Identification of G2607A mutation in EGFR gene with a significative rate in Moroccan patients with Bladder Cancer.

The epidermal growth factor receptor (EGFR) is involved in the regulation of several cellular processes and in the development of many human cancers. Somatic mutations of EGFR at tyrosine kinase domain have been associated with clinical response to tyrosine kinase inhibitors (TKIs) in lung cancer patients. In this study, we evaluated the frequency of point mutations in EGFR for future use of TKI in clinical treatment of bladder cancer. A total, 50 Moroccan patient specimens with bladder cancer and 48 healthy controls were analysed for EGFR mutations in the region delimiting exons 18-21 by PCR amplification and direct sequencing. Our results showed the absence of mutations in the EGFR kinase domain in these exons in all analysed specimens. However, sequence analysis of the EGFR-TK domain, revealed the presence of (G2607A) polymorphism at exon 20. Statistical analysis showed significant difference in the frequencies of G2607A polymorphism between cancer cases and healthy controls (p=0.0001) and the frequencies of the GG and GA/AA genotypes among the cancer cases were 28% and 72%, respectively. Moreover, allelic frequencies of G2607A polymorphism showed significant difference between cancer cases and healthy controls (p=0.0025). Data analysis showed no significant association between G2607A polymorphism and patients' age, clinical stage and tumor grade (p > 0.05). However, a significant difference was found between this polymorphism and patients' sex that could be a sampling bias due to the very limited number of women with bladder cancer. Our findings highlight that, mutations in EGFR kinase domain is a rare event in bladder cancer, suggesting, that treatment of bladder cancer patients with TKI may not be effective. However, the EGFR G2607A polymorphism in exon 20 is frequent in bladder cancer cases and must be further explored for its relevance in the treatment of this disease.

Association between GPX1 Pro189Leu polymorphism and the occurrence of bladder cancer in Morocco.

Worldwide, Bladder cancer is the most frequent male malignancy. It is the third most common male malignancy in Morocco. The risk factors for developing bladder cancer are multiples including dietary conditions, environmental exposure and oxidative stress. GPX1 gene encoding for the human cellular antioxidant enzyme glutathione peroxidase1 is a key factor in the cell detoxification process. GPX1 Pro198Leu polymorphism is associated with a decrease of enzyme activity and may contribute to bladder cancer susceptibility. The present case-control study was planned to assess the presence of GPX1 Pro198Leu polymorphism in Moroccan population to determine whether it is associated with the risk of developing bladder cancer in Moroccan patients. A total of 32 patients with bladder cancer and 40 healthy controls were enrolled. Genotyping of the GPX1 Pro198Leu polymorphism was carried out by PCR amplification and DNA sequencing. Pro198Leu polymorphism was observed in both bladder cancer patients and healthy controls. No significant association between the polymorphism and bladder cancer occurrence was found (Pro/Leu vs. Pro/Pro: p=0.425; Leu vs. Pro: p=0.435). For the analysis of Pro198Leu polymorphism and progression of bladder cancer, no association was observed neither for stages (Pro/Leu vs. Pro/Pro: p=0.500; Leu vs. Pro: p=0.500) nor grades (Pro/Leu vs. Pro/Pro: p=0.415; Leu vs. Pro: p=0.427). Our results clearly showed no significant association between Pro198Leu polymorphism and risk of bladder cancer in our population, suggesting that the effect of this polymorphism on bladder cancer development might be a result of a combination with other genetic alterations and/or non-genetic variables such as diet and lifestyle factors.

Epigenetic alterations of adenomatous polyposis coli (APC), retinoic acid receptor beta (RARß) and survivin genes in tumor tissues and voided urine of bladder cancer patients.

The CpG promoter methylation has been reported to occur frequently in bladder cancer. Moreover, analysis of gene methylation has been shown to be feasible from voided urine and can be detected with a high degree of sensitivity. The aim of this present study is to determine how methylation patterns of APC, RARβ and Survivin genes change during bladder carcinogenesis and to evaluate whether DNA methylation could be detected in urine sediment. Using the sensitive assay of MSP, we explored the promoter methylation status for the three genes in tumor specimens and urine sediment DNA from 32 bladder cancer patients. Methylation frequencies of the tested genes in tumor specimens were 100%, 75% and 84.4% for APC, RARβ and Survivin, respectively. Hypermethylation of APC was found in all pathological grades and stages of bladder cancer. More frequent promoter hypermethylation of RARβ and Survivin was observed in high grade tumors and the hypermethylation increased from low to high stages, but there was no significant correlation between stages/grades and hypermethylation of these two gene promoters. In order to investigate clinical usefulness for noninvasive bladder cancer detection, we further analyzed the methylation status in urine samples of bladder cancer patients. Methylation of the tested genes in urine sediment DNA was detected in the majority of cases that were hypermethylated in tumor samples (93.7%) and the frequencies were 79.3% 70.8% and 96.3% for APC, RARβ and Survivin, respectively. Our results indicate that methylation of APC, RARβ and Survivin gene promoters is a common finding in patients with bladder carcinoma. The ability to detect methylation not only in bladder tissue, but also in urine sediments, suggests that methylation markers are promising tools for noninvasive detection of bladder cancer.

Rapid screening and diagnosis of tuberculosis: a real challenge for the mycobacteriologist.

Tuberculosis (TB) is an infectious, devastating and contagious disease, which infects third of the global population worldwide with high rates of incidence in the developing countries, where the health care providers face a serious problem and a real challenge during their clinical practice for controlling and preventing the transmission of this illness. Indeed the first step of control is the correct diagnosis and the initiation of the drug treatment regimen at the early stage of infection, which mandate the rapidity of screening and the accuracy of laboratory testing. In this paper we aim to highlight the different actual techniques, regarding the rapid screening and diagnosis of tuberculosis.

In vitro antiproliferative effect and induction of apoptosis by Retama monosperma L. extract in human cervical cancer cells.

The antiproliferative effect of different extracts obtained from Retama monosperma L. was investigated on human SiHa and HeLa cervical cancer cell lines using a MTT colorimetric assay. The Retama monosperma L. dichloromethane fraction (Rm-DF) was the most active extract, exhibiting a significant cytotoxic activity on both cell lines in a dose-dependent manner, after 72 h of treatment. IC50 values obtained were 14.57 ± 4.15 µg/ml and 21.33 ± 7.88 µg/ml, for SiHa and HeLa cell lines respectively. The morphological features assessment of apoptosis in Rm-DF-treated cells showed a condensation of chromatin and apoptotic bodies, accompanied by a decrease in mitochondrial membrane potential (ΔΨm) and an increase in reactive oxygen species in both cell lines. The induction of apoptosis was further confirmed by Western blotting pro-caspase 3, Bcl2 and PARP; caspase 3 activity assay; and Annexin V labelling. Analysis of Rm-DF by CG/MS revealed the presence of five known quinolizidine alkaloids as well as, sparteine (10,97%), L-methyl cytisine (9.11%), 17-oxosparteine (3.49%), lupanine (0.93%) and anagyrine (39.63%). This study shows that Retama monosperma L. extract exhibits a potential anticancer activity against cervical cancer cell lines in vitro through the inhibition of proliferation and induction of apoptosis, which may involve a mitochondria-mediated signaling pathway.

EGFR, p16INK4a and E-cadherin immuno-histochemistry and EGFR point mutations analyses in invasive cervical cancer specimens from Moroccan women.

The involvement of human papillomavirus in the development of cervical cancer has been firmly established. However, better management of cervical cancer rests on good diagnosis and an effective therapy. In this study we evaluated the frequency of point mutations in epidermal growth factor receptor (EGFR) for future use of tyrosine kinase inhibitors in clinical treatment and to assess the use of EGFR, p16INK4a and E-cadherin as biomarkers in cervical cancer diagnosis with immunohistochemistry. Fifty-three patient specimens of cervical cancer were analysed for HPV infection, for EGFR mutations in exons 18 through 21, and for expression of EGFR, p16INK4a and E-cadherin by immunostaining. Results showed that 79.24% of the cases (42/53) are HPV positive and the HPV types more closely associated with risk are HPV 16 and 18. In all 53 analysed specimens, any mutation affecting the EGFR kinase domain in exons 18 through 21 was observed. Expressions of EGFR, p16INK4a and E-cadherin were detected in 88,67% (47/53), 92,45% (49/53) and 79,24% (42/53) of analysed specimens respectively. Thus, EGFR, p16INK4a and E-cadherin would be excellent tools for IHC analysis during the cervical cancer development. EGFR and p16INK4a can be used for early diagnosis and E-cadherin for cancer progression and cell migration. However, treatment of cervical cancer with TKIs may not be effective and the identification of other EGFR inhibitors is needed.

Identification of G2607A mutation in EGFR gene with a significative rate in Moroccan patients with nasopharyngeal carcinoma.

The epidermal growth factor receptor (EGFR) is involved in the regulation of several cellular processes and in the development of many human cancers. Somatic mutations of EGFR at tyrosine kinase domain have been associated with clinical response to tyrosine kinase inhibitors (TKIs) in lung cancer patients. In this study, we evaluated the frequency of point mutations in EGFR for future use of TKI in clinical treatment of nasopharyngeal carcinoma (NPC). Sixty Moroccan patient specimens of NPC were analysed for EGFR mutations in the region delimiting exons 18 and 21 by direct sequencing. Our results showed the absence of mutations in the EGFR kinase domain in these exons in all 60 analysed specimens. Sequence analysis of the EGFR—TK domain, revealed the presence of (G2607A) polymorphism at exon 20. The genotypes AA and GA were found respectively in 39 (65%) and 16 (26.6%) cases. Statistical analysis showed no difference between the polymorphism and either gender or age of patients. Mutations in EGFR kinase domain are rare events in NPC biopsies, suggesting, that treatment of NPC patients with TKI may not be effective. However, EGFR G2607A polymorphism at exon 20 is frequent in NPC cases and could be associated to clinical response to TKI therapy.

p53 codon 72 polymorphism and risk of cervical carcinoma in Moroccan women.

Human papillomavirus (HPV) has been implicated in cervical carcinoma and the p53 gene is polymorphic at amino acid 72 of the protein that it encodes. The association between p53 polymorphisms and risk for HPV-associated cervical cancer has been examined, but the results have been conflicting. It has been reported that patients with the arginine form have a higher risk of developing cervical cancer than those with the proline form. The purpose of this study was to examine whether p53 Arg at the polymorphic position 72 could represents a risk factor for women with high-risk HPV-associated malignant cervical lesions. In this study, the polymorphism was examined by both allele-specific PCR and RFLP analysis in 113 patients with cervical cancer and in 100 healthy controls. There was no statistical difference in the subtype distribution between the cervix cancer and the control groups. There was no significant association between genotype distribution of the p53 codon 72 polymorphism and HPV infection. Thus, polymorphism of the p53 itself as well as in combination with HPV infection may not be a genetic risk for cervical cancer and therefore much attention should be paid to other risk factors such as sexual behavior and smoking.

[Impact of flour fortification with elemental iron on the prevalence of anaemia among preschool children in Morocco]. / Impact de l'enrichissement de la farine en fer élémentaire sur la prévalence de l'anémie chez les enfants en âge préscolaire au Maroc.

Anaemia is a public health problem in Morocco. To limit this problem, Morocco developed a national programme based on fortification of flour with electrolytic elemental iron. To evaluate the programme's impact on the prevalence of anaemia in children between 2 and 5 years, 4 surveys were conducted, between 2006 and 2008, throughout the country. The results showed a significant improvement in the mean rate of haemoglobin accompanied by a significant decrease in the prevalence of anaemia. This improvement appears to be the result of several mutually reinforcing actions in addition to the fortification of flour with iron, including the promotion of a diversified diet rich in micronutrients and the promotion of public health measures.

Impact of flour fortification with elemental iron on the prevalence of anaemia among preschool children in Morocco

Anaemia is a public health problem in Morocco. To limit this problem, Morocco developed a national programme based on fortification of flour with electrolytic elemental iron. To evaluate the programme's impact on the prevalence of anaemia in children between 2 and 5 years, 4 surveys were conducted, between 2006 and 2008, throughout the country. The results showed a significant improvement in the mean rate of haemoglobin accompanied by a significant decrease in the prevalence of anaemia. This improvement appears to be the result of several mutually reinforcing actions in addition to the fortification of flour with iron, including the promotion of a diversified diet rich in micronutrients and the promotion of public health measures

Molecular characterisation of rifampicin-resistant Mycobacterium tuberculosis strains from Morocco.

Mutations in the rpoB gene associated with rifampicin (RMP) resistance were studied in 47 RMP-resistant and 147 RMP-susceptible clinical strains of Mycobacterium tuberculosis from Morocco using probe-based assay and DNA sequencing. RMP-resistant mutations were identified in 85% of RMP-resistant isolates. No mutations were observed among the 147 RMP-susceptible strains. Sequence analysis identified 10 alleles, including two deletions not previously reported. Nucleotide changes at codons 531, 526 and 516 were the most prominent, accounting for 74.4% of our RMP-resistant strains. These results demonstrate that resistance genotyping at these codons would be a good marker for the rapid detection of RMP resistance.

Detoxification of olive mill wastewaters by Moroccan yeast isolates.

A total of 105 yeast strains were isolated from Moroccan olive oil production plants and evaluated for their ability to grow in olive oil mill wastewaters (OMW). The 9 isolates that grew best on OMW were selected for further study to evaluate their effect on removal of organic pollutants and OMW phytotoxicity (barley seed germination test). The results showed that at least four yeast isolates effectively lowered the toxicity of this effluent in addition to providing very useful materials in terms of both yeast biomass (6 g/l DW) and an irrigation fluid. This group of yeast isolates significantly reduced the concentration of total phenols (44% removal) and Chemical Oxygen Demand, COD (63% removal). The best germination rate of 80% for undiluted OMW was obtained for strain Candida holstii that also increased the pH from 4.76 to 6.75. Principal component analysis of the results obtained for the best yeast strains confirmed the importance of COD and total phenol reduction along with increase of organic nitrogen and final pH for the improvement of germination rates and phytotoxic reduction. This study has highlighted the potential of indigenous yeasts in detoxification of olive mill wastewaters.

Human papillomavirus DNA in cervical lesions from Morocco and its implications for cancer control.

To determine the types of human papillomaviruses (HPV) in northern Morocco, information which is needed for the design and use of HPV vaccines, we have analysed 129 cervical biopsies from this region. In our study, 91 cases were HPV positive, 45 cases had HPV-16 DNA, and 20 cases had HPV-18 DNA. This distribution of virus type was similar in inflammatory cervical lesions and in invasive cervical carcinomas. In conclusion, the HPV type distribution in Morocco is similar to that in other African Mediterranean countries, where the proportion of HPV-18 cases is significantly higher than in Europe. Determination of virus-type distribution is essential for vaccination programs.

Evaluation of a flow cytofluorometric method for rapid determination of amphotericin B susceptibility of yeast isolates.

A rapid-flow cytofluorometric susceptibility test for in vitro amphotericin B testing of yeasts was evaluated and compared to the National Committee for Clinical Laboratory Standards (NCCLS) M27-T reference broth macrodilution method. The flow cytofluorometric method is based on the detection of decreased green fluorescence intensity of cells stained with DiOC5(3), a membrane potential-sensitive cationic dye, after drug treatment. Testing was performed on 134 clinical isolates (Candida spp. and Torulopsis glabrata). From the dose-response curve obtained for each isolate, three endpoints were calculated by computer analysis (the concentrations at which the fluorescence intensity was reduced by 50, 80, and 90%, i.e., 50% inhibitory concentration [IC50], IC80, and IC90, respectively). A regression analysis correlating these endpoints with the M27-T MICs showed that the best agreement was obtained with IC80. The flow cytofluorometric method showed good reproducibility with control strains. These initial results suggest that the flow cytofluorometric method is a valid alternative to the NCCLS reference method.

The Salmonella sulA-test: a new in vitro system to detect genotoxins.

The Salmonella sulA-test is a newly developed colorimetric assay to detect genotoxins. This technique is based on the ability of DNA-damaging agents to induce the sulA gene, one of the SOS response genes. A constructed plasmid, pEM1968, carrying a fused sulA'::'lacZ was introduced into Salmonella typhimurium TA1538. Monitoring sulA gene expression was performed by assaying the beta-galactosidase activity in the transformed strain S. typhimurium TA1538/pEM1968. A simple, fast and sensitive liquid incubation procedure has been developed after optimization of the S9 mix composition and beta-galactosidase assay. The SOS-inducing potency (SOSIP, microM-1) was defined as the slopes of the non-linear dose-response relationships. Twenty-one chemicals with different modes of action were examined for a preliminary evaluation of the test. Nineteen chemicals were genotoxic in the Salmonella sulA-test. The SOSIP ranged from 1.2 x 10(-4) microM-1 (ethyl methanesulfonate) to 419.9 microM-1 (bleomycin). Sodium azide and 5-fluorouracil were not genotoxic. Frameshift, base-pair and oxidative genotoxins were detected by the tester strain. The calculated SOSIP and the minimum concentrations detected (MCD) in the Salmonella sulA-test were compared to the reported values obtained with two similar assays: the SOS Chromotest and umu-test. The SOSIP values of 12 compounds were the highest in this new assay. Five chemicals tested in the Salmonella sulA-test gave similar SOSIP values with those of one of the two other tests. ICR-191 had the highest SOSIP with the SOS Chromotest and 3-methylchloranthrene showed the highest SOSIP with the umu-test. Similarly, the lowest MCD values were found for 12 compounds in the Salmonella sulA-test. Four compounds had close MCD values in this assay and one of the two other techniques. The SOS Chromotest remained the most sensitive assay for cisplatin and ICR 191. The umu-test was the technique of choice for 3-methylchloranthrene.