Anti-proliferating Cell Nuclear Antigen Research Articles

Ovarian changes and development of the branchial placenta occurring in Jenynsia lineata (Cyprinodontiformes, Anablepidae).

In viviparous teleosts, intraovarian gestation occurs intrafollicularly, as in poeciliids, or intraluminally, as in goodeids and anablepids. Furthermore, there are two different forms of embryonic nutrition: lecithotrophy and matrotrophy; depending on the species, these can be exclusive or coexist during gestation. In matrotrophic species, nutrients are transmitted from the mother to the embryo and are especially important in species with intraluminal gestation. Jenynsia lineata is a South American viviparous teleost with intraluminal gestation, characterized by eggs with scarce yolk, which is resorbed when embryos are 6 mm long, thus developing a branchial placenta. Using histological, histochemical, and immunohistochemical techniques, the present study describes the characteristics and changes of the ovarian mucosa in J. lineata during gestational and nongestational phases, and analyzes the embryonic pharyngeal epithelium in the branchial placenta. The ovaries of 30 adult female specimens were processed using histological techniques and stained with hematoxylin-eosin, Masson's trichrome, and Alcian Blue pH 2.5/periodic acid Schiff reagent. To detect cell proliferation, we used antiproliferating cell nuclear antigen antibody. In nonpregnant females, eosinophilic granular cells (EGCs) and lymphocytes were identified in the lamina propria of the tunica mucosa, and melanomacrophage centers (MMCs) and fibroblasts were identified adjacent to tissue debris in the ovarian folds'. In the cellular debris, an embryo in resorption was observed. In pregnant females, the ovarian mucosa has thin vascularization branches entering the opercular chamber of the embryos, in close contact with the forming gill processes, thereby establishing a branchial placenta. Active cell replacement was observed in these ovarian branches. The identification of fibroblasts, lymphocytes,EGCs, and MMCs adjacent to tissue debris could indicate that these cell types are involved in the embryonic resorption process. Considering the new data obtained in this study on the branchial placenta of J. lineata, we conclude that cell proliferation could be involved in the development of maternal-embryonic interaction.

Machine learning identifies clusters of longitudinal autoantibody profiles predictive of systemic lupus erythematosus disease outcomes

ObjectivesA novel longitudinal clustering technique was applied to comprehensive autoantibody data from a large, well-characterised, multinational inception systemic lupus erythematosus (SLE) cohort to determine profiles predictive of clinical outcomes.MethodsDemographic, clinical...

Disseminated Tuberculosis with Poncet’s Disease Masquerading as Systemic Lupus Erythematosus – A Case Report

Tuberculosis (TB) rheumatism (Poncet’s disease) is defined as a polyarthritis associated with extrapulmonary TB in which there is no evidence of bacteriological involvement of the joints. We report a case of 21-year-old female whose initial clinical and laboratory investigations were suggestive of inflammatory arthropathy. The case was complicated by weak anti-nuclear antibody positivity of anti-proliferating cell nuclear antigen (+) antibodies, suggestive of systemic lupus erythematosus. However, after clinical examination and investigations, the patient was diagnosed as a case of disseminated TB with Poncet’s disease. The patient was started on anti-tubercular treatment and showed improvement at the end of 1st month of treatment. The case highlights that Poncet’s disease should be considered as one of the differentials while dealing with cases of unexplained or prolonged arthralgia.

New strategy of using double-network hydrogel extravascular stent for preventing venous graft restenosis after coronary artery bypass grafting.

Effective therapies for the prevention of vein graft failure, which frequently occurs in coronary artery bypass grafting (CABG) due to intimal hyperplasia (IH), are still lacking. Here, we investigated the effects of the perivenous application of double-network hydrogel on vein grafts in carotid artery bypass grafting in a rabbit model. Healthy New Zealand white rabbits were randomized into the following groups: no graft, graft, or graft + Double-network hydrogel external stent (DNHES). The rabbits' carotid artery was bypassed via the jugular vein. Double-network hydrogel external stent was wrapped around the jugular graft after the anastomoses were completed. Blood flow parameters and tissue histology of the vein grafts were evaluated. Compared with the untreated vein grafts at 12 weeks after the surgery, the DNHES significantly improved graft flow, attenuated intimal and medial thickening, reduced the anti-proliferating cell nuclear antigen proliferation index of the vein grafts, decreased the mRNA and protein expression of Mitogen-Activated Protein Kinase (MAPK) and Transforming Growth Factor-β (TGF-β), and increased the mRNA and protein expression of endothelial Nitric Oxide Synthase (eNOS). The perivenous application of DNHES exerts beneficial effects on vein grafts, reduces the inflammatory response in carotid artery bypass grafting in a rabbit model, and appears to be a safe and promising strategy to prevent vein graft failure.

Comparative Analysis of Line Immunoassay with Immunofluorescence Assay for the Identification of Autoantibodies in Patients with Suspected Systemic Autoimmune Disorders: A Cross-sectional Study

Introduction: Systemic Autoimmune Diseases (SAD) are the diseases, including Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), sjogren’s syndrome, polymyositis, dermatomyositis, etc. where multiple organs are involved in the presence of a large variety of autoantibodies directed against one’s own immune system at various levels. They are one of the major causes of death and disability in all age groups and are characterized by the presence of Antinuclear Antibodies (ANA) in the blood of patients. Aim: To comparatively evaluate the utility of Line Immunoassay (LIA) with Indirect Immunofluorescence Assay (IIFA) for the detection and identification of ANA in suspected SAD patients. Materials and Methods: The present observational, crosssectional study was conducted on the collected samples of 560 clinically suspected SAD patients attending the Outpatient and Inpatient Departments (Medicine, Paediatrics, Orthopaedics, Dermatology and General Surgery) at Shri Ram Murti Smarak Institute of Medical Sciences, Bareilly, Uttar Pradesh, India (tertiary care hospital), from March 2016 to March 2020. Samples were subjected to ANA testing by IIFA and LIA. All variables anti-dsDNA, anti-Smd1, SSA/Ro and U1 SnRNP, SSA/ Ro, anti Proliferating Cell Nuclear Antigen (PCNA) and PO/RPP, SSB/La, anti CENP-B, Scl70, AMA-M2 and Mi-2 included in the study were presented in the form of percentages. Performance of LIA was reported in terms of sensitivity and specificity at 95% confidence interval. Data was analyzed using Statistical Package for the Social Sciences (SPSS) version 22.0 (Chicago, IL, USA) for windows. Results: Samples of both male (n=229) and female (n=331) patient of age group ranging from 0-90 years with mean age of 38.2 years were studied. Out of 560 samples, 197 (35.17%) patients were found to be ANA positive by IIFA. The LIA was positive in 175 (31.25%) patients. Of these 175 positive patients, 92 (52.57%) had a recognised autoimmune disease with the most common diagnosis was SLE, found in 38 (41.30%). In comparison to IIFA the sensitivity and specificity of LIA was found to be 82.13% and 98.62% respectively with 92.63% accuracy. Conclusion: The combined analysis of IIFA and LIA can be very useful for rapid identification of ANA which strengthen the initiation of treatment at the earliest to reduce disease morbidity and mortality.

Immunohistochemical expression of PCNA, STRO-1 and CD 44 in the healing of experimentally induced periapical lesions in rats.

The aim of the study was to determine the expression of cell proliferating marker, anti-proliferating cell nuclear antigen (anti-PCNA) and mesenchymal stem cell (MSC) markers (anti-STRO-1 and anti-CD44) in periapical periodontitis and their role in the healing of periapical lesion in periapical periodontitis. Ninety Sprague-Dawley male rats (100 g) were divided into 3 groups: Experimental group I (EG I: n = 30), experimental group II (EG II: n=30) and control group (CG: n = 30). Periapical lesions were experimentally developed by leaving the dental pulp of maxillary first molars mesial root open to oral environment for 4 weeks. Conventional root canal treatment was performed in EG II. Maxillary first molars along with alveolar bone were resected and fixed. The processed samples were stained with routine hematoxylin and eosin (H&E), and evaluated immunohistochemically using antibodies against anti-PCNA, anti-STRO-1, and anti-CD44 polyclonal antibodies. Data were analyzed using Chi-square test and a p-value of <0.05 was considered significant. Immunostaining of anti-PCNA showed 30%, 70% and 53.3% positive staining in CG, EG I, and EG II, respectively (p<0.001). Moreover, the CD44 staining was 20% in CG in contrast to 63.6% in EG I and 43.3 in EG II. STRO-1 staining in CG was 10%, 50% in the EG I and 36.6% in the EG II (p<0.001). Periapical inflammatory tissues expressed significant proliferative cell marker PCNA and mesenchymal stem cell markers STRO-1, and CD44. These findings further reaffirm the promising role of mesenchymal stem cells in the healing of periapical periodontitis.

Cell-Free Fat Extract Prevents Vaginal Atrophy in an Ovariectomized Model by Promoting Proliferation of Vaginal Keratinocytes and Neovascularization.

Most perimenopausal and postmenopausal women experience estrogen deficiency-induced vaginal atrophy. However, estrogen replacement therapy has contraindications and side effects, which makes it unsuitable for most women. Cell-free fat extract (CEFFE) has pro-proliferative and proangiogenic tissue regeneration activities. The purpose of this study was to evaluate the effect of topical application of CEFFE in the vagina and the effect of CEFFE on vaginal keratinocytes. Ovariectomized mice were treated with CEFFE via vaginal topical application for 2 weeks. The vaginal mucosal cell layer number, mucosal thickness, and vaginal collagen volume were determined by histologic analyses. Vaginal mucosa proliferation and lamina propria angiogenesis were evaluated with anti-proliferating cell nuclear antigen and anti-CD31 staining, respectively. For in vitro analysis, VK2/E6E7 cells were administered, increasing the CEFFE concentration. Cell proliferation and cell-cycle distribution were analyzed by Cell Counting Kit 8 assay and flow cytometry, respectively. Mucosal migration was evaluated with a wound-healing assay. The expression of Ki-67 and estrogen-related proteins was detected by western blotting. CEFFE-treated mice showed increased mucosal thickness and number of vaginal mucosal cell layers and reduced vaginal atrophy compared to ovariectomized mice. The number of proliferating cell nuclear antigen-positive cells and CD31-positive capillaries also increased. In addition, CEFFE promoted the proliferation and migration of VK2/E6E7 cells, upregulated the expression of Ki-67, and inhibited the expression of estrogen-related proteins and the PI3K/AKT pathway. CEFFE prevents estrogen deficiency-induced vaginal atrophy by promoting vaginal mucosal proliferation and increasing neovascularization, but not through the estrogen/estrogen receptor pathway, in an ovariectomized mouse model.

Preemptive Stem Cells Ameliorate Neuropathic Pain in Rats: A Central Component of Preemptive Analgesia.

The present study aims to investigate the efficacy of intravenously injected mesenchymal stem cells (MSCs) in treating neuropathic pain either before or after its induction by a chronic constriction injury (CCI) model. Rats were divided into four groups: control group, neuropathic group, and treated groups (pre and postinduction) with i.v. mononuclear cells (106 cell/mL). For these rats, experimental testing for both thermal and mechanical hyperalgesia was evaluated. The cerebral cortex of the rats was dissected, and immunohistochemical analysis using anti-proliferating cell nuclear antigen (PCNA), CD117, nestin, and glial fibrillary acidic protein was performed. Our results showed that a single injection of MSCs (either preemptive/or post-CCI) produced equipotent effects on allodynia, mechanical hyperalgesia, and thermal response. Immunohistochemical analysis showed that the stem cells have reached the cerebral cortex. The injected group with MSCs before CCI showing few stem cells expressed PCNA, CD117, and nestin in the cerebral cortex. The group injected with MSCs after CCI, showing numerous recently proliferated CD117-, nestin-, PCNA-positive stem cells in the cerebral cortex. In conclusion, our findings demonstrate that the most probable effect of i.v. stem cells is the central anti-inflammatory effect, which opens concerns about how stem cells circulating in systemic administration to reach the site of injury.

Erythropoietin Plays a Protective Role in Submandibular Gland Hypofunction Induced by Irradiation

This study aims to explore the radioprotective effects of recombinant human erythropoietin (rhEPO) on rats' submandibular gland hypofunction induced by irradiation (IR). Thirty rats were divided into 3 groups: 1) control group, 2) IR group, and 3) IR+rhEPO group. The IR group and IR+rhEPO group received a single dose of 15Grays (Gy) (0.98Gy/min), plus, the IR+rhEPO group also received subcutaneous administration of rhEPO at a dose of 3,000 IU/kg body weight 3days before irradiation and then repeated every 24hours for the first 2 weeks after irradiation. Immunohistochemistry analysis to erythropoietin receptor was performed to detect the levels of erythropoietin receptor in submandibular glands with or without radiation. Ninetydays after irradiation, the salivary flow rates were assessed, and the submandibular gland of every rat was subjected to hematoxylin and eosin staining and immunohistochemical staining with antiaquaporin 5 and anti-proliferating cell nuclear antigen antibodies. Apoptosis was examined by the terminal deoxynucleotidyl transferase biotin-dUDP nick end-labeling assay. In addition, to examine the protective role of rhEPO on human submandibular gland cells, the apoptotic and proliferation rate of cells under a radiation dose of 8Gy was detected. One-way analysis of variance was carried out to analyze the results of each group, and the P value was set at 0.05. Erythropoietin receptor was expressed in the submandibular glands at a low level under normal conditions but upregulated after irradiation. rhEPO administration remarkably alleviated gland atrophy, increased salivary flow rates with upregulation of aquaporin-5 compared with the IR group. In addition, fewer apoptotic cells and more proliferative cells were observed in the IR+rhEPO group compared with the IR group, both invivo and invitro. rhEPO administration may be a useful countermeasure to mitigate submandibular gland hypofunction after therapeutic radiation exposure.

The possible protective role of pumpkin seed oil in ameliorating tongue mucosal damage induced by orlistat in adult male albino rats: A light and scanning electron microscopic study

Background: Orlistat is an effective anti-obesity drug by reducing fat absorption. The oral cavity and its associated structures are target organs for many abnormalities that develop from orlistat. Pumpkin seed oil (PSO) is a valuable nutritional and health protective agent with prominent pharmacological properties.Aim: Evaluation of the possible protective role of pumpkin seed oil in ameliorating tongue mucosal damage induced by orlistat in adult male albino rats.Materials and Methods: Fifty adult male albino rats were used as a control group, an orlistat group and an orlistat-pumpkin group. Both orlistat (32mg/kg) and PSO (1.5ml/kg) were given orally once daily for four weeks. Specimens of the tongue were processed for light and scanning electron microscopic studies. Immunohistochemical study was performed using anti-proliferating cell nuclear antigen (PCNA) antibodies.Results: Specimens of the orlistat group showed an obvious distortion in the filiform and the fungiform papillae and in the covering epithelium of the tongue mucosa. There was a focal loss of the papillae and the epithelial ridges. The epithelial cells showed vacuolated cytoplasm and nuclear alteration. The lamina propria contained congested blood vessels and severe inflammatory cellular infiltration. There was a statistically significant decrease in the epithelial thickness, papillae height, papillae width and in the PCNA-immunoreaction of the epithelium. Scanning electron microscopy showed disfigurement and a focal atrophy of the filiform and the fungiform papillae. In contrast, minimal changes appeared in orlistat-pumpkin group that received PSO before orlistat.Conclusion: Orlistat induced significant structural alterations in the tongue mucosa of albino rats. PSO attenuated these effects and preserved the structure of the tongue mucosa.

Evaluation of the impact of platelet rich plasma and hyaluronic acid on ketamine-induced cystitis in albino rats : A histological and immunohistochemical study.

Background: ketamine induced cystitis (KIC) is commonly occurred in ketamine abusing individuals. It is associated with damage in the lining of the bladder and lower urinary tract symptoms. Platelets rich plasma (PRP) is attractive therapeutic line in regenerative medicine. Hyaluronic acid (HA) has many beneficial therapeutic effects in treatment of different types of cystitis.Objective: to evaluate the effect of PRP and HA on ketamine induced cystitis in adult male rats.Materials and Methods: Forty- four adult male rats were divided into five groups: Group I: control rats. Group II: Ketamine group. Group III: Recovery group. Group IV: Ketamine and PRP treated group. Group V: Ketamine and HA treated group. Specimens from the body of the urinary bladder were processed and examined histologically and immunohistochemically. Morphometrical studies and statistical analysis were conducted.Results: Groups II and III showed focal areas of degeneration and ulceration with apparently decreased thickness of the urothelium. Cellular infiltration and dilated congested blood vessels of the lamina propria were also observed. A significant increase of the collagen fibers in the lamina propria and between the muscle bundles. Nuclear factor kappa B (NF-κB) immunoreaction was significantly increased but there was a non-significant increase of antiproliferating cell nuclear antigen (PCNA) immunopositive nuclei among urothelium and a significant decrease of desmin, compared to group I. Groups III and IV showed improvement of most of the histological and immunohistochemical changes described before.Conclusion: The intravesical injection of PRP and HA provide a positive impact on treatment of KIC. HA therapy is a more efficient mean as it provides a better improvement in healing of urothelium and promotes more rapid tissue regeneration.

A study on hematological and immunological profile of patients with systemic Lupus Erythematosus

Background: Systemic lupus erythematosus (SLE) is a chronic, relapsing, inflammatory and often febrile multisystem disorder of the connective tissue, characterized principally by involvement of the skin, joints, kidneys, and serosal membranes. Hematological findings may be present at the onset of the disease or can develop during the course of the disease. Since blood and blood vessels together contain more diverse number of antigens than any other organ in the body, it is only natural to expect hematological manifestations more often than the other systems. Objectives: To study the haematological and immunological Profile of Patients with Systemic Lupus Erythematosis. Methodology: An Ambispective study was conducted at Kasturba Medical College and hospital, Manipal from January 2011 to December 2013. A total of One hundred twenty seven cases of SLE were classified as having SLE according to the revised American College of Rheumatology (ACR) classification criteria (1997). Results: Hematological manifestations were frequently seen in the cases studied, which included anemia, neutropenia, lymphopenia, thrombocytopenia, pancytopenia, increased Erythrocyte sedimentation rate (ESR), presence of spherocytes, DCT positivity etc.Anemia was the most common hematological abnormality observed in the patients accounting to 87.4% (111/127) of cases. Hemoglobin of patients varied from a minimum of 3.8g/dl up to 13.5g/dl (Median = 9.6±2.2). The erythrocyte sedimentation rate (ESR) was assessed in all except one.The PCNA (Anti proliferating cell nuclear antigen) antibodies were positive in 44% of the cases. Conclusion: Hematological abnormalities are the commonest among all other anifestations in SLE, and their treatment is challenging. Bone marrow examination should be considered in all cases of severe or persistent leukopenia or thrombocytopenia in SLE, to exclude dug-induced myelotoxicity in susceptible patients.

Role of bone marrow-derived stem cells versus insulin on filiform and fungiform papillae of diabetic albino rats (light, fluorescent and scanning electron microscopic study)

BackgroundDiabetes mellitus (DM) is a chronic metabolic disease characterized by high blood glucose levels. DM affects many body’s organs and caused by insulin production deficiency or by the ineffectiveness of the produced insulin. Administration of exogenous insulin is required for management of type I DM; however, it does not cure the disease. Bone marrow-mesenchymal stem cells (BM-MSCs) have been highlighted to offer a novel cell based approach for treatment of diabetes because of their anti-diabetic effect, direct differentiation into a variety of cell types, or release of paracrine factors. AimTo examine the effect of BM-MSCs versus insulin on true filiform and fungiform papillae of diabetic rats. Materials and methodsFifty six male Wistar albino rats weighing 200–250 g were equally divided into: Control group (Gp I): Rats did not receive any drug. Diabetic group (Gp II): Rats received a single intra-peritoneal injection of streptozotocin (40 mg/kg). BM-MSCs treated diabetic group (Gp III): After DM confirmation; rats received a single intravenous injection of BM-MSCs (million units) through tail vein. Insulin treated diabetic group (Gp IV): After DM confirmation; rats received a daily subcutaneous injection of insulin (5IU/kg). After four weeks, half of the tongue specimens were processed and stained by Hematoxyline & Eosin and Anti-proliferating cell nuclear antigen (Anti-PCNA) then examined by light microscope. Fluorescent microscope was used to detect homing of injected labeled BM-MSCs in rats’ filiform and fungiform papillae. While the other half were examined by scanning electron microscope. ResultsTrue filiform and fungiform papillae of Gp II showed significant histological and morphological alterations. In treated groups, Gp III and Gp IV, both papillae showed marked improvements, being more noticeable in Gp IV. There was a significant increase in the number of Anti-PCNA positive cells and a significant decrease in fasting blood glucose level in Gp III and Gp IV in comparison to Gp II. ConclusionsDM had degenerative effects on true filiform and fungiform papillae. Administration of BM-MSCs reduced the deleterious effects of DM on both papillae. Insulin injection caused more obvious improvements in both papillae of diabetic rats than BM-MSCs.

Effect of developmental lead exposure on neurogenesis and cortical neuronal morphology in Wistar rats

Lead (Pb) is a neurotoxic heavy metal that largely affects the developing nervous system. The present study examined the temporal effect of perinatal Pb exposure on neurogenesis and cortical neuronal morphology. Wistar pregnant rats were exposed to 0.5% lead acetate throughout pregnancy and to postnatal day (PD) 28. Offspring were grouped as gestational day (GD) 18 and 21 and PD 7, 14, 21, and 28 in both control and experimental groups. Brain sections were processed for immunohistological staining with anti-proliferating cell nuclear antigen (PCNA) or glial fibrillary acidic protein (GFAP). Brains from 14, 21, and 28 PDs pups were processed for Golgi-Cox stain. Pb exposure significantly increased PCNA-positive nuclei in the ventricular and subventricular zones of the lateral ventricle at 18 and 21 GDs. Postnatally, the Pb-treated groups showed a significant decrease in PCNA-positivity and neuron density compared to control. This reduction was associated with an increase in damaged or apoptotic cell profiles in the experimental groups. At PD 21, there was a significant increase in GFAP immunoreactivity in Pb-exposed groups compared with control. Furthermore, the total apical and basal dendritic length of pyramidal neurons in layer 2-3 of the Golgi-Cox stained sensorimotor cortex was comparable in both control and Pb-exposed groups. Spine density per 10 µm was significantly increased at PD 14 and 21 on the apical dendrites but not basal dendrites of Pb-treated groups. In conclusion, developmental Pb exposure in rats induces a toxic effect on neurogenesis and on cortical neurons, which may be related to cognitive disabilities observed in children exposed to lead.

Inhibitory effects of CP on the growth of human gastric adenocarcinoma BGC-823 tumours in nude mice.

ObjectiveTo investigate the potential antitumour effects of [2-(6-amino-purine-9-yl)-1-hydroxy-phosphine acyl ethyl] phosphonic acid (CP) against gastric adenocarcinoma.MethodsHuman BGC-823 xenotransplants were established in nude mice. Animals were randomly divided into control and CP groups, which were administered NaHCO3 vehicle alone or CP dissolved in NaHCO3 (200 µg/kg body weight) daily, respectively. Tumour volume was measured weekly for 6 weeks. Resected tumours were assayed for proliferative activity with anti-Ki-67 or anti-proliferating cell nuclear antigen (PCNA) antibodies. Cell apoptosis was examined using terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assays and with caspase-3 immunostaining. Proteins were measured by Western blotting.ResultsThere was a significant reduction in tumour volume and a reduced percentage of Ki-67-positive or PCNA-positive cells in the CP group compared with the control group. The percentage of TUNEL-positive or caspase 3-positive cells significantly increased following CP treatment compared with the control group. Tumours from the CP group had higher levels of phosphorylated-extracellular signal-regulated kinase (p-ERK) and phosphorylated-AKT (p-AKT) compared with control tumours.ConclusionCP treatment inhibited tumour growth and induced tumour cell apoptosis in a nude mouse model of BGC-823 gastric adenocarcinoma. Activation of the AKT and ERK signalling pathways may mediate this antitumour activity.

Effects of stem cells applications on oxidative stress and apoptosis during implantation

Objective: To investigate the effects of bone marrow derived mesenchymal stem cell (BMSC) application into the rat endometrium on oxidative stress, cell proliferation and apoptosis. Methods: The female rats selected in estrous cycle were divided into three groups (saline, media and BMSC group). The intrauterine and intraperitoneal injections were performed using the saline (200 μL), culture media (200 μL) and 1×106 BMSCs/200 μL culture media, and then they were mated with male rats. On the 7th day of the pregnancy, uterine samples were harvested and dyed with heamatoxylin-eosin histochemically, anti-endothelial nitric oxide synthase and anti-inducible nitric oxide synthase, and anti-proliferating cell nuclear antigen immunohistochemically, with terminal deoxynucleotidyl transferas dUTP nick end labeling for apoptosis. The stainings were evaluated by H-score and the results were analyzed using one-way ANOVA test statistically. Results: It was found that BMSCs increased the endometrial thickness, endometrial epithelium thickness and number of endometrial glands compared to control and sham groups. The intrauterine BMSC application decreased both anti-endothelial nitric oxide synthase and anti-inducible nitric oxide synthase immunoreactivities and the number of apoptotic cells compared to the intraperitoneal applications whereas the immunoreactivity of proliferating cell nuclear antigen was increased. Conclusions: In current study, we define that stem cells do not cause any structural damages. Also they change the distribution of oxidative stress and cell proliferation marker. These findings support the reliability of stem cells in clinical use in the case of infertility.

Effects of different rearing temperatures on muscle development and stress response in the early larval stages of Acipenser baerii.

The present study aims at investigating muscle development and stress response in early stages of Siberian sturgeon when subjected to different rearing temperatures, by analysing growth and development of the muscle and by assessing the stress response of yolk-sac larvae. Siberian sturgeon larvae were reared at 16°C, 19°C and 22°C until the yolk-sac was completely absorbed. Sampling timepoints were: hatching, schooling and complete yolk-sac absorption stage. Histometrical, histochemical and immunohistochemical analyses were performed in order to characterize muscle growth (total muscle area, TMA; slow muscle area, SMA; fast muscle area, FMA), development (anti-prolif erating cell nuclear antigen -PCNA or anticaspase) as well as stress conditions by specific stress biomarkers (heat shock protein 70 or 90, HSP70 or HSP90). Larvae subjected to the highest water temperature showed a faster yolk-sac absorption. Histometry revealed that both TMA and FMA were larger in the schooling stage at 19°C while no differences were observed in the SMA at any of the tested rearing temperatures. PCNA quantification revealed a significantly higher number of proliferating cells in the yolk-sac absorption phase at 22°C than at 16°C. HSP90 immunopositivity seems to be particularly evident at 19°C. HPS70 immunopositivity was never observed in the developing lateral muscle.

Ex vivo regenerative effects of a spring water.

Previous experiments by our group have indicated the regenerative effects of a spring water (Comano), which was possibly associated with the native non-pathogenic bacterial flora. The present study aimed to confirm these regenerative properties in a human ex vivo experimental model in the context of physiological wound healing. Human 6-mm punch skin biopsies harvested during plastic surgery sessions were injured in their central portion to induce skin loss and were cultured in either conventional medium (controls) or medium powder reconstituted with filtered Comano spring water (treated samples). At 24, 48 and 72 h the specimens were observed following staining with hematoxylin and eosin, Picrosirius Red, orcein and anti-proliferating cell nuclear antigen. Compared with the controls, the treated samples exhibited reduced overall cell infiltration, evidence of fibroblasts, stimulation of cell proliferation and collagen and elastic fiber regeneration. In the spring water, in addition to 12 resident non-pathogenic bacterial strains exhibiting favorable metabolic activities, more unknown non-pathogenic species are being identified by genomic analysis. In the present study, the efficacy of this ‘germ-free’, filtered spring water in wound regeneration was indicated. Thus, the Comano spring water microbiota should be acknowledged for its regenerative properties.

Knockdown of HIF-1α inhibits the proliferation and migration of outer root sheath cells exposed to hypoxia in vitro: An involvement of Shh pathway

AimsOuter root sheath (ORS) is a highly proliferative component of a hair follicle. This study is performed to investigate whether hypoxia-induced elevation of hypoxia-inducible factor (HIF)-1α, a transcriptional activator, contributes to the outgrowth of ORS cells in vitro. Main methodsHair follicles with intact ORS collected from 4-month old male American minks were cultured in normoxic or hypoxic condition (3% oxygen) for 7days. Primary ORS cells isolated from the mink hair follicles were exposed to hypoxia for 12, 24 or 48h, and their proliferation was analyzed with immunofluorescence assay using anti-proliferating cell nuclear antigen (PCNA) antibody. The migratory ability of ORS cells was detected via the transwell chamber. The endogenous HIF-1α was knocked down with its specific siRNA in ORS cells. Key findingsHypoxic exposure induced an elevation of HIF-1α in ex vivo cultured hair follicles. The mRNA and protein levels of sonic hedgehog (Shh), Shh receptor Patched 1, Smoothened and glioma-associated oncogene homologue 1 were upregulated. In vitro, hypoxia induced an increase in HIF-1α in ORS cells. Further, under hypoxic condition, the number of PCNA-positive cells was increased, and more cells migrated towards high serum media. Hypoxia-enhanced proliferation and migration of ORS cells were suppressed either by HIF-1α siRNA or by pharmacological inhibitors of Shh pathway, cyclopamine and GANT61. The activation of Shh pathway was attenuated in HIF-1α-silenced ORS cells under hypoxic condition. SignificanceOur work demonstrates a direct role of activated HIF-1/Shh biological axis in sustaining the development of ORS in vitro.

Hypothermia inhibits the proliferation of bone marrow-derived mesenchymal stem cells and increases tolerance to hypoxia by enhancing SUMOylation.

Hypothermia therapy has a positive effect on patients with severe brain injury. Recent studies have shown that mild hypothermia increases the survival of bone marrow-derived mesenchymal stem cells (BMSCs) in a hypoxic environment; however, the underlying mechanisms are not yet fully understood. Small ubiquitin-like modifiers (SUMOs) are sensitive to temperature stress reactions and are considered to exert a protective effect. In this study, we examined the protective effects of hypothermia on BMSCs in terms of SUMO protein modification. First, we found that mild hypothermia inhibited the proliferation and differentiation of BMSCs and increased cell tolerance to a hypoxic environment. Second, hypothermia significantly increased the levels of SUMO modification of multiple proteins in BMSCs. The knockdown of SUMO1/2/3 induced the rapid aging of the BMSCs, while the inhibition of the SUMO-conjugating enzyme, Ubc9, reduced cell proliferation and increased the proportion of BMSCs differentiating into nerve cells. Moreover, the tolerance of BMSCs to the hypoxic environment was significantly decreased. Lastly, we investigated 4 reported SUMO target proteins, anti-proliferating cell nuclear antigen, octamer-binding transcription factor 4, p53 and hypoxia-inducible factor-1α, to confirm that SUMO modification was indeed involved in maintaining the proliferation, inhibiting differentiation and enhancing the resistance of BMSCs against adverse conditions. Taken together, our results indicate that the SUMO pathway is involved in the response to hypothermic stress, and that SUMOylation may be an important protective mechanism against hypothermia for the survival of BMSCs under unfavorable conditions.