miRNA-559 and MTDH as possible diagnostic markers of psoriasis: Role of PTEN/AKT/FOXO pathway in disease pathogenesis.

Psoriasis is a persistent, inflammatory, autoimmune skin disorder which can be elicited by genetic and environmental factors. Several microRNAs (miRNAs) that are abnormally expressed in psoriasis have emerged as an interesting candidate in psoriasis pathogenesis. However, the expression profile and function of miRNA-559, and its direct target metadherin (MTDH), in psoriasis need to be further illuminated. This study intended to assess miRNA-559 and MTDH levels in skin and sera of psoriatic patients and to investigate their clinical significance in an attempt for developing novel distinct tools for early diagnosis of psoriasis. Moreover, this study aimed at exploring participation of miRNA-559 in regulating MTDH/PTEN/AKT pathway in psoriasis. Expression levels of miRNA-559, AKT, FOXO1 and PTEN were measured by real-time qRT-PCR, whereas MTDH and p27 levels were assessed by ELISA in lesional, non-lesional tissues and serum of 20 psoriatic patients and 20 matching controls. Correlation study was conducted between different parameters. The diagnostic performance of miRNA-559 and MTDH in psoriasis was estimated by receiver operating characteristic (ROC) curve analysis. Expression of miRNA-559 in psoriatic patients was significantly downregulated in both lesional tissues and serum as compared to controls. Conversely, MTDH protein level showed significant increase in both tissues and serum of psoriatic patients and was inversely correlated with miRNA-559 level. Meanwhile, levels of PTEN, AKT and FOXO1 were dramatically changed in psoriatic patients compared to controls. Furthermore, serum miRNA-559 and MTDH displayed comparable diagnostic accuracy in discriminating psoriatic patients from controls. Yet, miRNA-559 demonstrated superior diagnostic performance than MTDH in psoriasis diagnosis. Together, the current findings provide the first suggestion of a new mechanism by which downregulation of miRNA-559 might induce proliferation in psoriasis through modulating PTEN/AKT/FOXO1 pathway by positive regulation of MTDH. Thus, miRNA-559 and MTDH might be proposed as promising diagnostic biomarkers of psoriasis.

Silencing of the cytokine receptor TNFRSF13B: A new therapeutic target for triple-negative breast cancer.

BACKGROUND: TNFRSF13B, TACI, is a member of the TNF receptor superfamily; it plays a key role in cancer cell proliferation and progression. METHOD: Influence of silencing of human cytokine receptors on cell viability was screened by Luminescent Cell Viability Assay, after transfection of the siRNA library to find the maximum cell death superhits in both triple-negative MDA-MB-231 and double-positive MCF7 breast cells. The mode of cell death was investigated by dual DNA fluorescence staining. The expression of mRNAs of TACI, BAFF, BAFF-R, and APRIL was explored by qPCR. Immunocytofluorescence analysis was used to evaluate changes in TACI, Bcl-2, TNFR2, cyclin-D2, and PCNA. NF-kB p65, cell cycle, and necrosis/apoptosis (late and early) were analyzed by flow cytometry. RESULTS: TACI is the most potent cytotoxic superhit resulted from high-throughput screening of the siRNA library, in both types of cells. Our findings indicated that silencing receptor TACI in both types of breast cancer cells led to significant cell death, after different intervals from siRNA transfection. Cell death mediators (TNFR2, Bcl-2, and NF-κB) were significantly decreased after TACI silencing. The key factors for cell division (Cyclin-D2 and PCNA) were significantly increased in silenced cells of both types but the cell cycle was arrested before the completion of mitosis. Expression of BAFF, BAFF-R and APRIL mRNA in TACI-silenced cells showed significant upregulation in MDA-MB-231 cells, while only BAFF-R and APRIL showed significant downregulation in MCF7 cells. CONCLUSION: TACI silencing can be a new and promising therapeutic target for mesenchymal-stem like triple-negative breast cancer subtype.

Molecular and phenotypic spectrum of ASPM-related primary microcephaly: Identification of eight novel mutations.

Autosomal recessive primary microcephaly (MCPH) is an abnormal proliferation of neurons during brain development that leads to a small brain size but architecturally normal in most instances. Mutations in the ASPM gene have been identified to be the most prevalent. Thirty-seven patients from 30 unrelated families with a clinical diagnosis of MCPH were enrolled in this study. Screening of ASPM gene mutations was performed by targeted linkage analysis followed by direct sequencing. Thirteen protein truncating mutations of the ASPM were identified in 15 families (50%), eight of which were novel mutations. The mutations detected were eight nonsense, four frameshift, and one splice site. Two of these mutations (p.R1327* and p.R3181*) were recurrent and shared similar haplotypes suggesting founder effect. Patients with ASPM mutations had mild to severe intellectual disability and variable degrees of simplified gyral pattern and small frontal lobe. In addition, hypoplasia of corpus callosum (18 patients), mildly small cerebellar vermis (10 patients), and relatively small pons (13 patients) were found in 85.7%, 47.6%, and 61.9%, respectively. Furthermore, one patient had porencephaly and another had a small midline cyst. Epilepsy was documented in two patients (9.5%). Non-neurologic abnormalities consisted of growth retardation (four patients), and co-incidental association of oculo-cutaneous albinism (one patient). Our study expands the mutation spectrum of ASPM. Moreover, the simplified gyral pattern and small frontal lobe together with hypoplastic corpus callosum, small cerebellum and pons enable ASPM mutated patients to be distinguished. © 2016 Wiley Periodicals, Inc.

Ozone Therapy in Ethidium Bromide-Induced Demyelination in Rats: Possible Protective Effect.

Multiple sclerosis, an autoimmune inflammatory disease of the central nervous system, is characterized by excessive demyelination. The study aimed to investigate the possible protective effect of ozone (O3) therapy in ethidium bromide (EB)-induced demyelination in rats either alone or in combination with corticosteroids in order to decrease the dose of steroid therapy. Rats were divided into Group (1) normal control rats received saline, Group (2) Sham-operated rats received saline, Group (3) Sham-operated rats received vehicle (oxygen), Group (4) EB-treated rats received EB, Group (5) EB-treated rats received O3, Group (6) EB-treated rats received methylprednisolone (MP), and Group (7) EB-treated rats received half the dose of MP concomitant with O3. EB-treated rats showed a significant increase in the number of footfalls in the grid walk test, decreased brain GSH, and paraoxonase-1 enzyme activity, whereas brain MDA, TNF-α, IL-1ß, INF-γ, Cox-2 immunoreactivity, and p53 protein levels were increased. A significant decline in brain serotonin, dopamine, norepinephrine, and MBP immunoreactivity was also reported. Significant improvement of the above-mentioned parameters was demonstrated with the administration of either MP or O3, whereas best amelioration was achieved by combining half the dose of MP with ozone.

Evaluation and screening of mRNA S100A genes as serological biomarkers in different stages of bladder cancer in Egypt.

Calcium-binding proteins S100A are multifunctional proteins that show altered expression in various diseases and cancers. This study aimed at validating an easier and less time-consuming technique to evaluate the value of combined use of messenger RNA (mRNA) S100A genes in comparison and combination with voided urine cytology in detection of bladder cancer patients. Blood and urine specimens were collected from patients (n = 120) with histologically confirmed bladder carcinoma who are classified according to bladder cancer stage into four groups and from healthy volunteers (n = 30). Histopathology examination, bilharzias antibody detection, urine cytology, and mRNA expression of S100A genes were estimated for all subjects by real time polymerase chain reaction (RT-PCR). Results indicate that each of the investigated S100A genes can be used as diagnostic marker for bladder cancer. Both S100A4 and S100A6 can be used to differentiate between different stages of bladder cancer. S100A7 can be used for the diagnosis of squamous cell carcinoma. Both S100A8 and S100A9 can be used for detection of invasive bladder carcinoma while S100A11 can be used for early detection of superficial bladder carcinoma. The overall sensitivity and specificity for the studied S100A genes ranged from 73 to 90 and 84 to 92, respectively. The combined use of urine cytology with the investigated S100A genes increased sensitivity from 56 % up to a range of 87-96 %. In conclusion, serum S100A genes can be useful as potential serological biomarkers for bladder cancer, and combined use of urine cytology with S100A genes can improve the sensitivity for detection of bladder cancer.

Molecular biomarkers for prediction of response to treatment and survival in triple negative breast cancer patients from Egypt.

BACKGROUND: Triple negative breast cancer (TNBC) is an aggressive phenotype of breast cancer with reduced survival and poor prognosis. Increased VEGF-A, IGF-I, IGF-IR and TGF-ß1 expressions were detected in breast cancer. However, little is known about their prognostic and predictive roles in TNBC. AIM: We assessed the possible prognostic and predictive values of VEGF-A, IGF-I/IGF-IR and TGF-ß1 in TNBC cases by measuring their protein and mRNA expression in TNBC and non-TNBC cases. METHODS: VEGF-A, IGF-I, IGF-IR and TGF-ß1 RNA and their corresponding proteins were assessed in 43 TNBCs, 53 non-TNBCs and 30 normal breast tissues (NBT) by real time PCR (qPCR) and immunohistochemistry (IHC); respectively. Results were related to clinico-pathological factors, response to treatment and survival rates. RESULTS: Increased mRNA expression of VEGF-A, IGF-I, and IGF-IR was significantly higher in TNBC (65.1%, 65.1%, and 72.1%) than non-TNBC (28.1%, 33.96% and 28.3%) and NBT (0.00%) (P<0.001). Similarly, TNBC patients were significantly associated with high expression of VEGF-A, IGF-I, and IGF-IR proteins (67.44%, 62.79% and 83.72%) than non-TNBC (20.75%, 35.86% and 20.75%) and NBT (0.00%) (P<0.001). Protein and RNA expression levels of all studied markers showed high concordance in all investigated patients (correlation coefficient exceeding 0.5 and 0.4, respectively). In the TNBC group, metastasis and poor response to treatment were significantly associated with VEGF-A (P<0.001, P=0.007, respectively), IGF-I (P<0.001, P<0.001, respectively), IGF-IR (P=0.001, P=0.015, respectively) and TGF-ß1 (P<0.001, P=0.007, respectively) protein levels. Multivariate logistic regression showed that IGF-I was the only independent prognostic factor for reduced OS (P=0.034) and DFS (P=0.026) in the TNBC patients. CONCLUSIONS: VEGF-A, IGF-I and IGF-IR play an important role in the development and progression of TNBC compared to non-TNBC. Therefore, they could be used as prognostic and predictive biomarkers as well as candidates for targeted therapy. However, only IGF-I can predict survival in those patients.

Transforming growth factor-ß, insulin-like growth factor I/insulin-like growth factor I receptor and vascular endothelial growth factor-A: prognostic and predictive markers in triple-negative and non-triple-negative breast cancer.

In the current study, the prognostic and predictive values of serum transforming growth factor-ß1 (TGF-ß1), insulin-like growth factor I (IGF-I)/IGF-I receptor (IGF-IR) and vascular endothelial growth factor-A (VEGF-A) were evaluated in triple-negative and non-triple-negative breast cancer (TNBC and non-TNBC). The aim was to identify a group of serological biomarkers and to identify possible candidates for targeted therapy in patients with TNBC and non-TNBC. Protein levels of TGF-ß1, IGF-I/IGF-IR and VEGF-A in the serum were measured in 43 TNBC, 53 non­TNBC and 20 normal control participants using quantitative ELISA assays. Results were correlated against standard prognostic factors, response to treatment and survival. TNBC was identified to be associated with poor prognosis and serum levels of VEGF-A and IGF/IGF-IR were significantly higher in the TNBC group compared with the non-TNBC group. IGF-IR and VEGF-A overexpression was observed to be correlated with TGF-ß1 expression and all of the markers investigated were associated with metastasis and disease progression. In the multivariate analysis, VEGF-A, IGF-I and IGF-IR were observed to be independent predictors for overall survival, whereas TGF-ß1 and lymph node status were identified as independent predictors for disease-free survival. The overall response rate was significantly lower in patients with TNBC and those with high levels of TGF-ß1, IGF-I/IGF-IR and VEGF-A. In view of the present results, it was concluded that TGF-ß1, IGF-I/IGF-IR and VEGF-A overexpression is associated with the presence of aggressive tumors, which exhibit an increased probability of metastasis, a poor response to treatment and reduced survival rate. This indicates that VEGF-A, IGF-IR and IGF-I have the potential to be used as surrogate biomarkers and are promising candidates for targeted therapy, particularly in patients with TNBC.

Simple molecular diagnostic method for fragile X syndrome in Egyptian patients: pilot study.

BACKGROUND: Poor knowledge about Fragile X syndrome (FXS) may be a major barrier to early diagnosis that could improve quality of life and prognosis especially in the developing countries. AIM: The aim of this study was to evaluate simple and reproducible method for premutation detection in females of fragile X families for the first time in Egypt. SUBJECTS AND METHODS: We have developed a rapid modified polymerase chain reaction (PCR)-based screening tool for expanded Fragile X mental retardation 1 (FMR1) alleles. This method utilizes betaine as additive to facilitate FMR 1 gene amplification. We screened fifty three males, thirty two first-degree females; twenty normal healthy controls in addition to six reference samples. RESULTS: Simple PCR method showed 16 males with abnormal CGG repeats, where 10 of their mothers and four sisters had FMR 1 premutation. Consanguineous marriage was present in 66.6% percent of the studied families. Studying the correlation between genotype and clinical manifestations showed premature ovarian failure in 40% and learning disability in 50% of the studied female carriers. CONCLUSION: FXS has to be ruled out in families with consanguineous parents, before assuming that familial mental retardation is due to autosomal recessive gene defects. Early carrier detection may reduce the number of affected children. In conclusion, more studies are still needed of much larger sample size with known allele sizes in order to guarantee the accuracy of the method used.

Deltamethrin-induced genotoxicity and testicular injury in rats: comparison with biopesticide.

Deltamethrin is a synthetic pyrethroid insecticide used extensively in pest control. Aim of the current study was to investigate the ability of deltamethrin-based commercial formulation to induce genotoxicity and testicular injury in rats in comparison to the use of the biopesticide; Bacillus thuringiensis. Rats were divided into three groups: Group I (DEL) received deltamethrin, 5 mg/kgb.w./day orally, in corn oil. Group II (Biopesticide, B. thuringiensis) received oral suspension of the biopesticide at daily dose of 8400 mg/kgb.w./day. Group III (Control) received appropriate volume of corn oil. After 4 weeks, deltamethrin-treated rats showed decreased serum testosterone, luteinizing and follicle-stimulating hormone levels. Testicular total oxidant capacity (TOC), poly (ADP-ribose) polymerase (PARP), lactate dehydrogenase (LDH) and DNA damage were significantly increased. Significant increase in bone marrow chromosomal aberrations, induced by deltamethrin, including chromatid breaks, deletions, fragments and gaps was also observed. RT-PCR demonstrated significant up-regulation in testicular mRNA for glutathione-s-transferase and heat-shock protein-70 (HSP-70) whereas steroidogenic acute regulatory (StAR) mRNA was down-regulated after deltamethrin exposure. Oral administration of the biopesticide, under the condition of our study, was found to be safe when compared to the deleterious effect of deltamethrin in rats.

Diagnostic evaluation of apoptosis inhibitory gene and tissue inhibitor matrix metalloproteinase-2 in patients with bladder cancer.

Bladder carcinoma is an important worldwide health problem. Both cystoscopy and urine cytology used in detecting bladder cancer suffer from drawbacks where cystoscopy is an invasive method and urine cytology shows low sensitivity in low-grade tumors. This study validates easier and less time-consuming techniques for the estimation of survivin and TIMP-2 in urine of bladder cancer patients to evaluate them in comparison with cytology. This study includes malignant (bladder cancer patients, n = 42), benign (patients with bilharzial cystitis, n = 22) and healthy (n = 21) groups. The studied groups were subjected to cystoscopic examination, detection of bilharzial antibodies, urine cytology, and estimation of urinary survivin by qualitative RT-nested PCR and TIMP-2 by ELISA. Significantly higher positivity rates of urinary survivin and TIMP-2 were observed in the malignant group compared with benign and healthy groups. On associating the two urinary markers with different clinicopathological factors, only TIMP-2 exerted significantly higher positivity rate in invasive stage (100%) than superficial stage (82.3%). Survivin showed 78.6% sensitivity, 95.3% specificity, 94.3% PPV, 82% NPV, and 87% accuracy. When combined with urine cytology, the sensitivity increased to 83.3%. While on applying the cutoff value of urinary TIMP-2 (< or =639.5 pg/mg protein), it showed 93% sensitivity, 83.7% specificity, 85% PPV, 92.3% NPV, and 88.2% accuracy. When combined with urine cytology, the TIMP-2 sensitivity remained 93%. On combining cytology with both urinary survivin and TIMP-2, the highest sensitivity was reached (98%). Survivin and TIMP-2 can be considered as potentially useful urine markers in early detection of bladder cancer.

Detection of HER-2/neu, c-myc amplification and p53 inactivation by FISH in Egyptian patients with breast cancer.

Breast cancer is a leading cause of cancer-related deaths in women worldwide. The clinical course of this disease is highly variable and clinicians continuously search for prognostic parameters that can accurately predict prognosis, and indicate a suitable adjuvant therapy for each patient. Amplification of the two oncogenes HER-2/neu and c-myc and inactivation of the tumor suppressor gene p53 are frequently encountered in breast carcinomas. The purpose of this study was to use the fluorescence in situ hybridization (FISH) for the assessment of HER-2/neu and c-myc amplification and p53 inactivation and to relate these molecular markers with the commonly used clinical and pathological factors. The study was conducted on 34 tissue samples obtained from 33 females and 1 male with breast carcinomas and 17 samples obtained from 16 females and 1 male with benign breast lesions. Results revealed that the level of HER-2/neu, c-myc and p53 in the malignant group was significantly increased as compared to the benign group. On relating the level of the molecular markers to clinicopathological factors, p53 was significantly associated with increased patient's age. The sensitivity of the investigated markers significantly increased with larger tumor size. Concerning tumor grade, HER-2/neu and p53 showed a significant increase in low-grade tumors whereas c-myc showed a highly significant increase in high-grade tumors. With regard to disease staging, HER-2/neu and c-myc were the only markers that showed significant increase at late stages of disease. p53 and HER-2/neu were significantly associated with positive lymph nodal status. A significant correlation was obtained between the levels of the three biomarkers to each other. Conclusively, the combination of HER-2/neu, c-myc and p53 can stratify patients into different risk groups.