LncRNA ANRIL Promotes Glucose Metabolism and Proliferation of Colon Cancer in a High-Glucose Environment and is Associated with Worse Outcome in Diabetic Colon Cancer Patients.

BACKGROUND: The potential involvement of type 2 diabetes mellitus (T2DM) as a risk factor for colon cancer (CC) has been previously reported. Epigenetic changes, such as deregulation of long non-coding RNA (lncRNA) and microRNA (miR), have been linked to the advancement of CC; however, the effects of high glucose levels on their deregulation and, in turn, colon cancer remain unexplored. METHODS: Fifty patients had a dual diagnosis of CC and T2DM, and 60 patients with CC without diabetes mellitus were included in the study. qRT-PCR was used to examine the expression of lncRNA ANRIL and miR-186-5p in tissue samples. ANRIL, miR-186-5p, and their downstream target genes HIF-1α, PFK, HK, Bcl-2, and Bax were also determined in CC cell lines under various glucose conditions. Glucose uptake, lactate production and cells proliferation were estimated in CC cell lines. RESULTS: A significant upregulation of ANRIL expression levels (p<0.001) and a significant downregulation of miR-186-5p expression (p<0.001) in diabetic colon cancer specimens compared to those in non-diabetic colon cancer group were observed. MiR-186-5p expression levels were inversely correlated with ANRIL expression levels, blood glucose levels and HbA1c%. Concerning in vitro model, a significant upregulation of ANRIL, downregulation of miR-186-5p, upregulation of HIF-1α, glycolytic enzymes and activation of antiapoptotic pathway was detected in higher glucose concentrations than lower one. There was a significant increase of glucose uptake, lactate accumulation and proliferation of the Caco2 and SW620 cell lines in a dose dependent manner of glucose concentrations. Moreover, a significant positive correlation between glucose uptake and ANRIL expression was shown. CONCLUSIONS: A high-glucose environment can increase the tumor-promoting effect of ANRIL. ANRIL can promote glucose metabolism and colon cancer proliferation by downregulating miR-186-5p with subsequent upregulation of glycolysis enzymes expression and inhibition of apoptosis.

Hepatitis B Immunization Status in Children with Chronic Kidney Disease: Experience at a Single Center, Egypt.

Background: Children with chronic kidney disease (CKD), particularly those who require hemodialysis (HD), are at high risk of hepatitis B virus (HBV) infection. The HBV vaccine non-/hypo-response rate among HD children remains high, and it is critical to investigate the influencing factors and their linkages. The aim of this study was to identify the pattern of HB vaccination response in HD children and to analyze the interference of various clinical and biomedical factors with the immunological response to HB vaccination. Methods: This cross-sectional study included 74 children on maintenance hemodialysis, aged between 3 and 18 years. These children were subjected to complete clinical examination and laboratory investigations. Results: Out of a total of 74 children with HD, 25 (33.8%) were positive for the HCV antibody. Regarding the immunological response to hepatitis B vaccine, 70% were non-/hypo-responders (≤100 IU/mL) and only 30% mounted a high-level response (more than 100 IU/mL). There was a significant relation between non-/hypo-response and sex, dialysis duration, and HCV infection. Being on dialysis for more than 5 years and being HCV Ab-positive were independent variables for non-/hypo-response to HB vaccine. Conclusions: Children with CKD on regular HD have poor seroconversion rates in response to the HBV vaccine, which were influenced by dialysis duration and HCV infection.

Impact of cytokines genes polymorphisms and their serum levels on childhood asthma in Egyptian population.

BACKGROUND: Asthma is chronic immune-mediated airway inflammation, and it is affected by a complex network of interacting cytokines. To date, the exact role of each cytokine and its genetic polymorphisms in childhood asthma development and its severity has remained poorly understood. The purpose of this study was to explore potential roles of four cytokine genes polymorphism and serum levels l [(T helper-2 (Th2) cytokine); Interleukin-4 (IL-4) 590, (Th3 cytokine); and transforming growth factor ß1 (TGF-ß1) 509T; (Th17) including tumor necrosis factor-alpha (TNF-α), and IL17A rs8193036] in childhood asthma risk and control in Egyptian children, for the 1st time. MATERIALS AND METHODS: This case-control study included two children subgroups; Group1 included 216 non-asthmatic controls and (Group 2) 216 cases diagnosed with asthma (clinically and spirometry-based) were classified as controlled, partly controlled, and uncontrolled. Polymorphisms of TGF-ß1-509, IL-4 590, and TNF-α-308 genes were detected using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). IL-17 was genotyped using tetra-primer amplification refractory mutation system polymerase chain reaction (ARMS-PCR). Serum cytokines levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Serum total IgE, TGF-ß1, IL4, TNF-α, and IL17A levels were significantly higher in asthmatic compared to controls. Also, significant increases in serum total IgE, IL-4, TGF-ß1, and TNF-α levels are combined with poor asthma control, while no significant IL17A changes. There were significant changes of IL-4-590, TNF-α-308, and IL17A genotypes and allele distributions between asthmatic and controls groups as well as different asthma control levels; while no impact of TGF-ß1 SNP on asthma risk and control level. Four cytokines SNPs affected their serum levels among asthmatic patients. CONCLUSION: There are impacts of cytokine gene polymorphisms (IL-4-590, TNF-α-308, and IL17A); but not TGF-ß1 on asthma susceptibility and poor asthma control in Egyptian children.

Spinal cord-wide structural disruption in type 2 diabetes rescued by exenatide "a glucagon-like peptide-1 analogue" via down-regulating inflammatory, oxidative stress and apoptotic signaling pathways.

The mechanisms of spinal cord-wide structural and functional disruption in diabetic patients remain elusive. This study evaluated histopathological alterations of the spinal cord cytoarchitecture in T2DM model of rats and assessed the potential ameliorating effect of exenatide "a potent GLP-1 analogue". Thirty male rats were allocated into three groups; I (control), II (Diabetic): T2DM was induced by high fat diet for 8 weeks followed by a single I.P injection of STZ (25 mg/kg BW) and III (Diabetic/Exenatide): T2DM rats injected with exenatide (10 µg/Kg, S.C. twice daily for 2 weeks). Neurobehavioral sensory and motor tests were carried out and glycemic control biomarkers and indices of insulin resistance and sensitivity were measured. In addition, the spinal cord was processed for histological and immunohistochemical studies besides assessing its tissue homogenate levels of pro-inflammatory/anti-inflamatory cytokines and oxidant/antioxidant biomarkers. Moreover, RT-qPCR was performed to measure the expression of proapoptotic/antiapoptotic and neurotrophic genes. The diabetic rats exhibited thermal hyperalgesia, mechanical allodynia and decreased locomotor activity along with increased serum glucose, insulin, HbA1c, HOMA-IR while, quantitative insulin sensitivity check index (QUICKI) was decreased. Also, IL-1ß NF-kB, MDA increased while IL-10, SOD activity and ß-endorphin decreased in the spinal tissue. Up regulation of caspase-3 and down regulation of Bcl-2, nerve growth factor (NGF) and glial cell-derived neurotrophic (GDNF) in diabetic rats. Also, they exhibited histopathological changes and increased CD68 positive microglia and Bax immunoreactivity in the spinal cord. Subsequent to exenatide treatment, most biomolecular, structural and functional impairments of the spinal cord were restored in the diabetic rats. In conclusion, the neuro-modulating effect of exenatide against diabetic-induced spinal cord affection warrants the concern about its therapeutic relevance in confronting the devastating diabetic neuropathic complications.

Osteoprotegerin expression and serum values in obese women with type 2 diabetes mellitus.

BACKGROUND: Obesity and diabetes prevalence are increasing worldwide. We aimed to detect the possible association of osteoprotegerin (OPG) gene expression with visceral adiposity indices and cardiometabolic risk factors among obese women. METHODS AND RESULTS: The study enrolled 150 controls and 150 obese cases subdivided into two subgroups non-diabetic (n = 70) and 80 patients with type 2 diabetes mellitus (T2DM). Circulating OPG gene expression levels were figured out by real time PCR (Polymerase Chain Reaction). Serum OPG levels were assessed by Enzyme Linked Immunosorbent Assay. Our results explored that OPG serum levels were lower in the obese women compared to control group (p < 0.001) and obese diabetics had higher serum levels of OPG in comparison to obese non-diabetic patients (p < 0.001). Expression levels of OPG were higher in obese women than controls (p < 0.001). Moreover, the blood expression levels of OPG gene were higher in diabetic obese patients than non-diabetics. We found positive correlations between parameters of metabolic syndrome and obesity indices. After adjustment of the traditional risk factors, stepwise linear regression analysis test revealed that OPG expression levels were independently correlated with glycated hemoglobin, high-density lipoprotein-cholesterol, and waist-to-hip ratio. CONCLUSIONS: OPG mRNA levels were associated with surrogate markers of insulin resistance in Egyptian obese women.

Urinary exosomal microRNA-96-5p and microRNA-183-5p expression as potential biomarkers of bladder cancer.

Because of low sensitivity and specificity of the currently available urine biomarkers of bladder cancer (BC) detection and painful cystoscopy procedure. Our study aimed to evaluate expression of urinary exosomal miR-96-5p and miR-183-5p as probable non-invasive and accurate biomarkers for the diagnosis and follow up of BC. Using quantitative real-time polymerase chain reaction; expression of exosomal microRNA (miR)-96-5p and miR- 183-5p in the urine samples of 51 patients with BC, 21 patients with benign urinary bladder lesions and in 24 normal individuals as control group was done. Our study results showed higher expressions of both miR-96-5p and miR-183-5p in urine of BC patients in comparison with control group (P < 0.001 for each). Receiver operating characteristic curve (ROC) analysis showed that each microRNA had good sensitivity and specificity to differentiate BC from non-BC patients miR-96-5p 80.4% and 91.8% and miR-183-5p 78.4% and 81.6% respectively compared to cytology (37.3% and 100%). In addition, it was obvious that the sensitivity of combined miR-96-5p and miR-183-5p for the diagnosis of BC reached 88.2%% and specificity reached 87.8%, which were higher than each one alone. We also found that expression of miR-96-5p and miR-183-5p with high grade, and pathological stage was significantly increased. After surgery, collected urine samples showed significantly lower expression of miR-96-5p-: P < 0.001; and miR-183-5p: P = 0.002. In conclusion, urine miR-96-5p and miR-183-5p are promising tumor biomarkers of BC diagnosis; particularly, when they combined with each other or with urinary cytology.

Promoter methylation and expression of intercellular adhesion molecule 1 gene in blood of autoimmune thyroiditis patients.

Growing data supported that epigenetic modifications, including altered DNA methylation have a potential role in the pathogenesis of autoimmune thyroid diseases (AITD). In the present study we aimed to investigate the methylation status of ICAM-1 gene promoter in patients with AITD. Forty patients with Graves' disease (GD), 40 patients with Hashimoto thyroiditis (HT) and 40 normal controls were included. DNA extraction from blood samples was done to analyze the ICAM-1 methylation status using methylation-specific PCR method. RNA was also extracted to determine the ICAM-1 expression values by real time PCR. We found that the differences in the frequency of ICAM-1 methylation status between GD or HT and healthy individuals were statistically significant (p = 0.04, 0.018 respectively) whereas there was no significant difference between GD and HT patients (p > 0.05). There was a correlation between decreased ICAM-1 methylation and exophthalmos (p = 0.01) and the high TSI level (p < 0.002) in GD patients. However, there was no correlation between ICAM-1 methylation and other clinicopathological features or other laboratory parameters in GD or HT. Furthermore, ICAM-1 mRNA expression showed significant up-regulation in ICAM-1 un-methylated samples compared to methylated samples in both GD and HT patients (p < 0.001 for each). Results provided the evidence of association of the hypo-methylation status of ICAM-1 gene promoter with GD and HT patients. In addition, DNA methylation may have a critical role in the ICAM-1 expression regulation of AITD patients.

Circulating long non-coding RNA GAS5 and SOX2OT as potential biomarkers for diagnosis and prognosis of non-small cell lung cancer.

Early diagnosis of non-small cell lung cancer (NSCLC) is essential for patient treatment and prognosis. Long noncoding RNA (lncRNA) have potential roles in tumor initiation and differentiation. The objective of this study was to investigate whether the circulating lncRNA, growth arrest-specific transcript 5 (GAS5) and SOX2 overlapping transcript (SOX2OT), could be used as noninvasive biomarkers for NSCLC diagnosis. Moreover, we aimed at evaluating the association between lncRNA and the clinicopathological features of NSCLC in order to predict the cancer prognosis. The results showed significant downregulation of GAS5 expression and upregulation of SOX2OT in NSCLC patients compared with controls (P < 0.001). Furthermore, the expression level of GAS5 was declined in stage IV of NSCLC, but SOX2OT expression was increased sharply in stages III and IV. The expression levels of lncRNAs were used to distinguish NSCLC patients from control with an area under curve of 0.81 (sensitivity 82.5% and specificity 80%) for GAS5 and 0.73 (sensitivity 76.3% and specificity 78.6%) for SOX2OT. The combination of GAS5 and SOX2OT showed differentiation NSCLC patients from controls with increased sensitivity (83.8) and specificity (81.4). In conclusion, the newly developed diagnostic panel involving of circulating GAS5 and SOX2OT could be perfect biomarker for diagnosis and prognosis of NSCLC.

Beneficial effects of metformin on muscle atrophy induced by obesity in rats.

AIM: A growing interest to understand the signaling pathways mediating obesity-induced muscle atrophy is given. Metformin (Met) was reported to possess positive effects on preventing muscle damage and promoting muscle mass maintenance. The aim of the present study to investigate pathways involved in Met effect on obesity induced muscle atrophy. METHODS: Thirty adult male albino rats were assigned into two groups: normal chew diet fed group as control group (C; n = 10) and high-fat-diet (HFD) fed group ( n = 20). After 16 weeks, the HFD-fed animals were subdivided into two groups; HFD group ( n = 10) and HFD fed treated with oral Met (320 mg/day) treatment (Met, n = 10) for 4 weeks. At the end of the experiment; final body weight, visceral fat weight, fasting blood glucose, insulin, lactate, total cholesterol, triglycerides were measured and calculated homeostatic model assessment insulin resistant (HOMA-IR) for all groups. Soleus muscle weight, histopathlogical examination and expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), forkhead box O3 (FoxO3), atrogin-1/MAFbx, and muscle RING finger 1 (MuRF-1) were performed. RESULTS: HFD-fed animals showed significant increase in final body weight, visceral fat mass, fasting blood glucose, insulin, calculated HOMA-IR, lactate, total cholesterol and triglycerides with significant decrease in soleus muscle weight, PGC-1α and significant increase in FoxO3, atrogin-1/MAFbx, and MuRF-1 expression. Also, there was significant decrease in fiber diameter, myosin heavy chain (MHC) I content while collagen content and myosin heavy chain IIa were increased compared with control group. Met-treated group showed a significant decrease in the measured parameters compared with the HFD group. It also restored the gene expression, morphometric measures and MHC composition toward normal. CONCLUSION: The current study is the first to provide evidence that Met could ameliorate muscle atrophy in high-fat diet induced obesity and this effect may be in part due to regulation of PGC-1α-FoxO3 pathway.

Fluoxetine pretreatment enhances neurogenic, angiogenic and immunomodulatory effects of MSCs on experimentally induced diabetic neuropathy.

Being one of the most debilitating complications among diabetic patients, diabetic polyneuropathy (DPN) is a paramount point of continuous research. Stem cell therapies have shown promising results. However, limited cell survival and paracrine activities hinder its transfer from bench to bedside. We designed this study to evaluate fluoxetine-pretreatment technique of mesenchymal stem cells (MSCs) as an approach to enhance their paracrine and immunomodulatory properties in DPN. Effects of fluoxetine treatment of MSCs were tested in vitro. Forty-two adult Wistar male albino rats were utilized, further subdivided into control, diabetic, MSC-treated and fluoxetine-pretreated MSC groups. Sciatic nerve sections were prepared for light and electron microscope examination and immunohistochemical detection of neurofilament (NF) protein. Also, we assessed in vitro survival and paracrine properties of fluoxetine-pretreated MSCs. Real time PCR of BDNF, VEGF, IL-1ß, and IL-10 expression in tissue homogenate was performed. Our results showed restoration of normal neuronal histomorphology and ultrastructure, moreover, immunohistochemical expression of anti-neurofilament protein was significantly elevated in MSC-treated groups compared to the diabetic one. Fluoxetine enhanced the MSC survival and their paracrine properties of MSCs in vitro. Furthermore, the fluoxetine-pretreated MSC group revealed a significant elevation of mRNA expression of BDNF (neurotrophic factor) and VEGF (angiogenic factor), denoting ameliorated MSC paracrine properties. Similarly, improved immunomodulatory functions were evident by a significant reduction of interleukin-1ß mRNA expression (pro-inflammatory) and a reciprocal significant increase of interleukin-10 (anti-inflammatory). We concluded that fluoxetine-pretreatment of MSCs boosts their survival, paracrine, and immunomodulatory traits and directly influenced neuronal histomorphology. Hence, it presents a promising intervention of diabetic polyneuropathy. Graphical Abstract.

Vitamin K2 alleviates type 2 diabetes in rats by induction of osteocalcin gene expression.

OBJECTIVES: The biological mechanisms behind the association between vitamin K (Vit K) and glucose metabolism are uncertain. We aimed to analyze the expression of insulin 1 (Ins 1), insulin 2 (Ins 2) and cyclin D2, the expression of adiponectin and UCP-1 . In addition, we aimed to estimate the doses of Vit K2 able to affect various aspects of glucose and energy metabolism in type 2 diabetes. METHODS: Thirty adult male rats were allocated equally into five groups: control group, diabetes mellitus group, and groups 3, 4, and 5, which received Vit K2 at three daily dose levels (10, 15, and 30 mg/kg, respectively) for 8 wk. At the end of the study, blood samples were collected to quantify total osteocalcin, fasting plasma glucose, fasting insulin, and relevant variables. The expression of OC, Ins 1, Ins 2, cyclin D2, adiponectin, UCP-1 genes was analyzed by real-time polymerase chain reaction. RESULTS: After administration of Vit K2, a dose-dependent decrease in fasting plasma glucose, hemoglobin A1c and homeostatic model assessment method insulin resistance, and a dose-dependent increase in fasting insulin and homeostatic model assessment method ß cell function levels, when compared with diabetes mellitus rats, were detected. There was significant upregulation of OC, Ins 1, Ins 2, or cyclin D2 gene expression in the three treated groups in a dose-dependent manner when compared with the diabetic rats. However, expression of adiponectin and UCP-1 were significantly increased at the highest dose (30 mg/kg daily) only. CONCLUSIONS: Vit K2 administration could improve glycemic status in type 2 diabetic rats by induction of OC gene expression. Osteocalcin could increase ß-cell proliferation, energy expenditure, and adiponectin expression. Different concentrations of Vit K2 were required to affect glucose metabolism and insulin sensitivity.

Serum miRNA-27a and miRNA-18b as potential predictive biomarkers of hepatitis C virus-associated hepatocellular carcinoma.

Hepatitis C virus (HCV) infection remains the main risk factor for chronic hepatitis (CHC), liver cirrhosis, and hepatocellular carcinoma (HCC). Changes in microRNA (miRNA) profiles can be associated with HCV infection and may either favor or inhibit the virus and/or its complication. Moreover, miRNAs have emerged as key regulators of various cancers including HCC. The aim of this work was to investigate the potentail role of miRNA-27a and miRNA-18b expression levels as non-invasive predictive biomarkers of hepatitis C virus-associated HCC. Furthermore, we aimed to explore potential association of these miRNAs expressions with HCC clinicopathological features' in Egyptian cases. This case control study included 200 participants [60 CHC patients, 39 post-HCV cirrhosis patients, 51 HCC cases], and 50 healthy volunteers. The serum miRNA-27a and miRNA-18b expression profiles were measured using quantitative real time-polymerase chain reaction (qRT-PCR). miRNA-27a and miRNA-18b expression levels were significantly increased in post-hepatitis C cirrhosis cases compared to control and CHC groups. In HCC group, only miRNA-27a expression levels were significantly increased. Moreover, miRNA-27a and miRNA-18b expression levels were positively correlated with distant metastasis, Child-Pugh grade, and lymph node metastasis. Logistic regression analysis revealed that miRNA-27a expression was an independent predictor of cirrhosis among CHC. Receiver operating characteristic (ROC) analyses showed that miRNA-27a and miRNA-18b expression levels were useful biomarkers discriminating cirrhosis from CHC (AUC were 0.672 and 0.487, respectively), and in differentiating HCC from post-hepatitis C cirrhosis (AUC were 0.897 and 0.723, respectively). By combined ROC analysis, power of miRNA-27a and miRNA-18b expression levels as discriminator between HCC from post-hepatitis C cirrhosis was high (AUC = 0.0.821). Serum microRNA-27a and miRNA-18b expression levels are promising diagnostic and non-invasive biomarkers of CHC, post-CHC cirrhosis, and HCC.

Promoter methylation and expression of DNA repair genes MGMT and ERCC1 in tissue and blood of rectal cancer patients.

Rectal cancer involves one-third of colorectal cancers (CRCs). Recently, data supported that DNA methylation have a role in CRC pathogenesis. In the present study we aimed to analyze the methylation status of MGMT and ERCC1 promoter regions in blood and tissue of patients with benign and malignant rectal tumors. We also studied the methylated MGMT and ERCC1 genes and their relations with clinicopathological features. Furthermore, we suggested that methylation may play a critical function in the regulation of MGMT and ERCC1 expression. Fifty patients with non-metastatic cancer rectum and 43 patients with benign rectal lesions were involved in the study. DNA extraction from blood and rectal specimens was done to analyze the methylation status of MGMT and ERCC1 genes by methylation-specific PCR method. RNA was extracted also to determine the expression levels of these genes by real time-PCR. The frequency of MGMT and ERCC1 methylation was significantly higher in rectum cancers than in benign tumors both for the tissue and the blood (p<0.001). There was no relation between MGMT or ERCC1 methylation and clinicopathological features; while they were correlated with the response to therapy. An interesting finding that the agreement of the methylation levels in the blood and rectal tissue was classified as good (κ=0.78) for MGMT gene and as very good (κ=0.85) for ERCC1. Lastly, the MGMT and ERCC1 genes methylation was associated with down-regulation of their mRNA expression when compared with the non-methylated status. Our findings provided evidence that both blood and tumor tissue MGMT and ERCC1 methylation were associated with cancer rectum. MGMT or ERCC1 methylation in blood could be suitable non-invasive biomarkers differentiating benign and malignant rectal tumors. Furthermore, the methylation of the MGMT and ERCC1 promoter regions was associated with down-regulation of their mRNA expression.

Mechanistic effect of human umbilical cord blood derived mesenchymal stem cells on the submandibular salivary gland in ovariectomized rats.

We performed this study to understand the effect of human umbilical cord blood derived mesenchymal stem cells (hUCB-MSCs) on the submandibular gland after bilateral ovariectomy. For this, 21 adult female rats were distributed equally among 3 groups: the sham-operated group (SHAM); the ovariectomized group (OVX); and the OVX group that received repeated intravenous injections of the hUCB-MSCs (OVX + hUCB-MSCs). We used reverse transcription - PCR to analyze for the gene expression of AQPs 3, 4, 5, and BMP-6. The cellular localization and expression of human CD105, human CD34, proliferating nuclear antigen (PCNA), single-stranded DNA (ss-DNA), caspase 3, AQP1, and α smooth muscle actin (α-SMA) were determined immunohistochemically. In the OVX group, a significant decrease in the gene expression of AQP3, AQP4, and BMP6, as well as the acinar area % was detected, while area % of granular convoluted tubules (GCTs) showed a significant increase. A significant decrease in area % staining positively for AQP1 and α-SMA was noted. An obvious improvement in the structure of the submandibular gland was demonstrated in the group injected with hUCB-MSCs, as well as a significant increase in the gene expression of AQP3, AQP4, and BMP6. The acinar and GCT area %, as well as the different measured markers, were relatively normal. This demonstrates that E2-deficiency induces structural changes to the submandibular gland. Moreover, a definite amelioration of the structure and function of the submandibular gland was detected after the administration of hUCB-MSCs.

Morphological alterations in the jejunal mucosa of aged rats and the possible protective role of green tea.

INTRODUCTION: Gastrointestinal disorders become more prevalent with ageing. This study is aimed to describe morphological changes that occur in the jejunal mucosa of male albino rats as a result of ageing and the protec-tive effect of green tea (GT) extract. MATERIAL AND METHODS: The experiment was performed on sixty rats: thirty young-adult (6-month old, body mass 200-220 g) and thirty old (24-month-old, body mass 220-260 g) animals. Each group was further divided into two subgroups (n = 15 each): control rats and GT-treated rats that received 1.5 mL (300 mg/kg/day) of GT extract for 14 weeks by oral gavage. Sections of the jejunum were stained with hematoxylin and eosin, periodic acid Schiff, toluidine blue and Mallory trichrome methods. The presence of proliferating cell nuclear antigen (PCNA)- and CD68-positive cells was evaluated by immunohistochemical staining. Ultrathin sections were prepared and examined by a transmission electron microscope (TEM). RESULTS: Jejunal sections of the old control rats showed distortion of submucosa and attenuated muscularis externa with decreased height of intestinal villi. The villi also showed partial loss of acidophilic brush border with wide spaces between enterocytes. Swollen, short, blunt or broad villi with abundant mononuclear cell infiltration of lamina propria and congested blood vessels were evident both by light and electron microscopy. The number of PCNA- and CD68-positive cells in jejunal mucosa of old rats was higher than in young rats. The activity of glutathione peroxidase (GPX) and total antioxidant capacity (TAC) in the mucosa of old control rats were lower, whereas malondialdehyde (MDA) levels were higher in the jejunal homogenates of old rats as compared to young control rats. Administration of GT extract protected the jejunal mucosa from age-related changes by restoring its histological structure. The treatment of old rats with GT extract significantly decreased MDA levels in the jejunum and increased TAC and GPX activity. CONCLUSIONS: The age-related changes of the morphology of rat jejunum could be ameliorated by prolonged supplementation of the green tea extract.

Association of incretin receptors genetic polymorphisms with type 2 diabetes mellitus in Egyptian patients.

BACKGROUND: Incretins have opened a new era in type 2 diabetes mellitus (T2DM) pathogenesis. The present study aimed to assess whether there is an association between GIPR rs2302382, GIPR rs1800437 and GLP-1R rs367543060 polymorphisms with T2DM or not and also to determine the effect of these polymorphisms on gastric inhibitory polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) levels. METHODS: One hundred and fifty T2DM patients and 150 healthy controls were included in the study. Polymorphisms of GIPR rs1800437, GIPR rs2302382 and GLP-1R rs367543060 were genotyped using restriction fragment length polymorphism (RFLP)-polymerase chain reaction (PCR), multiplex allele-specific PCR and RFLP-PCR respectively. GIP and GLP levels were measured by an enzyme-linked immunosorbent assay. RESULTS: We found a significant association of both the homozygous AA and the minor allele A of GIPR rs2302382 with T2DM. The frequency of haplotype C(rs2302382) G(rs1800437) was significantly higher in controls than in diabetics; odds ratio (95% confidence interval): 1.99 (1.44-2.75) (p < 0.001), whereas the haplotype A(rs2302382) C(rs1800437) was significantly higher in patients than controls. We did not find any association of GLP-1R rs367543060 polymorphism with T2DM. We found a significant increase in serum total GIP and a significant decrease of GLP-1 levels in T2DM patients. CONCLUSIONS: We reveal for the first time an association between the GIPR rs2302382 polymorphism and T2DM in Egyptians. Yet, there was no significant association of GIPR rs1800437 or GLP-1R rs367543060 with T2DM risk. The haplotype A (rs2302382) C (rs1800437) was associated with an increased risk of T2DM. Furthermore, there was a significant increase of GIP and a significant decrease of GLP-1 levels when diabetic patients were compared with controls. An important finding was that there was a relationship between both GIPR rs2302382 and rs1800437 variants and their cognate ligand levels.

Assessment of neutralizing interleukin-4 effect on CD133 gene expression in colon cancer cell line.

Colorectal cancer may be maintained by cancer stem-like cells (CSCs) that express the cell surface marker CD133. CSCs (CD133+cells) exhibits greater resistance to the chemotherapy and this resistance may be mediated in part by an autocrine response to IL4. The aim of the study was to assess the effect of anti-IL4 antibody alone or in combination with chemotherapy on the CD133 expression andthe tumor growth. We used Caco cell line in our experiments and the samples were as the following; untreated colorectal cell line, cells treated by chemotherapy, cells treated by anti-IL4 antibody in 3doses (2.5, 5, 10µg/ml), cells treatedby combination of chemotherapy and anti-IL4 antibody in 3 doses. Results of our in vitro studies demonstrated that anti-IL4 inhibited growth of Caco cell line in a dose-dependent manner revealing a 32.11% inhibition at the highest concentration (10µg/ml). There was further significant inhibition by combination of anti IL4 and chemotherapy in a dose response manner when compared to group treated by chemotherapy only. These effects were associated with decreased expression of CD133 in tumor cells also. Lastly, anti-IL4 antibody stimulated apoptosis. Our study suggested that neutralizing of IL4 by anti IL4 antibody affect the CD133+ cells may be by increasing their apoptosis. The effects of anti IL4 antibody either, alone or in combination with chemotherapy, inhibited the tumor growth and decreased the viable tumor cells. Furthermore, neutralizing of IL4 increased the efficacy of chemotherapy treatment.

Combined Wharton's jelly derived mesenchymal stem cells and nerve guidance conduit: A potential promising therapy for peripheral nerve injuries.

BACKGROUND: Peripheral nerve injuries represent a clinical problem with insufficient or unsatisfactory treatment options. Functional outcome with nerve guidance conduits was unsatisfactory in nerve defects with increased gap size. So, cell therapy may benefit as a tool for optimizing the regeneration process. The aim of the present study was to evaluate the impact of combination of cell therapy and nerve guidance conduits on the nerve regeneration and on the expression of the factors aiding the regeneration in a rat model of sciatic nerve injury. METHODS AND RESULTS: Sixty Wistar rats were randomly divided into four groups: Group I: normal control group; Group II: sciatic nerve injury (SNI) with a 10mm long sciatic nerve gap; Group III: SNI with using a nerve conduit (NC) for nerve gap bridging; and Group IV: SNI with using a NC associated with Wharton's jelly derived mesenchymal stem cells (WJ-MSCs). The results showed that the combination therapy NC+WJ-MSCs caused much better beneficial effects than NC alone evidenced by increasing sciatic nerve index and pin-prick score. The histopathological analysis found that the use of the NC combined with WJ[HYPHEN]MSCs resulted in a structure of the sciatic nerve comparable to the normal one with better nerve regeneration when compared with NC only. There was no differentiation of WJ-MSCs into nerve structure. Lastly, there was an upregulation of expression for netrin-1, ninjurin, BDNF, GDNF, VEGF and angiopoitin-1 rat genes in NC+WJ-MSCs group than NC alone. CONCLUSION: The addition of WJ-MSCs to the nerve guidance conduits seems to bring significant advantage for nerve regeneration, basically by increasing the expression of neurotrophic and angiogenic factors establishing more favorable environment for nerve regeneration.

Murine hepatocytes DNA changes as an assessment of the immunogenicity of potential anti-schistosomal vaccines experimentally.

Hepatic affection by granulomatous inflammation in schistosomiasis suggested that a potential anti-pathology vaccine could be generated based on limiting the presence of hazardous hepatocytes induced apoptosis and caused reduction of granulomas number and size . So, this work is concerned with experimental assessment of the efficacy of different Schistosoma mansoni antigens (SEA, SWAP and combined SEA and SWAP) on murine liver after challenge by Schistosoma infection, histopathological, histochemical and molecular investigations were performed on sixty male laboratory bred Swiss Albino mice. A schedule of vaccination and challenge infection was followed and performed on 6 mice groups (each of ten); control normal (G1), control infected (G2), adjuvant received then infected (G3), SEA + adj. received then infected (G4), SWAP + adj. received then infected (G5) and SEA + SWAP + adj. received then infected (G6).Animals were euthanized 10 weeks post infection.Vaccination efficacy was assessed by histopathological, histochemical and molecular studies on murine hepatic tissues.Results showed that:The combined (SEA + SWAP) antigens were better in reducing the number and diameter of the hepatic granulomas, with more protection of the hepatocytes DNA, in addition to more decrease of hepatocytes induced apoptosis and fragmentation as demonstrated by molecular assay.

Effect of human umbilical cord blood mesenchymal stem cells administered by intravenous or intravitreal routes on cryo-induced retinal injury.

Traumatic optic neuropathy is an important cause of severe vision loss. So, many attempts were performed to transplant stem cells systemically or locally to regenerate the injured retina. In this study, we investigated the effect of human umbilical cord blood mesenchymal stem cells (hUBMSCs) on histological structure, apoptotic, antiapoptotic, oxidant and antioxidant markers in an experimental model of cryo-induced retinal damage in mice. Forty-eight mice were included with 4 major groups; group I contained 18 mice as controls. The others included 30 mice exposed to cryo-induced retinal injury and were subdivided into three equal groups: group II received no treatment after injury. Group III was intravenously injected with hUCBMSCs after injury and group IV received an intravitreal injection with hUCBMSCs into both eyes. Retinal tissues were used for histopathological, immunological and gene expression studies. Real time-PCR was performed to assess B-cell lymphoma 2 (bcl2), Bcl2-associated X protein (bax), heme oxygenase-1 (hmox-1) and thioredoxin-2 (tnx-2) expression and to assess the differentiation of the stem cells into the retinal tissue. Immunohistochemical analysis was performed to assess caspase-3, 3-nitrotyrosine (3-NT) and basic fibroblast growth factor (bFGF). Disturbed retinal structure was seen in cryo-injured mice while hUCBMSCs treated groups showed nearly normal structure. By real time-PCR, significantly reduced mRNA expressions of Bax and notably enhanced mRNA expression of Bcl-2, hmox-1 and txn-2 were demonstrated in retinal injured mice with hUCBMSCs treatment compared to those without. In addition, immunohistochemical analysis confirmed downregulation of 3-NT and caspase-3 and upregulation of bFGF after hUCBMSCs injection in injured retina. Furthermore, there was no differentiation of transplanted stem cells into the retinal tissue. In conclusions, hUCBMSCs could improve the morphological retinal structure in cryo-induced retinal damage model by modulation of the oxidant-apoptotic status and by increased the expression of bFGF. © 2017 IUBMB Life, 69(3):188-201, 2017.