of (E )‐Resveratrol: Its Therapeutic Potential in Spinal Muscular Atrophy

Spinal muscular atrophy is an autosomal recessive motor neuron disease that is caused by mutation of the survival motor neuron gene (SMN1) but all patients retain a nearly identical copy, SMN2. The disease severity correlates inversely with increased SMN2 copy. Currently, the most promising therapeutic strategy for spinal muscular atrophy is induction of SMN2 gene expression by histone deacetylase inhibitors. Polyphenols are known for protection against oxidative stress and degenerative diseases. Among our candidate prodrug library, we found that (E )‐resveratrol, which is one of the polyphenolic compounds, inhibited histone deacetylase activity in a concentration‐dependent manner and half‐maximum inhibition was observed at 650 μm. Molecular docking studies showed that (E )‐resveratrol had more favorable free energy of binding (−9.09 kcal/mol) and inhibition constant values (0.219 μm) than known inhibitors. To evaluate the effect of (E )‐resveratrol on SMN2 expression, spinal muscular atrophy type I fibroblast cell lines was treated with (E )‐resveratrol. The level of full‐length SMN2 mRNA and protein showed 1.2‐ to 1.3‐fold increase after treatment with 100 μm (E )‐resveratrol in only one cell line. These results indicate that response to (E )‐resveratrol treatment is variable among cell lines. This data demonstrate a novel activity of (E )‐resveratrol and that it could be a promising candidate for the treatment of spinal muscular atrophy.

Expression of matrix metalloproteinases in vasculitic neuropathy

The aim of this study was to investigate the expression pattern and cellular source of matrix metalloproteinases (MMP) in vasculitic neuropathy. Matrix metalloproteinases are endopeptidases degrading components of extracellular matrix proteins, and they have been implicated in the pathogenesis of inflammatory demyelination. They are induced by cytokines, secreted by inflammatory cells, and enhance T cell migration. Vasculitic neuropathy occurs as a component of systemic vasculitis or as an isolated angiitis of the peripheral nervous system, and T cell-mediated inflammation is detected in its pathogenesis. Nerve biopsy sections of eight patients with nonsystemic vasculitic neuropathy (NSVN) and four with systemic vasculitic neuropathy were examined for the presence of CD4+, CD8+, and CD68+ cells and immunohistochemically for MMP-2 and MMP-9 expression. Nerve biopsies of eight patients with noninflammatory neuropathy were used as a control group. Semiquantitative polymerase chain reaction analysis was performed to detect MMP-2 and MMP-9 mRNA. The predominant cells were CD8+ and CD68+ T cells. Expression of MMP-9, but not MMP-2, was increased in perivascular inflammatory infiltrate in nerve tissues of vasculitic neuropathy patients. This MMP-9 expression correlated positively with immunostaining of CD8+ T cells. No difference was detected between immunostaining patterns of nonsystemic and systemic vasculitic neuropathies with the antibodies used, except in MMP-9 immunostaining, which was found to be enhanced in NSVN group. Polymerase chain reaction analysis revealed elevated mRNA levels of MMP-9 and MMP-2 compared with controls, but this did not reach statistical significance. Our results imply a pathogenic role for MMP-9 secreted from CD8+ cells in vasculitic neuropathy.

Assessment of housekeeping genes for use in normalization of real time PCR in skeletal muscle with chronic degenerative changes

The real time PCR technique requires the normalization of the gene of interest to reference genes that are accepted to be ubiquitously expressed. The choice of the reference gene(s) needs to be determined by researchers according to the particular tissue or model of interest. The best normalization gene is not easy to decide, particularly if the investigated tissue displays architectural changes and structural reorganization. We have investigated the expression of four housekeeping genes that are widely used for the normalization purposes (TATA binding protein, beta actin, hypoxanthine-guanine phosphorybosyl transferase and glyceraldehyde-3-phosphate dehydrogenase) in a skeletal muscle degeneration model applied by the release of the Achilles tendon which leads to a time-course degeneration of the soleus and gastrocnemius muscles. This study indicates that the TATA binding protein and the beta actin gene to be the least effected in the course of degeneration induced by tenotomy in rat soleus and gastrocnemius muscle.

Periostin is temporally expressed as an extracellular matrix component in skeletal muscle regeneration and differentiation

The transcriptional events and pathways responsible for the acquisition of the myogenic phenotype during regeneration and myogenesis have been studied extensively. The modulators that shape the extracellular matrix in health and disease, however, are less understood. Understanding the components and pathways of this remodeling will aid the restoration of the architecture and prevent deterioration under pathological conditions such as fibrosis. Periostin, a matricellular protein associated with remodeling of the extracellular matrix and connective tissue architecture, is emerging in pathological conditions associated with fibrosis in adult life. Periostin also complicates fibrosis in degenerative skeletal muscle conditions such as dystrophies. This study primarily addresses the spatial and temporal involvement of periostin along skeletal muscle regeneration. In the acute skeletal muscle injury model that shows recovery without fibrosis, we show that periostin is rapidly disrupted along with the extensive necrosis and periostin mRNA is transiently upregulated during the myotube maturation. This expression is stringently initiated from the newly regenerating fibers. However, this observation is contrasting to a model that displays extensive fibrosis where upregulation of periostin expression is stable and confined to the fibrotic compartments of endomysial and perimysial space. In vitro myoblast differentiation further supports the claim that upregulation of periostin expression is a function of extracellular matrix remodeling during myofiber differentiation and maturation. We further seek to identify the expression kinetics of various periostin isoforms during the differentiation of rat and mouse myoblasts. Results depict that a singular periostin isoform dominated the rat muscle, contrasting to multiple isoforms in C2C12 myoblast cells. This study shows that periostin, a mediator with deleterious impact on conditions exhibiting fibrosis, is also produced and secreted by myoblasts and regenerating myofibers during architectural remodeling in the course of development and regeneration.

Interleukin-10 Gene Transfer: Prevention of Multiple Organ Injury in a Murine Cecal Ligation and Puncture Model of Sepsis

IntroductionThe aim of this study was to determine the effect of immunoregulatory cytokine interleukin-10 (IL-10) gene therapy on multiple organ injury (MOI) induced by a cecal ligation and puncture (CLP) model of sepsis in mice.MethodsMale Balb/c mice subjected to CLP were treated with either an hIL-10-carrying vector or an empty control vector. We assessed the degree of lung, liver, and kidney tissue destruction biochemically by measuring myeloperoxidase (MPO) and malondialdehyde (MDA) activity. Histologic assessments were based on neutrophil infiltration in lung and liver tissue. IL-10 protein expression was examined immunohistochemically, and ultrastructural changes in the liver were studied by transmission electron microscopy. We analyzed the expression of tumor necrosis factor-α (TNFα) mRNA by reverse transcription polymerase chain reaction 3, 8, and 24 hours after CLP in all organs.ResultsOrgan damage was significantly reduced by hIL-10 gene transfer, which was associated at the tissue level with reduced MPO activity in the liver, lung, and kidney and decreased leukocyte sequestration and MDA formation in the lung. The liver MDA was not significantly higher in the hIL-10 gene therapy group than in the controls and seemed not to be affected by hIL-10 gene transfer. The reduced portal tract neutrophilic infiltration and preserved ultrastructure of the hepatocytes also showed that tissue function was not impaired. The lung and kidney TNFα mRNA expression was suppressed markedly in the hIL-10 gene therapy group, but liver TNFα mRNA expression varied over time.ConclusionsThese findings showed that IL-10 gene therapy significantly attenuated sepsis-induced MOI.

Statin Potentiates Human Platelet eNOS Activity without Enhancing eNOS mRNA and Protein Levels

Background/Aims: Experimental studies suggest an enhanced endothelial and platelet nitric oxide (NO) generation after statin treatment, possibly due to increased endothelial NO synthase (eNOS) activity and protein levels. In parallel with experimental research, statins were shown to increase the forearm blood flow independently of serum cholesterol in humans. However, it was not possible to correlate blood flow changes with eNOS levels in these studies due to limitations in obtaining arterial samples. Hence, we investigated changes in eNOS activity, mRNA and protein levels after statin treatment in human platelets, which are readily accessible unlike arteries. Methods: In vitro bleeding times were measured in 22 patients by stimulating platelets with collagen-epinephrine or collagen-ADP. To assess platelet eNOS activity, the bleeding times were also determined after incubating platelets with L-arginine. The measurements were repeated following 14 days of pravastatin (40 mg/day) treatment. Platelet-rich plasma was collected before and after statin treatment to evaluate eNOS mRNA (semiquantitative RT-PCR) and protein levels (Western blotting). Results: The basal bleeding time was prolonged by 24 ± 3% (mean ± SE) when the samples were incubated with 500 µM of L-arginine. The NOS inhibitor L-N5-(I-iminoethyl)ornithine reversed this effect, suggesting that it was mediated by NO. After statin treatment, the NO-mediated prolongation of the bleeding time with 500 µM of L-arginine was significantly potentiated (to 44 ± 10%). Despite enhanced eNOS activity, there was no significant change in platelet eNOS mRNA and protein levels after statin treatment. Conclusion: These data demonstrate that platelet eNOS activity is potentiated after statin treatment in humans in parallel with experimental studies.

Four-Month-Old Infant With Focal Segmental Glomerulosclerosis and Mitochondrial DNA Deletion

Mitochondrial cytopathies are a group of heterogeneous disorders characterized by multisystem involvement. Renal involvement in mitochondrial cytopathies is usually manifested as tubular dysfunction owing to impaired energy metabolism; however, a few cases with glomerular changes have also been reported. Herein we report the case of a 4-month-old Turkish girl with a mitochondrial DNA deletion and focal segmental glomerulosclerosis. (J Child Neurol 2005;20:83—84).

Heightened CXCR4 and CXCL12 expression in NF1-associated neurofibromas

BackgroundNeurofibromatosis type 1 (NF1) is a common autosomal dominantly inherited disorder that affects both the skin and the nervous system. NF1 occurs due to the mutations in the NF1 gene. Neurofibromas are the most common Schwann cell-based tumors in NF1 patients, which are mainly categorized into dermal and plexiform neurofibromas. Studies on different tumor types demonstrate that CXCR4 expression increases in tumor tissues and is linked to metastasis and cancer progression.PurposeIn the present study, we aimed to analyze the gene expression of CXCR4, and its ligand CXCL12, in human neurofibromas.MethodsEight NF1 patients aged between 5 and 37 (2 males, 6 females) were selected. The patient group comprised 1 plexiform neurofibroma, 1 pheochromocytoma, and 6 dermal neurofibromas. Following pathological examination and diagnosis, tumors were co-stained with antibodies against Schwann cell marker S100 and target molecule CXCR4. CXCR4 expression in Schwann cell-based tumors was detected at the protein level. RNA isolated from the same tumors was used for RT-PCR-based studies to measure the quantitative expression of CXCR4 and CXCL12.ResultsCXCR4 gene expression increased 3- to 120-fold and CXCL12 gene expression increased 33- to 512-fold in all human Schwann cell-based tumors.ConclusionIn order to validate the role of CXCR4 and its relationship with CXCL12 in NF1, future studies should be performed with additional tumors and different tumor types.

PReS-FINAL-2086: In vitro investigation of the sustained therapeutic effect of etanercept loaded microspheres on human rheumatoid arthritis fibroblast-like synoviocytes

Anti-TNFα treatment has become an important part of our systemic treatment in chronic inflammatory arthritis. Local application to the joint produces transient clinical improvement since the drug quickly leaves the joint.