Urinary BLACAT1 as a non-invasive biomarker for bladder cancer.

BACKGROUND: Bladder cancer (BC) is recorded as the fifth most common cancer worldwide with high morbidity and mortality. The most urgent problem in BCs is the high recurrence rate as two-thirds of non-muscle-invasive bladder cancer (NMIBC) will develop into muscle-invasive bladder cancer (MIBC), which retains a feature of rapid progress and metastasis. In addition, only a limited number of biomarkers are available for diagnosing BC compared to other cancers. Hence, finding sensitive and specific biomarkers for predicting the diagnosis and prognosis of patients with BC is critically needed. Therefore, this study aimed to determine the expression and clinical significance of urinary lncRNA BLACAT1 as a non-invasively diagnostic and prognostic biomarker to detect and differentiate BCs stages. METHODS AND RESULTS: The expression levels of urinary BLACAT1 were detected by qRT-PCR assay in seventy (70) BC patients with different TNM grades (T0-T3) and twelve (12) healthy subjects as control. BLACAT1 was downregulated in superficial stages (T0 = 0.09 ± 0.02 and T1 = 0.5 ± 0.1) compared to healthy control. Furthermore, in the invasive stages, its levels started to elevate in the T2 stage (1.2 ± 0. 2), and higher levels were detected in the T3 stage with a mean value of (5.2 ± 0.6). This elevation was positively correlated with disease progression. Therefore, BLACAT1 can differentiate between metastatic and non-metastatic stages of BCs. Furthermore, its predictive values are not like to be influenced by schistosomal infection. CONCLUSIONS: Upregulation of BLACAT1 in invasive stages predicted an unfavorable prognosis for patients with BCs, as it contributes to the migration and metastasis of BCs. Therefore, we can conclude that urinary BLACAT1 may be considered a non-invasive promising metastatic biomarker for BCs.

Urinary Urea, Uric Acid and Hippuric Acid as Potential Biomarkers in Multiple Sclerosis Patients.

Urine is a proven source of metabolite biomarkers and has the potential to be a rapid, noninvasive, inexpensive, and efficient diagnostic tool for various human diseases. Despite these advantages, urine is an under-investigated source of biomarkers for multiple sclerosis (MS). The objective was to investigate the level of some urinary metabolites (urea, uric acid and hippuric acid) in patients with MS and correlate their levels to the severity of the disease, MS subtypes and MS treatment. The urine samples were collected from 73 MS patients-48 with RRMS and 25 with SPMS- and age matched 75 healthy controls. The values of urinary urea, uric acid and hippuric acid in MS patients were significantly decreased, and these metabolites in SPMS pattern showed significantly decrease than RRMS pattern. Also showed significant inverse correlation with expanded disability status scale and number of relapses. Accordingly, they may act as a potential urinary biomarkers for MS, and correlate to disease progression.

The biochemical value of urinary metalloproteinases 3 and 9 in diagnosis and prognosis of bladder cancer in Egypt.

BACKGROUND: Matrix metalloproteinases (MMPs) have long been associated with cancer-cell invasion and metastasis. Few studies are available that describe this association with bladder cancer either related or unrelated to schistosoma infection.Evaluating the urinary levels of MMP3 and MMP9 as diagnostic and prognostic biomarkers in different stages of schistosomal and non schistosomal bladder cancer was the aim of the present study.Urine samples were collected from 70 patients with schistosomal and non schistosomal bladder cancer at early and advanced stages and also from 12 healthy volunteers as controls. Urinary levels of MMP-3 and MMP-9 was measured by ELISA technique. Sensitivity and specificity of both markers were determined. RESULTS: Urinary levels of both MMP-3 and MMP-9 were significantly elevated in all bladder cancer patients compared with controls. MMP-3 started to elevate in early stages of schistosomal bladder cancer ( 0.173 ng/ml) and non-schistosomal bladder cancer patients (0.308 ng/ml) compared to control (0.016 ng/ml) and remained elevated in advanced stages (0.166, 0.235 ng/ml) of both types of bladder cancer patients. In contrast, MMP-9 showed a significant elevation in advanced stages only of both schistosomal and non schistosomal bladder cancer patients (10.33, 21.22 ng/ml) compared to control (0.409 ng/ml) and this elevation of both markers was much higher in non schistosomal bladder cancer. Both Metalloproteinases were specific for the diagnosis of the disease but MMP-3 was more sensitive and this sensitivity was evident in the early stage (84.85% for MMP3, 27.28% for MMP9). CONCLUSIONS: MMP3 may be the recommended urinary metalloproteinases as early diagnostic biomarker in the early stages of both types of bladder cancer although both MMP9 and MMP3 can be used in the diagnosis of advanced stages. Further studies are required on large number of urine samples to confirm these results.

Circular RNAs 0064286 and 0000475: Potential Diagnostic Biomarkers in Hepatocellular Carcinoma.

OBJECTIVE: Hepatocellular carcinoma (HCC) is considered the highest recorded malignancy in Egypt. The shortage of appropriate biomarkers for early detection often results in the late diagnosis of the HCC. Circular RNAs (CircRNAs) are presented as long stranded non-coding RNA that combine covalently to make a sealed circular form which make them very stable. CircRNAs are known to have interpretative role in cancer development and metastasis. AIM: To examine the dysregulation of two new CircRNAs obtained from Circbase database (hsa_circ_0064286 and hsa_circ_0000475) in the serum of HCC patients as predictable diagnostic biomarkers of HCC and their correlation with some liver biochemical parameters. METHODS: Sixty clinically diagnosed HCC Egyptian patients and 25 healthy volunteers were enrolled in the study. Expression levels of the selected CircRNAs was evaluated in subjects' serum. Moreover, correlation with liver biochemical parameters, sensitivity, and specificity of studied CircRNAs were estimated. RESULTS: Both circular RNAs were significantly down regulated in HCC patients, which was negatively correlated with ALP, ALT, AST, AFP, and bilirubin levels. Circ_0064286 showed more sensitivity and specificity (88.3% and 96%, respectively). CONCLUSION: As far as we know, this is the first study that shed light on the expression levels of both circRNAs in Egyptian HCC patients. They may serve as potential biomarkers for HCC diagnosis. Moreover, those circRNAs draw attention as therapeutic targets for HCC through targeting their sponge miRNAs.

Hepatitis C virus recombinants are rare even among intravenous drug users.

Systematic studies of the circulation of hepatitis C virus (HCV) recombinants in different parts of the world have been initiated only recently, and no detailed information on this subject is available. The aim of the current investigation was to determine the frequency of HCV recombinants in intravenous drug users (IVDU) from two European countries. HCV RNA from serum samples was tested by RT-PCR with primers derived from the core and NS5B regions with subsequent sequencing and genotype assignment. The 118 samples from Germany (100%) and 45 out of 47 (96%) sera from Russia demonstrated concordant genotyping results. In the two genotype discrepant sera from Russia 2k/1b recombinants were identified. In order to test the hypothesis that the individuals from the IVDU group might be multiply exposed to various genotypes, 145 out of 165 genotyped serum samples, which were found to be positive for anti-NS4 antibodies, were serotyped with the Murex HCV serotyping kit that is based on detection of antibodies to type-specific peptides derived from the NS4 proteins of different HCV genotypes. Discrepancy in genotype and serotype attributions was observed in 11% cases. Retesting of 99 type 1a or 3a samples with a set of type- and subtype-specific primers revealed the presence of a mixed infection only in one case (1a/3a). Thus, the cases of the mixed infection with different HCV genotypes as well as the recombinant forms of HCV are very rare even in such a highly exposed group as IVDU.

The combined effect of IL-17F and CCL20 gene polymorphism in susceptibility to multiple sclerosis in Egypt.

BACKGROUND: Levels of CCL20 and its CCR6 receptor are elevated in many autoimmune diseases which help in the recruitment of T helper (Th17) cells to site of inflammation. OBJECTIVES: Determine the value of single nucleotide polymorphism of CCL20 (rs6749704) and IL-17F (rs763780) genes and their concomitant effect on the serum CCL20 level and susceptibility to MS in Egyptian patients. SUBJECTS AND METHODS: Blood samples were collected from 83 patients and 95 healthy subjects. Serum levels of CCL20 were measured by ELISA. The DNA was analyzed for rs6749704 and rs763780 using Genotyping Taqman assay. RESULTS: The mean serum levels of CCL20 in the MS group were significantly higher than healthy group (P < 0.001). Frequencies of CT genotype of rs6749704 in CCL20 gene and C allele in MS patients were significantly higher compared to controls. Also significant increase of rs763780 in IL-17F gene was detected in MS patients. Concomitant polymorphism in both genes in MS patients showed an increase risk to MS rather than individual locus. CONCLUSION: CCL20 may play an important role in the pathogenesis of MS. Both allelic variation of (rs6749704) within CCL20 gene and (rs763780) within IL-17F gene can be considered risk factor for development of MS in Egyptian patients.

Consistency of quantitation of HCV genotype 4 from egypt across three HCV-RNA amplification assays.

Different quantitative assays for HCV-RNA are available that report test results in International Units (IU)/ml based on the WHO International Standard. Thus, assays have been calibrated with standard material containing HCV genotype 1, so the consistency of quantitation might differ between assays for other HCV genotypes. Three commercial HCV nucleic acid amplification techniques (HCV-NAT) were compared for quantitation consistency of genotype 4 and 1 specimens from an Egyptian blood donor panel (n = 92). Consistency of quantitation between HCV-NATs was higher for genotype 1 than for genotype 4. Most quantitative results reported by the assays were in the same range, but some genotype 4 samples were missed by two of the assays. The Abbott assay showed higher concentrations for genotype 4 than the two Roche assays. Follow-up investigations of individuals should use the same assay unless another assay has been validated properly. Standardization of HCV-NAT assays remains an issue.

PTEN and TRAIL genes loaded zein nanoparticles as potential therapy for hepatocellular carcinoma.

Gene therapy is one of the recent approaches in treatment of hepatocellular carcinoma (HCC). Development of a vector or vehicle that can selectively and efficiently deliver the gene to target cells with minimal toxicity is an urgent demand. In the present study, phosphatase and tensin homolog (PTEN) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) genes were loaded to zein nanoparticles (ZNPs). The formulated PTEN and TRAIL-loaded ZNPs were tested for their in vitro and in vivo potential antitumor efficacy using liver tumor cells (HepG2) and HCC-induced rats as animal model. Also, mRNA expression of p53, VGEF and MMP-2 were carried out as markers of apoptosis, angiogenesis and metastasis in animal liver tissues. The results of the study showed that both PTEN and TRAIL-loaded ZNPs proved anti-proliferative activity against HepG2 cell lines with IC50 values of 0.09, 0.25 µg/ml, respectively. In vivo assay confirmed decrease in mRNA expression of both VEGF and MMP-2 with increased in P53 expression level in liver tissues of the treated animals. Therefore, authors introduced new integration between gene therapy and nanotechnology in the form of PTEN and TRAIL-loaded ZNPs that proved potential to be used in gene therapy for the treatment of HCC.

Prognostic and biological significance of microRNA-221 in breast cancer.

INTRODUCTION: Breast cancer (BC) is the most notorious cancer between females with high rates of morbidity and mortality. The aim of this study was to determine the differential expression of breast tissues microRNA-221 (miR-221) and assess its prognostic and biological significance in breast cancer (BC). METHODS: A quantitative reverse transcription PCR (qPCR) assay was performed to detect the expression of breast tissue miR-221 in different subtypes of BC (n=76) and controls (n=36) and its correlations with clinicopathological factors of patients. Univariate and multivariate analyses using the Cox proportional hazards model were performed to analyze the prognostic significance of miR-221 expression. RESULT: Our data indicated that the relative level of miR-221 expression in BC tissues was significantly higher than that in noncancerous breast tissues (p<0.01). Of 76 BC patients, 62 (81.6%) were positive cases. By statistical analyses, high miR-221 expression was observed to be closely correlated with advanced clinical stage (p<0.01). Moreover, patients with high miR-221 expression had worse 5-year relapse free survival (p=0.0124). Univariate and multivariate analyses indicated that high miR-221 expression was an independent poor prognostic factor for BC patients. CONCLUSION: miR-221 is a potential biomarker for predicting the survival of BC patients and may be a molecular therapeutic target for BC.

Possible anticancer activity of rosuvastatine, doxazosin, repaglinide and oxcarbazepin.

BACKGROUND: Rosuvastatine, doxazosin, repaglinide and oxcarbazepin are therapeutic drugs available in the market for the treatment of different diseases. Potential to display antitumor activities has also been suggested. The aim of the current study was to evaluate their in vitro effects on some human transformed cell lines. MATERIALS AND METHODS: Cytotoxicity of the four drugs was tested in MCF-7, HeLa and HepG2 cells by the neutral red assay method and also the effect of rosuvastatine and doxazosin against Ehrlich Ascities Carcinoma Cells (EACC) by trypan blue assay. RESULTS: Rosuvastatine exerted the greatest cytotoxic effect against HepG2 cells with an IC50 value of 58.7±69.3; in contrast doxazosin showed least activity with IC50=104.4 ±115.7. Repaglinide inhibited the growth of both HepG2 and HeLa cells with IC50 values of 87.6±117.5 and 89.3±119.5, respectively. Oxcarbazepine showed a potent cytotoxicity against both HeLa (IC50=19.4±43.9) and MCF7 cancer cells ((IC50=22±35.7).On the other hand the growth of EACC was completely inhibited by doxazosine (100% inhibition) while rosuvastatine had weak inhibitory activity (11.6%) . CONCLUSIONS: The four tested drugs may have cytotoxic effects against hepatic, breast and cervical carcinoma cells; also doxazosine may inhibit the growth of endometrial cancer cells. Further investigations in animals are needed to confirm these results.

The potential value of plasma Circ-ITCH in hepatocellular carcinoma patients with current hepatitis C virus infection / El valor potencial de Circ-ITCH en plasma en pacientes con carcinoma hepatocelular con infección actual por el virus de la hepatitis C

Background: There is an obvious need to diagnose hepatocellular carcinoma using novel non-invasive and sensitive biomarkers. Circular RNAs have recently attracted great interest as promising biomarkers and treatment targets. However, their function in hepatocellular carcinoma whose etiology related to hepatitis C has been rarely studied. Aim of work: The current study was conducted to analyze differential expression of circ-ITCH in plasma of Egyptian HCC patients with concomitant HCV infection, compared to normal control subjects, to investigate its correlation with liver function parameters, and to determine the possible diagnostic ability of circ-ITCH in plasma as a non-invasive marker, compared to its linear counterpart. Results: The results showed that the relative expression of circ-ITCH was significantly higher in the plasma of HCC patients (P<0.05). Moreover, when comparing its expression in the metastatic and non-metastatic subgroups, it was significantly higher in the non-metastatic HCC group compared to control group (P<0.05). Circ-ITCH was positively correlated with liver enzymes AST, ALT (P<0.001), also was significantly higher in HCC child C patients. To evaluate the potential diagnostic value of circ-ITCH in plasma, a ROC curve was generated, the AUC was 0.661, (95% CI: 0.5433–0.778) with a sensitivity and specificity 65% and 70% respectively. Conclusion: The results revealed that circ-ITCH is-with no doubt-involved in the pathogenesis of HCC and its high level may be related to HCV infection, further researches in this area will certainly make great contributions in understanding. In conclusion our results suggested that circ-ITCH may be used as a noninvasive diagnostic marker and a promising therapeutic target for HCC.(AU)

Antecedentes: Existe una necesidad obvia de diagnosticar el carcinoma hepatocelular (CHC) utilizando nuevos biomarcadores no invasivos y sensibles. Los ARN circulares han atraído recientemente un gran interés como biomarcadores prometedores y dianas de tratamiento. Sin embargo, su función en el carcinoma hepatocelular, cuya etiología está relacionada con la hepatitis C, apenas ha sido estudiada. Objetivo: Este estudio se realizó para analizar la expresión diferencial de circ-ITCH en el plasma de pacientes egipcios con CHC con infección concomitante por VHC, en comparación con sujetos de control normales, para investigar su correlación con los parámetros de la función hepática y para determinar la posible capacidad diagnóstica de circ-ITCH en plasma como marcador no invasivo, en comparación con su contraparte lineal. Resultados: Los resultados mostraron que la expresión relativa de circ-ITCH fue significativamente mayor en el plasma de pacientes con CHC (p<0,05). Además, al comparar su expresión en los subgrupos metastásico y no metastásico, fue significativamente mayor en el grupo de CHC no metastásico en comparación con el grupo control (p<0,05). Circ-ITCH se correlacionó positivamente con las enzimas hepáticas AST y ALT (p<0,001), y también fue significativamente mayor en pacientes con CHC infantil con VHC. Para evaluar el valor diagnóstico potencial de circ-ITCH en plasma se generó una curva ROC, el AUC fue de 0,661 (IC95%: 0,5433-0,778), con una sensibilidad y una especificidad del 65% y del 70%, respectivamente. Conclusión: Los resultados revelaron que circ-ITCH está, sin duda, involucrado en la patogénesis del CHC, y su alto nivel puede estar relacionado con la infección por VHC, por lo que investigaciones adicionales en esta área ciertamente harán grandes contribuciones para su comprensión. En conclusión, nuestros resultados sugirieron que circ-ITCH puede usarse como un marcador de diagnóstico no invasivo y una diana terapéutica prometedora para el CHC.(AU)

Asc-dependent and independent mechanisms contribute to restriction of legionella pneumophila infection in murine macrophages.

The apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc) is an adaptor molecule that mediates inflammatory and apoptotic signals. Legionella pneumophila is an intracellular bacterium and the causative agent of Legionnaire's pneumonia. L. pneumophila is able to cause pneumonia in immuno-compromised humans but not in most inbred mice. Murine macrophages that lack the ability to activate caspase-1, such as caspase(-1-/-) and Nlrc4(-/-) allow L. pneumophila infection. This permissiveness is attributed mainly to the lack of active caspase-1 and the absence of its down stream substrates such as caspase-7. However, the role of Asc in control of L. pneumophila infection in mice is unclear. Here we show that caspase-1 is moderately activated in Asc(-/-) macrophages and that this limited activation is required and sufficient to restrict L. pneumophila growth. Moreover, Asc-independent activation of caspase-1 requires bacterial flagellin and is mainly detected in cellular extracts but not in culture supernatants. We also demonstrate that the depletion of Asc from permissive macrophages enhances bacterial growth by promoting L. pneumophila-mediated activation of the NF-κB pathway and decreasing caspase-3 activation. Taken together, our data demonstrate that L. pneumophila infection in murine macrophages is controlled by several mechanisms: Asc-independent activation of caspase-1 and Asc-dependent regulation of NF-κB and caspase-3 activation.