Topiramate affords neuroprotection in diabetic neuropathy model via downregulating spinal GFAP/inflammatory burden and improving neurofilament production.

The current study aimed to test the neuroprotective action of topiramate in mouse peripheral diabetic neuropathy (DN) and explored some mechanisms underlying this action. Mice were assigned as vehicle group, DN group, DN + topiramate 10-mg/kg and DN + topiramate 30-mg/kg. Mice were tested for allodynia and hyperalgesia and then spinal cord and sciatic nerves specimens were examined microscopically and neurofilament heavy chain (NEFH) immunostaining was performed. Results indicated that DN mice had lower the hotplate latency time (0.46-fold of latency to licking) and lower von-Frey test pain threshold (0.6-fold of filament size) while treatment with topiramate increased these values significantly. Sciatic nerves from DN control mice showed axonal degeneration while spinal cords showed elevated GFAP (5.6-fold) and inflammatory cytokines (∼3- to 4-fold) but lower plasticity as indicated by GAP-43 (0.25-fold). Topiramate produced neuroprotection and suppressed spinal cord GFAP/inflammation but enhanced GAP-43. This study reinforces topiramate as neuroprotection and explained some mechanisms included in alleviating neuropathy.

Expression of epidermal growth factor receptor and human epidermal growth factor receptor 2 in urothelial bladder carcinoma in an Egyptian cohort: Clinical implication and prognostic significance.

BACKGROUND: Bladder cancer (BC) has a particular importance in Egyptian patients due to aggressive behavior and absence of prognostic markers. OBJECTIVE: To evaluate the expression of gene and protein expression of HER2 and epidermal growth factor (EGFR) in Egyptian patients with BC and ultimately to investigate their clinical implication and prognostic significance. MATERIAL AND METHODS: The study was carried out on 46 patients with urothelial bladder BC. Tissue were obtained from transurethral resection (N = 22) and radical cystectomy (N = 24) specimens. The original hematoxylin and eosin slides were re-evaluated and the formalin fixed, paraffin-embedded (FFPE) tissues which had sufficient tumor tissue (>75%) and minimal or absent tumor necrosis were selected for immunohistochemistry (IHC) and RNA extraction. Furthermore, five control biopsies were obtained from patients with cystitis. Follow-up data were retrieved from the medical records which included the treatment regimen, disease recurrence and/or progression, and survival. RESULTS: EGFR and HER2 protein were overexpressed in 35% and 46% of patients respectively. EGFR was correlated with the tumor size, grade and pathological stage, with a similar trend for HER2. The recurrence rate was higher in patients with expression of any of the markers. Gene expression was significantly higher (10.6-folds) for EGFR and (21-folds) for HER2 in patients with BC in comparison to control patients. Survival analysis showed lower median disease-free survival in association with HER2 protein overexpression. CONCLUSIONS: Our data highlighted the prognostic significance of EGFR and HER in BC and proposed their possible use as predictive markers and potential therapeutic targets.

Association of IL-16 rs11556218 T/G polymorphism with the risk of developing type 2 diabetes mellitus.

INTRODUCTION: Diabetes mellitus is a chronic disorder that has serious complications. Type 2 diabetes mellitus is more widespread worldwide and in Egypt. Interleukin-16 is a pro-inflammatory factor that can lead to many inflammatory diseases by stimulating the secretions of cytokines. Inflammation and obesity are concomitant factors that may lead to insulin resistance and type 2 diabetes mellitus. In this study, we tried to focus on the relation between Interleukin-16 rs11556218 polymorphism and the risk of development of type 2 diabetes mellitus. PATIENTS AND METHODS: 128 type 2 diabetic patients and 128 healthy individuals as control were included in this case-control study. Interleukin-16 gene polymorphism (SNP rs11556218 T/G) was genotyped using a real-time polymerase chain reaction. RESULTS: Interleukin-16 rs11556218 T/G gene polymorphism has a statistically significant association with type 2 diabetes mellitus in the co-dominant, dominant and, over dominant genetic models. The genotype TG was presented in approximately 30 % of diabetic patients vs. control (p = 0.04) and patients with TG genotype have a 1.8 times higher risk for developing type 2 diabetes mellitus (OR1.87; 95 % CI = 1.04 to 3.39) and 9.5 times higher after risk-adjusted diabetes (OR9.58; 95 % CI = 1.50 to 61.25) (p = 0.031). We found an association between Interleukin-16 gene polymorphism with both body mass index and high density lipoprotein. CONCLUSION: This study is the first one in the Middle East and Africa which found a correlation between Interleukin-16 gene polymorphism rs11556218 and type 2 diabetes mellitus. Egyptians with TG genotypes have a higher risk to develop type 2 diabetes mellitus.

3'UTR enhances hCD47 cell surface expression, self-signal function, and reduces ER stress in porcine fibroblasts.

INTRODUCTION: Macrophages contribute to xenograft rejection by direct cytotoxicity and by amplifying T cell-mediated immune responses. It has been shown that transgenic expression of hCD47 protects porcine cells from human macrophages by restoring the CD47-SIRPα self-recognition signal. It has also been reported that the long 3' untranslated region (3'UTR) of the hCD47 gene, which is missing from constructs previously used to make hCD47 transgenic pigs, is critical for efficient cell surface expression in human cells. The aim of this study was to investigate the impact of a modified form of the 3'UTR on the expression, localization, and function of hCD47 in transfected porcine cells. METHODS: hCD47 constructs with and without the modified 3'UTR were knocked into the GGTA1 locus in porcine fetal fibroblasts using CRISPR. Flow cytometry of the transfected cells was used to analyze hCD47 localization. Endoplasmic reticulum (ER), mitochondrial, and oxidative stress were examined by gene expression analysis and confocal microscopy. Phagocytosis of transfected cells by human macrophages was measured by flow cytometry, and stimulation of human/non-human (NHP) primate lymphocytes by the cells was examined using a PBMCs proliferation assay. RESULTS: Cells transfected with the construct lacking the 3'UTR (hCD47(3'UTR-)) exhibited predominantly intracellular expression of hCD47, and showed evidence of ER stress, dysregulated mitochondrial biogenesis, oxidative stress, and autophagy. Inclusion of the 3'UTR (hCD47(3'UTR+)) decreased intracellular expression of hCD47 by 36% and increased cell surface expression by 53%. This was associated with a significant reduction in cellular stress markers and a higher level of protection from phagocytosis by human macrophages. Furthermore, hCD47(3'UTR+) porcine cells stimulated significantly less proliferation of human/NHP T cells than hCD47(3'UTR-) cells. CONCLUSION: Our results suggest the potential benefits of using hCD47 constructs containing the 3'UTR to generate genetically engineered hCD47-expressing donor pigs.

The protective effect of biochanin A against rotenone-induced neurotoxicity in mice involves enhancing of PI3K/Akt/mTOR signaling and beclin-1 production.

Rotenone is an insecticide that generates oxidative stress in the CNS and induces locomotor dysfunction and neurodegeneration in rodents. Biochanin A [BioA] is an isoflavone with antioxidant and anti-inflammatory actions. The antioxidant and the modulatory action of BioA on PI3K/Akt/mTOR signaling and autophagy were tested in rotenone-Parkinsonian mice. Mice were allocated into; Group I: oil control group, Group II: rotenone group [1-mg/kg/48h, subcutaneously], group III: rotenone and BioA [10-mg/kg]. Rotenone injection resulted in locomotor disturbances in mice, degeneration in dopaminergic neurons [tyrosine hydroxylase-immunoreactive cells], low striatal dopamine, increased malondialdehyde and decreased level of glutathione. Neuroinflammation was evidenced by upregulation of astrocytes [glia fibrillary acidic protein, GFAP] and elevated levels of cytokines. The phosphorylation of PI3K/Akt/mTOR and the autophagy-related protein, beclin-1, were decreased significantly as indicated by Western blot analysis. BioA treatment enhanced locomotor activity and afforded nigral neuroprotection. The mechanism by which BioA produced this effect includes increased antioxidant defenses, lessened proinflammatory cytokines, increased phosphorylation of PI3K/Akt/mTOR proteins and upregulated beclin-1. Importantly, BioA suppressed the striatal astrocyte marker [GFAP]. Overall, the currents study highlighted that BioA activates PI3K/Akt/mTOR signaling and enhances beclin-1 leading to neuroprotection for nigral dopaminergic neurons.

Supplement With Calcium or Alendronate Suppresses Osteopenia Due to Long Term Rabeprazole Treatment in Female Mice: Influence on Bone TRAP and Osteopontin Levels.

BACKGROUND AND PURPOSE: Rabeprazole, a proton pump inhibitor (PPIs) is much endorsed to patients with increased gastric acidity. PPIs were accused to have osteoporotic effects on patients who chronically use them. The point of the current investigation was to decide the impact of rabeprazole on osteoporosis and to explore the modulatory effects of dietary calcium or alendronate on this side effect. METHODS: 80 female mice were alienated into four groups maintained for 18 weeks: [1] Vehicle group: given distilled water in 12 ml/kg, P.O. [2] Rabeprazole control group: given rabeprazole in a dose equals 10 mg/kg every 48 h, P.O. [3] Rabeprazole + calcium: given rabeprazole (10 mg/kg every 48 h) along with calcium supplement. [4] Rabeprazole + alendronate: given rabeprazole (10 mg/kg every 48 h) and alendronate (1 mg/kg per week, i.p.). Serum calcium, phosphorus and parathyroid hormone were measured. Both femurs were kept in paraformaldehyde, and then the right one was used for X-ray examination with analysis by Digora software and the left one for histopathological examination (H&E) and immunohistochemical stains for osteopontin and tartrate resistant acid phosphatase (TRAP). RESULTS: Calcium supplementation or administration of alendronate along with rabeprazole significantly restored the mean bone density as shown by X-ray analysis. Femurs from mice received rabeprazole showed widely separated, thin-walled bone trabeculae and increased number of osteoclasts. Calcium or alendronate with rabeprazole showed thick bone trabeculae without full recovery from rabeprazole induced damage. Adding calcium supplementation to rabeprazole did not affect the histological abnormalities related to osteoclasts meanwhile alendronate produced inactivation of osteoclasts. Both calcium and alendronate decreased the rabeprazole-induced increment in the femur osteopontin level. CONCLUSION: Calcium or alendronate can be recommended for female patients on PPI therapy who are at risk of osteopenia.

Association of autoimmune regulator gene polymorphism with susceptibility to rheumatoid arthritis in Egyptian population.

The autoimmune regulator (AIRE) gene controls autoimmunity via its transcript AIRE protein that suppresses naïve T cells during central selection. The role of AIRE polymorphism in rheumatoid arthritis (RA) autoimmunity remains elusive. This study aimed to investigate the association of two selected SNPs, namely, rs760426 and rs2075876, with RA susceptibility in the Suez Canal Zone population. The study population included 100 RA patients, and the control group included 100 healthy subjects who were age- and sex-matched to the RA group. SNP genotyping was performed using real-time polymerase chain reaction-based allelic discrimination assay, the odds ratio was defined to assess the strength of the association. For rs760426, combining genotypes data revealed a significant increase for A/G genotype in the RA cases (47%, n = 47) than in the control group (27%, n = 27) in both co-dominant and over-dominant models (P = 0.013 and 0.003 respectively). In addition, rs760426 correlated to duration of RA (P = 0.031) and anti-cyclic citrullinated peptide antibody (P = 0.021). For rs2075876, there was a significant increase in the A/A genotype in RA patients compared with control subjects. In the co-dominant model, the frequency of A/A was 14% and 7% respectively (P = 0.02). In contrast to rs760426, rs2075876 associated with the risk of increased body mass index (P = 0.014) and the positivity of rheumatoid factor (RF) (P = 0.043). The frequency of minor alleles, G allele in rs760426 SNP, and A allele in rs2075876 was higher in RA patients than in control. The haplotype frequency of both G and A alleles in rs760426 and rs2075876 receptively was 11% in RA group with statistically significant difference (P = <0.001) between RA patients and healthy control. SNPs rs760426 and rs2075876 in the AIRE gene may contribute to the risk for RA susceptibility. These two polymorphisms were associated with variable risk factors and predictive biomarkers for RA. The mutant allele (G) of rs760426 SNP has significant indication of poor prognosis.

Promoter Methylation Status of Breast Cancer Susceptibility Gene 1 and 17 Beta Hydroxysteroid Dehydrogenase Type 1 Gene in Sporadic Breast Cancer Patients.

Epigenetic modifications are involved in breast carcinogenesis. Identifying genes that are epigenetically silenced via methylation could select target patients for diagnostic as well as therapeutic potential. We assessed promoter methylation of breast cancer susceptibility gene 1 (BRCA1) and 17 Beta Hydroxysteroid Dehydrogenase Type 1 (17ßHSD-1) in normal and cancer breast tissues of forty sporadic breast cancer (BC) cases using restriction enzyme based methylation-specific PCR (REMS-PCR). In cancerous tissues, BRCA1 and 17ßHSD-1 were methylated in 42.5% and 97.5%, respectively, while normal tissues had 35% and 95% methylation, respectively. BRCA1 methylation in normal tissues was 12.2-fold more likely to associate with methylation in cancer tissues (p < 0.001). It correlated significantly with increased age at menopause, mitosis, the negative status of Her2, and the molecular subtype "luminal A" (p = 0.048, p = 0.042, p = 0.007, and p = 0.049, resp.). Methylation of BRCA1 and 17ßHSD-1 related to luminal A subtype of breast cancer. Since a small proportion of normal breast epithelial cells had BRCA1 methylation, our preliminary findings suggest that methylation of BRCA1 may be involved in breast tumors initiation and progression; therefore, it could be used as a biomarker for the early detection of sporadic breast cancer. Methylation of 17ßHSD-1 in normal and cancer tissue could save patients the long term use of adjuvant antiestrogen therapies.

Female Sex Hormones Pattern and Its Relation to Disease Severity and Treatment in Pre- and Postmenopausal Patients with Chronic Hepatitis C Virus (Genotype 4) Infection.

Chronic hepatitis C (CHC) course revealed differences between men and women. Male gender and postmenopausal women are thought to be of the critical factors affecting HCV infection progression. The study aimed to assess female sex hormones and their relation to disease severity and treatment in HCV infected females. Subjects were divided to 2 groups: 44 CHC female patients and 44 controls. Both groups were classified to premenopausal and postmenopausal females. Serum estradiol (E2), progesterone (PRG), and total testosterone (TT) were assessed using chemiluminescent immunoassay. Our results showed that menopausal patients had significantly higher levels of estradiol, total testosterone, and progesterone compared to controls (P < 0.001). Reproductive aged patients had lower level of total testosterone compared to menopausal patients (P < 0.001). HCV infected females of reproductive age had higher level of progesterone compared to menopausal HCV infected females (P = 0.0014). Indicators of disease severity and treatment response were significantly worse in menopausal women compared to reproductive aged women (fibrosis: P < 0.001, activity: P = 0.045, and treatment: P < 0.001). We observed that lower estradiol level may be related to fibrosis severity in CHC females. Higher total testosterone and progesterone levels may be related to fibrosis severity and poor response to treatment in CHC menopausal females only.

Hypoxia biomarkers, oxidative stress, and circulating microparticles in pediatric patients with thalassemia in Upper Egypt.

This study aimed to investigate the oxidative stress, hypoxia biomarkers, and circulating microparticles (MPs) in ß thalassemia major. The study included 56 children with thalassemia and 46 healthy controls. Hypoxia biomarkers, oxidative stress biomarkers, and total plasma fragmented DNA (fDNA) were detected by the standard methods. The MPs were assessed by flow cytometry. Hypoxia and oxidative stress biomarkers, fDNA, and MPs were higher and total antioxidant capacity (TAC) was lower in patients with thalassemia than the controls. In splenectomized patients and those who had complications, vascular endothelial growth factor (VEGF), malondialdehyde, fDNA, endothelial, platelet, and activated platelet MP levels were higher while, TAC was lower than the nonsplenectomized patients. In conclusion, the increased tissue hypoxia, oxidative stress in ß thalassemia, and its relationship with DNA damage and MPs release could explain many complications of thalassemia and may have therapeutic implications. The VEGF could serve as an important indicator for adequacy of blood transfusion in thalassemia.

Hypoxia and oxidative stress markers in pediatric patients undergoing hemodialysis: cross section study.

BACKGROUND: Tissue injury due to hypoxia and/or free radicals is common in a variety of disease processes. This cross-sectional study aimed to investigate effect of chronic kidney diseases (CKD) and hemodialysis (HD) on hypoxia and oxidative stress biomarkers. METHODS: Forty pediatric patients with CKD on HD and 20 healthy children were recruited. Plasma hypoxia induced factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) were measured by specific ELISA kits while, total antioxidant capacity (TAC), total peroxide (TPX), pyruvate and lactate by enzymatic/chemical colorimetric methods. Oxidative stress index (OSI) and lactate/pyruvate (L/P) ratio were calculated. RESULTS: TAC was significantly lower while TPX, OSI and VEGF were higher in patients at before- and after-dialysis session than controls. Lactate and HIF-1α levels were significantly higher at before-dialysis session than controls. Before dialysis, TAC and L/P ratio were lower than after-dialysis. In before-dialysis session, VEGF correlated positively with pyruvate, HIF-1α and OSI correlated positively with TPX, but, negatively with TAC. In after-dialysis session, HIF-1α correlated negatively with TPX and OSI; while, OSI correlated positively with TPX. CONCLUSIONS: CKD patients succumb considerable tissue hypoxia with oxidative stress. Hemodialysis ameliorated hypoxia but lowered antioxidants as evidenced by decreased levels of HIF-1α and TAC at before- compared to after-dialysis levels.

EGFR activation and ultraviolet light-induced skin carcinogenesis.

The epidermal growth factor receptor (EGFR) regulates the proliferation of keratinocytes through multiple mechanisms that differ depending on the localization of the cell within the skin. Ultraviolet (UV) irradiation, the main etiologic factor in the development of skin cancer, also activates the receptor. In this review, we discuss how the UV-induced activation of EGFR regulates the response of the skin to UV. UV-induced EGFR activation increases keratinocyte proliferation, suppresses apoptosis, and augments and accelerates epidermal hyperplasia in response to UV. Pharmacological inhibition of the UV-induced activation of EGFR in a genetically initiated mouse skin tumorigenesis model suppresses tumorigenesis and the activation of mitogen-activated protein (MAP) kinases and phosphatidyl inositol-3-kinase (PI3K)/AKT signaling pathways. EGFR has pleiotropic, complex, and cell-type-specific functions in cutaneous keratinocytes; suggesting that the receptor is an appropriate target for the development of molecularly targeted therapies for skin cancer and other pathologies.

Ultraviolet irradiation induces keratinocyte proliferation and epidermal hyperplasia through the activation of the epidermal growth factor receptor.

Chronic exposure to ultraviolet (UV) irradiation induces skin cancer, in part, through epigenetic mechanisms that result in the deregulation of cell proliferation. UV irradiation also rapidly activates the epidermal growth factor receptor (EGFR). Since EGFR activation is strongly mitogenic in many cell types including keratinocytes of the skin, we hypothesized that UV-induced cutaneous proliferation results from EGFR activation. The role of EGFR activation in the response of the skin to UV was determined using Egfr-null and Egfr-wild-type skin grafted onto athymic nude mouse hosts, because Egfr-null mice survive only a few days after birth. EGFR was rapidly activated in mouse epidermis following exposure to UV, as detected by the phosphorylation of EGFR on tyrosine residues 992, 1045, 1068 and 1173. UV induced epidermal hyperplasia in Egfr-wild-type skin between 48 and 72 h post-UV. However, no epidermal hyperplasia occurred in Egfr-null skin. Baseline cell proliferation was similar in skin grafts of both genotypes. However, UV exposure increased cell proliferation, as measured by Ki67 immunohistochemistry and proliferating cell nuclear antigen immunoblotting, maximally at 48 h to a level more than three times higher in wild-type compared with Egfr-null skin. Apoptotic cell death, as measured by terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) analysis, was also increased in UV-exposed Egfr-null skin when compared with wild-type 1-2 days post-UV. These changes in cellular homeostasis after UV were accompanied by increased cyclin D expression in wild-type but not Egfr-null skin and increased expression of p53 and the cyclin-dependent kinase (CDK) inhibitor p21waf1 in Egfr-null skin when compared with wild-type. Collectively, these results demonstrate that the UV-induced activation of EGFR augments keratinocyte proliferation and suppresses apoptosis, leading to epidermal hyperplasia, associated with increased G1 cyclin expression and suppression of CDK inhibitor expression.

Chemoprevention of UV light-induced skin tumorigenesis by inhibition of the epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) is activated in skin cells following UV irradiation, the primary cause of nonmelanoma skin cancer. The EGFR inhibitor AG1478 prevented the UV-induced activation of EGFR and of downstream signaling pathways through c-Jun NH2-terminal kinases, extracellular signal-regulated kinases, p38 kinase, and phosphatidylinositol 3-kinase in the skin. The extent to which the UV-induced activation of EGFR influences skin tumorigenesis was determined in genetically initiated v-ras(Ha) transgenic Tg.AC mice, which have enhanced susceptibility to skin carcinogenesis. Topical treatment or i.p. injection of AG1478 before UV exposure blocked the UV-induced activation of EGFR in the skin and decreased skin tumorigenesis in Tg.AC mice. AG1478 treatment before each of several UV exposures decreased the number of papillomas arising and the growth of these tumors by approximately 50% and 80%, respectively. Inhibition of EGFR suppressed proliferation, increased apoptotic cell death, and delayed the onset of epidermal hyperplasia following UV irradiation. Genetic ablation of Egfr similarly delayed epidermal hyperplasia in response to UV exposure. Thus, the UV-induced activation of EGFR promotes skin tumorigenesis by suppressing cell death, augmenting cell proliferation, and accelerating epidermal hyperplasia in response to UV. These results suggest that EGFR may be an appropriate target for the chemoprevention of UV-induced skin cancer.