Microbial Diversity and Pathogenic Properties of Microbiota Associated with Aerobic Vaginitis in Women with Recurrent Pregnancy Loss.

Recurrent pregnancy loss (RPL) is a major reproductive problem that affects approximately 5% of couples. The objective of this study was to assess vaginal flora dysbiosis in women suffering from unexplained RPL and to investigate the pathogenic properties of the microbiota associated with aerobic vaginitis (AV). The study included one hundred fifteen women, 65 with RPL and 50 controls. The diversity of vaginal microbiota isolated was evaluated by molecular sequencing. Then, pathogenic factors, such as acid-resistance, antibiotics susceptibility, and biofilm formation were evaluated. The prevalence of AV was five-fold higher in the RPL group than in the controls (64.6% vs. 12.0%). The most prevalent isolates in the case group were Enterococcus spp. (52%) and Staphylococcus spp. (26%). All bacterial strains tolerate low pH. The prevalence of multidrug resistance (MDR) among all bacteria was 47.7%. Of all strains, 91.0% were biofilm producers. The presence of MDR was found to be related to biofilm formation. The results provide evidence supporting an increased presence of dysbiosis of the vaginal flora, especially AV, in women with RPL in Tunisia. The viability of the AV-associated bacteria and their persistence in the genitals may be due to their ability to resist low pH and to produce a biofilm.

Assessment of natural coagulants to remediate Tunisian textile wastewater by combining physicochemical, analytical, and toxicological data.

Due to the complexity and variability of textile wastewater composition, a constant search for new treatment strategies that are efficient, eco-friendly, and cost-effective is mandatory. In the present study, the efficiency of coagulation-flocculation using biocoagulants derived from cactus Opuntia ficus indica and eggplant Solanum melongena to remove toxic compounds from Tunisian textile wastewater samples was evaluated by combining assays to investigate physicochemical properties and in vitro (geno)toxicity with analytical chemistry. Both natural coagulants could significantly improve the physicochemical properties of the textile wastewater samples compared to the traditionally used chemical coagulant. The highest rate of decolorization was achieved after treatment with the cactus-derived coagulant. The analytical study revealed the presence of only crystal violet dye (CV) in only one sample. Both natural coagulants were able to remove CV, which may (partially) explain the decolorization of the treated samples. Only one untreated textile effluent induced a genotoxic response in the VITOTOX® assay. The genotoxic effect was not linked to the presence of CV and was no longer observed after treatment with each of the natural coagulants, suggesting the effectiveness of the remediation treatments to remove potentially genotoxic compound(s). However, in the other genotoxicity tests, no biologically relevant effects were observed for any of the tested samples. In conclusion, although the physicochemical data indicate that the use of natural coagulants (cactus and eggplant) could be an interesting alternative treatment process to the chemical coagulant for detoxifying textile effluents, these results were only partially supported by the toxicological and analytical data.

Association of FOXA1 and EMT markers (Twist1 and E-cadherin) in breast cancer.

The transcription factor FOXA1 (forkhead box A1) plays key roles in tumor development and progression. In the present study, we analyzed the expression of FOXA1 in 52 breast tumors and 10 normal tissues, and investigated the relationship between FOXA1 and two EMT markers, namely Twist1 and E-cadherin by RT-PCR and IHC respectively. The expression level of FOXA1 was higher in tumor compared to normal tissues but the difference was not statistically significant (P = 0.138). FOXA1 expression correlated with less aggressive behavior as SBR grade I (P = 0.04), small tumors size (P = 0.05), and longer survival (P = 0.001). Furthermore, estrogen and progesterone positive tumors exhibit high level of FOXA1 (P = 0.002 and P = 0.038 respectively). Survival analysis showed that patients with ER positive/FOXA1 positive (P log rank = 0.001), PR positive/FOXA1 positive (P log rank = 0.044) and HER-2 negative/FOXA1 positive (P log rank = 0.002) tumors have a significant prolonged overall survival. On the other hand, the expression of E-cadherin positively correlated with FOXA1 (P = 0.028), whereas negative association was seen between the expression of Twist1 and FOXA1 (P = 0.016). Kaplan-Meier plots showed that patients with Twist1negative/FOXA1positive tumors have a significant prolonged overall survival (P log rank = 0.001) and FOXA1 appeared as independent predictors of patient survival in multivariate analyses. Overall, our results indicate that FOXA1 could be a useful biomarker to predict prognosis in breast cancer patients.

Gastrointestinal parasites of canids, a latent risk to human health in Tunisia.

BACKGROUND: Although data on the parasite environmental contamination are crucial to implement strategies for control and treatment, information about zoonotic helminths is very limited in Tunisia. Contamination of areas with canid faeces harboring infective parasite elements represents a relevant health-risk impact for humans. The aim of this study was to assess the environmental contamination with eggs and oocysts of gastrointestinal parasites of dogs and wild canids in Tunisia with special attention to those that can be transmitted to humans. RESULTS: One thousand two hundred and seventy faecal samples from stray dogs and 104 from wild canids (red foxes and golden jackals) were collected from different geographical regions throughout Tunisia. The helminth eggs and protozoan oocysts were concentrated by sucrose flotation and identified by microscopic examination. The most frequently observed parasites in dog samples were Toxocara spp. (27.2%), E. granulosus (25.8%), and Coccidia (13.1%). For wild canid faeces, the most commonly encountered parasites were Toxocara spp. (16.3%) followed by Capillaria spp. (9.6%). The parasite contamination of dog faeces varied significantly from one region to another in function of the climate. CONCLUSION: To our knowledge, the study highlights for the first time in Tunisia a serious environmental contamination by numerous parasitic stages infective to humans. Efforts should be made to increase the awareness of the contamination risk of such parasites in the environment and implement a targeted educational program.

CpG methylation of APC promoter 1A in sporadic and familial breast cancer patients.

Tumour suppressor gene (TSG) silencing through promoter hypermethylation plays an important role in cancer initiation. The aim of this study was to assess the extent of methylation of APC gene promoter in 91 sporadic and 44 familial cases of Tunisian patients with breast cancer (BC) in. The frequency of APC promoter methylation is somewhat similar for sporadic and familial breast cancer cases, (52.1%, and 54.5% respectively). For sporadic breast cancer patients, there was a significant correlation of APC promoter hypermethylation with TNM stage (p = 0.024) and 3-year survival (p = 0.025). Regarding the hormonal status (HR), we found significant association between negativity to PR and unmethylated APC (p= 0.005) while ER and Her2/neu are not correlated. Moreover, unmethylated APC promoter is more frequent in tumours expressing at least one out the 3 proteins compared to triple negative cases (p= 0.053). On the other hand, aberrant methylation of APC was associated with tumour size (p = 0.036), lymph node (p = 0.028), distant metastasis (p = 0.031), and 3-year survival (p = 0.046) in the group of patients with familial breast cancer. Moreover, patients with sporadic breast cancer displaying the unmethylated profile have a significant prolonged overall survival compared to those with the methylated pattern of APC promoter (p log rank = 0.008). Epigenetic change at the CpG islands in the APC promoter was associated with the silence of its transcript and the loss of protein expression suggesting that this event is the main mechanism regulating the APC expression in breast cancer. In conclusion, our data showed that the loss of APC through aberrant methylation is associated with the aggressive behavior of both sporadic and familial breast cancer in Tunisian patients.

IFNGR2 genetic polymorphism associated with sex-specific paranoid schizophrenia risk.

BACKGROUND: Considering current scientific evidence about the significant role of chronic low grade inflammation in the physiopathology of schizophrenia, it has been hypothesized that changes in pro-inflammatory cytokines such as interferon gamma may have a significant role in the predisposition to schizophrenia. AIM: This study focuses on identifying whether the functional polymorphism of interferon gamma receptor 2 (IFNGR2) is a risk factor for the development of schizophrenia. METHODS: This study was conducted by the RFLP-PCR on a Tunisian population composed of 225 patients with different sub-types of schizophrenia and 166 controls. RESULTS: The IFNGR2 (Q64R) polymorphism analysis showed higher frequencies of minor homozygous genotype (RR) and allele (R) in all patients compared to controls (21.8% vs 10.2%; p = .006, OR = 2.54) and (44% vs 34.9%; p = .01; OR = 1.46), respectively. This correlation was confirmed only for males. This study also noted a significant increase of the mutated homozygous (RR) genotype and (R) allele frequencies of IFNGR2 in paranoid schizophrenics compared to controls (31.4% vs 10.2%; p = .001; OR = 3.34 and 47.2% vs 34.9%; p = .009; OR = 1.66, respectively). This increase remains significant after using binary logistic regression to eliminate confounding factors such as age and sex. Additionally, carriers of RR genotype have significant lower scores on the Scale of Assessment of Positive (SAPS) and negative (SANS) symptoms comparatively to the carrier of the QQ + QR genotypes, suggesting that the R recessive allele carriers could have milder symptoms. CONCLUSION: The IFNGR2Q64R polymorphism is correlated with male sex and paranoid schizophrenia. It is suggested that a chronic neuroinflammation may predispose to the paranoid schizophrenia development in men.

Association of the IFN-γ (+874A/T) Genetic Polymorphism with Paranoid Schizophrenia in Tunisian Population.

Since growing evidence suggests a significant role of chronic low-grade inflammation in the physiopathology of schizophrenia, we have hypothesized that functional genetic variant of the IFN gamma (IFN-γ; +874A/T; rs2430561) gene may be involved in the predisposition to schizophrenia. This research is based on a case-control study which aims to identify whether polymorphism of the IFN-γ gene is a risk factor for the development of schizophrenia. The RFLP-PCR genotyping of the IFN-γ gene was conducted on a Tunisian population composed of 218 patients and 162 controls. The IFN-γ (+874A/T) polymorphism analysis showed higher frequencies of minor homozygous genotype (TT) and allele (T) in all patients compared with controls (11.5 vs. 4.9%; p = 0.03, OR = 2.64 and 30.7 vs. 24.1%, p = 0.04, OR = 1.4, respectively). This correlation was confirmed for male but not for female patients. Also, the T allele was significantly more common among patients with paranoid schizophrenia when compared with controls (25.8 vs. 4.9%, p = 0.0001; OR = 6.7). Using the binary regression analysis to eliminate confounding factors as age and sex, only this last association remained significant (p = 0.03; OR = 1.76, CI = 1.05-2.93). In conclusion, our results showed a significant association between +874A/T polymorphism of IFN-γ and paranoid schizophrenia, suggesting that this single nucleotide polymorphism (SNP) or another at proximity could predispose to paranoid schizophrenia. Since the minor allele of this polymorphism was correlated with an increased expression of their product, our study validates the hypothesis of excessive pro-inflammatory cytokine in the physiopathology of paranoid schizophrenia.

Toxoplasma gondii infection in schizophrenia and associated clinical features.

The belief that latent toxoplasmosis is asymptomatic has been questioned, in particular due to the repeated highlighted link between the Toxoplasma gondii infection and an increased incidence of schizophrenia. However, to understand this relationship, the effect of infection with Toxoplasma gondii on the severity of schizophrenia has been poorly studied. Our work focused on comparing the prevalence of Toxoplasma infection between schizophrenic patients and healthy controls, as well as comparing the clinical features and the demographic characteristics between Toxoplasma-seronegative and Toxoplasma-seropositive patients with schizophrenia. The rate of IgG antibody in the schizophrenia patients was 74.8% compared 53.8% in controls. Patients with schizophrenia had a significantly higher mean of serum IgG antibodies to T. gondii compared to controls. The seropositive male patients had a higher age of disease onset, a higher BPRS score, a greater negative PANSS score and a lower GAF score than the seronegative male patients. These results suggest a higher severity of clinical symptoms in the male patients with schizophrenia. This study provides further evidence to the hypothesis that exposure to Toxoplasma may be a risk factor for schizophrenia. Moreover, toxoplasmosis in men with schizophrenia may lead to more severe negative and cognitive symptoms and a less favorable course of schizophrenia.

Loss of WIF-1 and Wnt5a expression is related to aggressiveness of sporadic breast cancer in Tunisian patients.

Activation of the Wnt/ß-catenin signaling pathway is common in various human cancers. The aim of this study was to investigate the expression of 2 members of the Wnt family (WIF-1 and Wnt5a) in sporadic and hereditary breast cancer tissues. WIF-1, is a secreted antagonist that binds Wnt ligands, and therefore inhibits the canonical Wnt/ß-catenin pathway. Wnt5a is one of the members of the noncanonical Wnt family that mainly acts through calcium signaling pathway. The expression of WIF-1 was analyzed by methylation-specific PCR and RT-PCR, and the level of Wnt5a ligand was quantified by RT-QPCR in breast cancer tissues. Methylation of WIF-1 was detected in 71.3 % and 81.8 % of sporadic and hereditary cases, respectively. Aberrant methylation of WIF-1 was associated with advanced TNM stage and triple negative cases in sporadic breast carcinoma (p=0.001 and p=0.037, respectively). In hereditary cases, methylation of WIF-1 correlated with age at diagnosis (p=0.027) and p53 status (p=0.035). Regarding patients' survival, WIF-1 methylated promoter conferred a reduced overall survival rate, and particularly in a group of patients with advanced TNM stage (p log rank=0.006). Furthermore, aberrant CpG methylation of the WIF-1 promoter was significantly associated with transcriptional silencing of this tumor suppressor gene in sporadic breast cancer tissues (p=0.036). On the other hand, in sporadic tumor tissues, the level of Wnt5a mRNA was significantly lower compared to normal tissues (p=0.031) and lower still in those showing more aggressive behavior, suggesting that Wnt5a, a ligand involved in the noncanonical Wnt/ß-catenin pathway, could act as a tumor suppressor gene in breast cancer.

Frequent CpG methylation of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) in sporadic and hereditary Tunisian breast cancer patients: clinical significance.

Aberrant methylation of the CpG islands in promoter regions is one of the mechanisms for inactivation of tumor suppressor genes in many human cancers including breast carcinoma. In this study, we aimed to assess, by methylation-specific PCR, the CpG methylation pattern of the UCHL1 promoter in 94 sporadic and 44 hereditary breast cancers from Tunisian patients. The percentage of UCHL1 methylation was 67 % in sporadic and 82 % in hereditary breast cancer cases. In sporadic cases, UCHL1 methylation correlated with poor response to treatment (P = 0.042) and progesterone receptor status (P = 0.036), whereas in patients with hereditary predisposition, the only significant association was found with Her2 expression (P = 0.024). Moreover, in patients with sporadic breast cancer, the UCHL1 unmethylated pattern conferred a prolonged overall survival time in particular in the group of patients with advanced TNM stage of the disease (P log rank = 0.04). Aberrant CpG methylation of the UCHL1 promoter was significantly associated with transcriptional silencing of this tumor suppressor gene in sporadic breast cancer tissues (P = 0.001). On the other hand, the UCHL1 unmethylated pattern correlated with P53 positivity in primary sporadic tumors (P = 0.032), supporting the functional link between the two tumor suppressors in breast tumorigenesis.

Negative/low HER2 expression alone or combined with E-cadherin positivity is predictive of better prognosis in patients with breast carcinoma.

The loss of E-cadherin expression leads to absence of tissue integrity, an essential step in tumor progression. Methylation of CpG islands in the promoter region of the CDH1 gene coding E-cadherin might be an alternative for gene silencing. In the present study, we investigate the expression of E-cadherin and hormone receptors in invasive ductal breast carcinoma (IDCs). Protein expression was analysed immunohistochemically in 87 cases, including 26 familial tumors. The most interesting results revealed a significantly reduced E-cadherin expression in cases with familial history compared to sporadic tumors (p=0.009), as well as with tumors ≤5 cm (p=0.022). Moreover, HER2 over-expression was associated with distant metastasis (p=0.011) and overall survival (p log rank=0.028). Tumors displaying negative/low HER2 expression combined with E-cadherin positivity confer better patient survival (p=0.052). Triple Negative tumors (TN) were more frequently found in patients with advanced grade (GIII) (p=0.001) and TNM (III+IV) (p=0.018) which supports the aggressive behavior of TN tumors. On the other hand, hypermethylation of CDH1 gene promoter was observed in 46% of hereditary cases and strongly associated with loss of E-cadherin expression (p=0.002). Furthermore, patients with unmethylated CDH1 pattern have a better 5-year disease free survival (p=0.021). In conclusion, in patients with hereditary breast cancer, the CpG methylation event contributes to the loss of E-cadherin expression. On the other hand, HER2 over-expression is predictive of worse prognosis, either alone or combined with loss of E-cadherin expression in Tunisian patients with breast cancer.

Methylation status and overexpression of COX-2 in Tunisian patients with ductal invasive breast carcinoma.

Inflammation and hormonal signalling induce the cyclooxygenase-2 (COX-2) expression in solid tumours including breast cancer, which in turn affects cell proliferation, apoptosis and metastasis. The aim of this study was to investigate the expression of COX-2 and its association with clinical parameters, patient's survival, hormones receptors (oestrogen, progesterone), ERBB2 and TP53 expression in 83 cases of infiltrating ductal breast carcinomas. Moreover, the methylation status at the CpG islands of the COX-2 gene promoter was also explored in 70 specimens. We showed that tumours exhibiting moderate to intense COX-2 immunostaining were significantly more frequent in patients over 45 years old (p = 0.027). Moreover, a high level of COX-2 expression correlated with a shorter survival time (p log-rank = 0.04) and was an independent prognostic factor (p = 0.022; HR 6.4; 95% CI = 1.3-31.4). On the other hand, hypermethylation of the COX-2 gene promoter was observed in 27% of cases and strongly associated with smaller tumours (<5 cm, p = 0.011). Furthermore, patients with methylated COX-2 pattern have a better 4-year disease-free survival (p = 0.022) as well as a prolonged overall survival (p log-rank test = 0.034). In conclusion, we showed that high COX-2 expression was associated with reduced survival and was an independent prognostic factor. However, hypermethylation of the COX-2 promoter correlated with a better overall survival in Tunisian patients with breast carcinoma.

Haplotype analysis of p53 polymorphisms: Arg72Pro, Ins16bp and G13964C in Tunisian patients with familial or sporadic breast cancer.

BACKGROUND: The p53 polymorphisms have been extensively studied as putative breast cancer susceptibility variants. The present study was undertaken to investigate the association of p53 Arg72Pro, Ins16bp and G13964C polymorphisms and their haplotypes with breast cancer risk in Tunisian women. METHODS: Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) on 159 patients and 132 controls. RESULTS: The G13964C intronic variant was significantly associated with familial breast cancer risk (p=0.0018) while the genotypic distribution was similar for p53 Arg72Pro and Ins16bp in patients and controls. Moreover, the (NoIns-C), (Arg-C) and (NoIns-Arg-C) haplotypes were significantly associated with familial breast cancer risk (p=0.0021, p=0.0096 and p=0.0084, respectively) while there was a trend of association between the (Ins-Arg) and (Ins-Arg-G) haplotypes and the risk of sporadic breast cancer. Only the G/C genotype as well as the (NoIns-C) haplotype remained significant after correction for multiple testing. CONCLUSION: Our data revealed an association between the G/C genotype and the (NoIns-C) haplotype and the risk of familial breast cancer in Tunisian women. However, these observations need to be confirmed due to the limited statistical power of our study and the small number of cases.

Aberrant methylation of RASSF1A is associated with poor survival in Tunisian breast cancer patients.

INTRODUCTION: Epigenetic gene silencing is one of the major causes of inactivation of tumor-suppressor genes in many human cancers. MATERIALS AND METHODS: The aim of the present study was to determine the methylation status of the promoter region CpG islands of four cancer-related genes RASSF1A, RARbeta2, CDH1, and p16 ( INK4a ) in 78 breast cancer specimens and to evaluate whether the methylation status is associated with estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2/neu) together with the major clinico-pathological parameters. RESULTS: We showed that the methylation frequencies ranged from 19.6% (p16 ( INK4a )) to 87% (RASSF1A) in primary breast tumors of Tunisian patients. Aberrant methylation of RARbeta2 was observed in 66.6% of cases and associated with age at diagnosis (P = 0.043), while CDH1 was methylated in 47.4% of tumors and was correlated with tumor size (P = 0.013). RASSF1A presented the highest percentage of methylation (87%) and was strongly associated with poor survival (P = 0.014), with age (P = 0.048), and tumor stage (P = 0.033). Loss of ER and PR was strongly associated with GIII tumors (P = 0.000 and 0.037 respectively) while HER2/neu was associated with lymph node involvement (P = 0.026) and 5-year survival rate (P = 0.028). CONCLUSIONS: Our preliminary findings suggested that aberrant methylation of RASSF1A and RARbeta2 occurs frequently in Tunisian breast cancer patients compared with others. Furthermore, RASSF1A hypermethylation could be used as a potential marker of poor prognosis.

Clinical significance of epigenetic inactivation of hMLH1 and BRCA1 in Tunisian patients with invasive breast carcinoma.

Aberrant hypermethylation of gene promoter regions is one of the mechanisms for inactivation of tumour suppressor genes in many human cancers including breast carcinoma. In the current study, we aimed to assess by MSP, the methylation pattern of two cancer-related genes involved in DNA repair: hMLH1 (mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli) and BRCA1 (breast cancer 1, early onset) in 78 primary breast cancers from Tunisian patients. The methylation frequencies were 24.36% for hMLH1 and 46% for BRCA1. BRCA1 methylation correlated with age at diagnosis (P = .015) and 5-years disease free survival (P = .016) while hMLH1 methylation was more frequent in larger tumors (P = .002) and in presence of distant metastasis (P = .004). Furthermore, methylation of hMLH1 significantly correlated with high level of P53 expression (P = .006) and with overall survival (P = .015) suggesting that silencing of hMLH1 through aberrant promoter methylation could be used as a poor prognosis indicator in breast cancer.