Ochratoxin a in breast milk in Morocco: the affecting dietary habits of the lactating mothers and the degree of exposure of newborns "CONTAMILK study".

The aim of the present study is to evaluate the level of contamination of breast milk (BM) by ochratoxin A, among Moroccan lactating mothers in the city of Rabat, and to identify the associated factors of exposure, also to estimate the degree of exposure of the breastfeed infant. The analysis of ochratoxin A (OTA) was accomplished by ELISA method on 82 colostrum samples. OTA was detectable (>0.08 ng/mL) in 55% of samples with a maximum concentration of 10.04 ng/mL, and the levels exceeded 0.5 ng /mL in 50 % of the samples. In addition, several factors and dietary habits affect significantly the level of OTA in the analyzed samples of breast milk including, the consumption of industrial dairy products, the frequency of consumption of canned foods, dried fruits and legumes, also the period of breast milk collection. Besides, OTA was higher than the tolerable daily intake for 49% newborns. However, these results need to be confirmed by multicenter studies to more broadly estimate the levels of exposition of Moroccan population to OTA. Furthermore, awareness campaigns are recommended to inform the public, especially pregnant women and lactating women about appropriate preventive measures to limit exposure to this mycotoxin.

Monoclonal Antibodies Application in Lateral Flow Immunochromatographic Assays for Drugs of Abuse Detection.

Lateral flow assays (lateral flow immunoassays and nucleic acid lateral flow assays) have experienced a great boom in a wide variety of early diagnostic and screening applications. As opposed to conventional examinations (High Performance Liquid Chromatography, Polymerase Chain Reaction, Gas chromatography-Mass Spectrometry, etc.), they obtain the results of a sample's analysis within a short period. In resource-limited areas, these tests must be simple, reliable, and inexpensive. In this review, we outline the production process of antibodies against drugs of abuse (such as heroin, amphetamine, benzodiazepines, cannabis, etc.), used in lateral flow immunoassays as revelation or detection molecules, with a focus on the components, the principles, the formats, and the mechanisms of reaction of these assays. Further, we report the monoclonal antibody advantages over the polyclonal ones used against drugs of abuse. The perspective on aptamer use for lateral flow assay development was also discussed as a possible alternative to antibodies in view of improving the limit of detection, sensitivity, and specificity of lateral flow assays.

Plasmid-based high-resolution melting analysis for accurate detection of rpoB mutations in Mycobacterium tuberculosis isolates from Moroccan patients.

BACKGROUND: Rapid diagnosis of drug resistance in tuberculosis (TB) is pivotal for the timely initiation of effective antibiotic treatment to prevent the spread of drug-resistant strains. The development of low-cost, rapid and robust methods for drug-resistant TB detection is highly desirable for resource-limited settings. METHODS: We report the use of an in house plasmid-based quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis for the detection of mutations related to rifampicin-resistant Mycobacterium tuberculosis (MTB) in clinical isolates from Moroccan patients. Five recombinant plasmids containing predominant mutations (S531L, S531W, H526Y and D516V) and the wild-type sequence of the Rifampicin Resistance-Determining Region (RRDR) have been used as controls to screen 45 rifampicin-resistant and 22 rifampicin-susceptible MTB isolates. RESULTS: The sensitivity and the specificity of the qPCR-HRM analysis were 88.8% and 100% respectively as compared to rifampicin Drug Susceptibility Testing (DST). The results of qPCR-HRM and DNA sequencing had a concordance of 100%. CONCLUSION: Our qPCR-HRM assay is a sensitive, accurate and cost-effective assay for the high-throughput screening of mutation-based drug resistance in TB reference laboratories.

Development and evaluation of an in-house single step loop-mediated isothermal amplification (SS-LAMP) assay for the detection of Mycobacterium tuberculosis complex in sputum samples from Moroccan patients.

BACKGROUND: Tuberculosis (TB) is a major global health problem and remains the leading cause of morbidity and mortality in developing countries. Routinely used TB diagnostic methods, in most endemic areas, are time-consuming, often less-sensitive, expensive and inaccessible to most patients. Therefore, there is an urgent need for the development of early, easy to use and effective diagnosis tools of TB, which can be effectively integrated into resource limited settings, to anticipate the early treatment and limit further spread of the disease. Over the last decade, Loop-mediated isothermal amplification (LAMP) assays have become a powerful tool for rapid diagnosis of infectious diseases because of the simplicity of device requirements. Indeed, LAMP is a simple, quick and cost effective Isothermal Nucleic Acid Amplification diagnostic test (INAAT) that has the potential to be used in TB endemic settings of resource-poor countries. METHODS: In the present study, we have developed a simple and rapid TB molecular diagnostic test using a Single-Step Loop-mediated isothermal DNA amplification (SS-LAMP) method for the detection of Mycobacterium tuberculosis complex (MTBC) strains, with a simplified sample preparation procedure, eliminating DNA extraction prior to LAMP amplification, DNA initial denaturation and enzymatic inactivation steps during the amplification process. To perform our in-house SS-LAMP assay, a set of six specific primers was specifically designed to recognize eight distinct regions on the MTBC species-specific repetitive insertion sequence 6110 (IS6110). The amplification of the targeted DNA was carried out under isothermal conditions at 65 °C within 1 h. Our protocol was firstly optimized using 60 of confirmed MTBC isolates and a recombinant pGEMeasy-IS6110 vector for sensitivity testing. Thereafter, the assay was evaluated on liquefied sputum specimens collected from 157 Moroccan patients suspected of having TB. RESULTS: Our SS-LAMP developed assay was able to detect MTBC DNA directly from liquefied sputum samples without any prior DNA extraction, denaturation nor the final enzymatic inactivation step. When compared to routinely used Löwenstein Jensen (LJ) Culture method, our SS-LAMP assay is rapid and showed specificity and sensitivity of 99.14 % and 82.93 % respectively which are within the international standards. In addition, the limit of detection of our assay was found to be as little as 10 copies of bacterial DNA. CONCLUSION: To our knowledge, this is the first study using a single step LAMP (SS-LAMP) procedure as a rapid, easy to perform and cost effective testing for TB early detection. This innovative assay could be suitable for low-income countries with restricted health equipment facilities.

Assessment of anti-hyperglycemic and anti-hyperlipidemic effects of thiazolidine-2,4-dione derivatives in HFD-STZ diabetic animal model.

Type 2 diabetes mellitus (T2DM) is a chronic endocrine/metabolic disorder characterized by elevated postprandial and fasting glycemic levels that result in disturbances in primary metabolism. In this study, we evaluated the metabolic effects of thiazolidine-2,4-dione derivatives in Wistar rats and Swiss mice that were fed a high-fat diet (HFD) for 4 weeks and received 90 mg/kg of streptozotocin (STZ) intraperitoneally as a T2DM model. The HFD consisted of 17% carbohydrate, 58% fat, and 25% protein, as a percentage of total kcal. The thiazolidine-2,4-dione derivatives treatments reduced fasting blood glucose (FBG) levels by an average of 23.98%-50.84%, which were also improved during the oral starch tolerance test (OSTT). Treatment with thiazolidine-2,4-dione derivatives also improved triglyceride (TG), low-density lipoprotein cholesterol (LDL-c), and total cholesterol levels (P < 0.05). The treatment intake has also shown a significant effect to modulate the altered hepatic and renal biomarkers. Further treatment with thiazolidine-2,4-dione derivatives for 28 days significantly ameliorated changes in appearance and metabolic risk factors, including favorable changes in histopathology of the liver, kidney, and pancreas compared with the HFD/STZ-treated group, suggesting its potential role in the management of diabetes. Thiazolidine-2,4-dione derivatives are a class of drugs that act as insulin sensitizers by activating peroxisome proliferator-activated receptor-gamma (PPAR-γ), a nuclear receptor that regulates glucose and lipid metabolism. The results of this study suggest that thiazolidine-2,4-dione derivatives may be a promising treatment option for T2DM by improving glycemic control, lipid metabolism, and renal and hepatic function.

Strategy for Developing a Stable CHO Cell Line that Produces Large Titers of Trastuzumab Antibody.

BACKGROUND: Trastuzumab (Herceptin®) is currently the main treatment option for breast cancer patients that overexpress the human epidermal growth factor receptor 2 (HER2). This antibody binds specifically to HER2, blocks cancer cell growth, and promotes effective cell death. In the present study, we sought to develop a robust and efficient process for the development of a stable Chinese hamster ovary (CHO) cell line with high trastuzumab expression and production. METHODS: We adapted a process that combines transposon system-based vector construction, suspension cell culture, and a high selection process. The latter, involved enhanced green fluorescent protein (eGFP) expression, fluorescence-activated cell sorting (FACS), and semi-solid methylcellulose media. RESULTS: The construction of trastuzumab as a humanized monoclonal antibody was achieved by subcloning the synthesized light and heavy chain sequences into a suitable piggyBac expression vector. The optimized piggyBac vector used for the expression of trastuzumab in CHO cells resulted in the production of trastuzumab and reached 4.24 g/L in the T1A7 clone after a 7-day batch culture. The T1A7 clone was selected after screening over 1500 clones. CONCLUSIONS: The current simple workflow ensures strict monoclonality and relatively high production of trastuzumab. This workflow could potentially be implemented in Research and Development (R&D) laboratories, including in developing countries for the production of recombinant monoclonal antibodies in a cost-effective manner.

Biological, toxicological and molecular docking evaluations of isoxazoline-thiazolidine-2,4-dione analogues as new class of anti-hyperglycemic agents.

In this work, three isoxazoline-thiazolidine-2,4-dione derivatives were synthesized and characterized by FT-IR, 1H-NMR, 13C-NMR and ESI-MS spectrometry. All compounds have been investigated for their α-amylase and α-glucosidase inhibitory activities. In vitro enzymatic evaluation revealed that all compounds were inhibitory potent against α-glucosidase with IC50 values varied from 40.67 ± 1.81 to 92.54 ± 0.43 µM, and α-amylase with IC50 in the range of 07.01 ± 0.02 to 75.10 ± 1.06 µM. One of the tested compounds were found to be more potent inhibitor compared to other compounds and standard drug Acarbose (IC50 glucosidase= 97.12 ± 0.35 µM and IC50 amylase= 2.97 ± 0.01 µM). All compounds were then evaluated for their acute toxicity in vivo and shown their safety at a high dose with LD > 2000mg/kg BW. A cell-based toxicity evaluation was performed to determine the safety of compounds on liver cells, using the MTT assay against HepG2 cells, and the results shown that all compounds have non-toxic impact against cell viability and proliferation compared to reference drug (Pioglitazone). Furthermore, the molecular homology analysis, SAR and the molecular binding properties of compound with the active site of α-amylase and α-glucosidase were confirmed through computational analysis. This study has identified the inhibitory potential of a new class of synthesized isoxazoline-thiazolidine-2,4-dione derivatives in controlling both hyperglycemia and type 2 diabetes mellitus without any hepatic toxicity.Communicated by Ramaswamy H. Sarma.

Synthesis, α-glucosidase and α-amylase inhibitory activities, acute toxicity and molecular docking studies of thiazolidine-2,4-diones derivatives.

In the present study, a series of thiazolidine-2,4-diones derivatives (3a-3e) and (4a-4e) were synthesized and characterized by 1H NMR, 13C NMR and ESI-MS spectrometry. All compounds were screened for their α-glucosidase and α-amylase inhibitory activities. In vitro biological investigations revealed that most of compounds were active against α-glucosidase with IC50 values in the range of 43.85 ± 1.06 to 380.10 ± 1.02 µM, and α-amylase with IC50 in the range of 18.19 ± 0.11 to 208.10 ± 1.80 µM. Some of the tested compounds were found to be more potent inhibitors than the clinical drug Acarbose (IC50glucosidase = 97.12 ± 0.35 µM and IC50amylase = 2.97 ± 0.004 µM). The lead compounds were evaluated for their acute toxicity on Swiss mice and found to be completely non-toxic with LD > 2000 mg/kg BW. Furthermore, the Structure-activity relationship (SAR) and the binding interactions of all compounds with the active site of α-glucosidase and α-amylase were confirmed through molecular docking and stabilizing energy calculations. This study has identified the inhibitory potential a new class of synthesized thiazolidine-2,4-diones in controlling both hyperglycemia and type 2 diabetes mellitus. Furthermore, the theoretical binding mode of the target molecules was evaluated by molecular docking studies against the 3D Crystal Structure of human pancreatic α-amylase (PDB ID: 1B2Y) and α-glucosidase (PDB ID: 3W37)Communicated by Ramaswamy H. Sarma.

Benzodiazepines use in Morocco: A nation wide consumption database study between 2004 and 2017.

Although information exists regarding the rate of benzodiazepines (BZDs) use in different countries, little information is available concerning the BZDs consumption in Morocco. To describe prescription rate in Morocco, a retrospective descriptive analysis of BZDs and their agonists use with the instituteIQIVIA database was performed during the period 2004-2017. The obtained data provide a dynamic approach to total BZDs consumption using an annual collection of sales data in Morocco and were expressed in terms of daily defined doses/ 1000 inhabitants / day. Data analysis showed that the major BZDs sold in Morocco were Alprazolam, Bromazepam, Nordazepam, Lorazepam, Parazepam, Diazepam and two benzodiazepine agonists, Zolpidem and Zopiclone. The Bromazepam was the molecule the most consumed during 2004-2016. In 2017, Alprazolam was the most consumed followed by Bromazepam, Nordazepam, Zolpidem, Lorazepam, Parazepam, Diazepam, Dipotassium clorazepate, Dipotassium Clorazypate and Zopiclone with 0.94, 0.91, 0.6, 0.55, 0.45, 0.32, 0.18, 0.18, 0.07 and 0.05 daily defined doses/ 1000 inhabitants / day respectively. The total amount consumed each year for all BZDs and their agonists in Morocco was 2.69, 2.77, 3, 3.17, 3.32, 3.54, 3.61, 3.81, 4.06, 4.30, 4.06, 3.94, 3.78 and 3.66 daily defined doses/ 1000 inhabitants / day, respectively during 2004-2017. To our knowledge, this is the first study that describes the consumption of BZDs and their agonists (Zolpidem, Zopiclone) in Morocco. This data may help the analytical toxicology laboratory and health organizations operating in the field of analytical biochemistry to develop specific BZDs quantification and detection methods needed for the Moroccan population.

Aflatoxin M1 prevalence in breast milk in Morocco: Associated factors and health risk assessment of newborns "CONTAMILK study".

The aim of this study was to assess the contamination of breast milk by aflatoxin M1 among nursing mothers from Rabat, Morocco, and to explore its association with several maternal parameters and dietary habits. In addition, the health risk assessment of the newborns by the estimation of the daily intake. A competitive Enzyme Linked Immunosorbent Assay method was used for the analysis of aflatoxin M1 in breast milk samples. Analytical results indicate that out of 82 total samples, 43 samples (52.4%) of milk were positive. Aflatoxin M1 levels ranged from undetectable to 13.33 ng/L, while the mean level was 5.75 ± 3.44 ng/L. Besides, several factors and foodstuffs seem to increase the level of AFM1 in breast milk. As regards the estimated daily intake of aflatoxin M1, it varies between immeasurable and a maximum of 1.16 ng/kg.bw. The degree of exposure to AFB1 and the levels of its metabolite AFM1 in breast milk were low, compared to some studies from other countries. Further investigations and periodic monitoring programs are recommended in large samples and in many cities of morocco to assess the level of exposure of the Moroccan population.

Analytical Methods Used for the Detection and Quantification of Benzodiazepines.

The prescription of psychotropic drugs, especially benzodiazepines (BZDs), occupies a preponderant place in the management of mental illnesses. Indeed, the BZDs have been used in different therapeutic areas including insomnia, anxiety, seizure disorders, or general anesthesia. Unfortunately, these drugs are present in the illegal street market, leading to a lot of drug abuse amongst some addicted users, road insecurity, and suicide. Hence, it has become essential to analyze the BZDs drugs in human biological specimens for drug abuse in forensic sciences. The present review provides a summary of sample preparation techniques (solid-phase extraction and Liquid-liquid phase extraction) and the methods for the detection and quantification of BZDs molecules in the commonly used biological specimens over the ten last years which may potentially lead to better and accurate evaluation of the physiological state of a given person. The commonly used methods for the detection and quantification of BZDs include nuclear magnetic resonance (NMR), chromatography (GC-MS, HPLC, and TLC), immunoassay (ELISA, RIA, LFA, CEDEA, FPIA, and KIMS), and electroanalytical methods (voltammetry and potentiometry).

Development and evaluation of a novel RT-qPCR based test for the quantification of HER2 gene expression in breast cancer.

Accurate measurement of Human epidermal growth factor receptor (HER2) gene expression is central for breast or stomach cancer therapy orientation and prognosis. The current standards testing methods for HER2 expression are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). In the current study, we explored the use of quantitative real time reverse transcription-PCR (RT-qPCR) as a potential method for the accurate relative quantification of the HER2 gene using formalin fixed paraffin embedded (FFPE) breast cancer biopsy samples. The main aim of the current study is to measure the level of concordance of RT-qPCR based quantification of HER2 overexpression with both IHC and FISH. Accordingly, an endogenous control gene (ECG) is required for this relative quantification and should ideally be expressed equivalently across tested samples. Stably expressed ECGs have been selected from a panel of seven genes using GenEx V6 software which is based on geNorm and NormFinder and statistical methods. Quantification of HER2 gene expression was performed by our RT-qPCR-based test and compared to the results obtained by both IHC and FISH methods. HER2 gene quantification using RT-qPCR test was normalized using the two ECGs (RPL30 and RPL37A) that were successfully identified and selected from a panel of seven genes as the most stable and reliable ECGs. We evaluated a total of 216 FFPE tissue samples from breast cancer patients. The results obtained with RT-qPCR in the current study were compared to both IHC and FISH data collected for the same patients. In addition to an internal evaluation, an external evaluation of this assay was also performed in a recognized pathology center in Europe (Clinic Barcelona Hospital Universitari, Spain) using 116 FFPE breast cancer tissue samples. The results demonstrated a high concordance between RT-qPCR and either IHC (98%) or FISH (72%) methods. Accordantly, the overall concordance was 85%. To our knowledge, this is the first study using the specific combination of RPL30 and RPL37 as reference genes for an accurate HER2 gene quantification in FFPE biopsy samples. Although further clinical validation regarding evolution and therapeutic response using RT-qPCR for the quantification of HER2 expression are still needed, the present study constitutes definitely a factual element that the RT-qPCR based assay may constitute a valid complementary test to accurately measure HER2 expression for a better treatment orientation.

Evaluation of Combined Quantification of PCA3 and AMACR Gene Expression for Molecular Diagnosis of Prostate Cancer in Moroccan Patients by RT-qPCR

Prostate cancer (PCa) remains one of the most widespread and perplexing of all human malignancies. Assessment of gene expression is thought to have an important impact on cancer diagnosis, prognosis and therapeutic decisions. In this context, we explored combined expression of PCa related target genes AMACR and PCA3 in 126 formalin fixed paraffin embedded prostate tissues (FFPE) from Moroccan patients, using quantitative real time reverse transcription-PCR (RT-qPCR). This quantification required data normalization accomplished using stably expressed reference genes (RGs). A panel of twelve RG was assessed, data being analyzed using GenEx V6 based on geNorm, NormFinder and statistical methods. Accordingly, the hnRNP A1 gene was identified and selected as the most stably expressed RG for reliable and accurate gene expression quantification in prostate tissues. The ratios of both PCA3 and AMACR gene expression relative to that of the hnRNP A1 gene were calculated and the performance of each target gene for PCa diagnosis was evaluated using receiver-operating characteristics. PCA3 and AMACR mRNA quantification based on RT-qPCR may prove useful in PCa diagnosis. Of particular interesting, combining PCA3 and AMACR quantification improved PCa prediction by increasing sensitivity with retention of good specificity.

Alloreactivity following in utero transplantation of cytokine-stimulated hematopoietic stem cells: the role of recipient CD4(-) cells.

OBJECTIVE: We have previously reported immunity to donor antigens following in utero transplantation (IUT) of cytokine-stimulated allogeneic hematopoietic stem cells (sca(+)/lin(-)) (day 9 of gestation). Transplanted mice showed accelerated rejection of donor skin grafts and high anti-donor cytotoxic response, a finding not seen in the control mice. This was accompanied by an enhancement of Th1 over Th2 cytokine production and persistent donor microchimerism. In order to assess the role of the thymus in allograft rejection, prenatal transplants were performed under similar experimental conditions at a later gestational age, when the thymus is more developed (day 13). MATERIALS AND METHODS: Cytokine-stimulated stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF)-purified sca(+)/lin(-) cells of C57BL/6 (H-2b, 1E(+)) background were injected into MHC-mismatched BALB/c (H-2d, 1E(-)) fetal mouse recipients at day 13 of gestation. Chimerism was determined by highly sensitive (0.001%) semiquantitative polymerase chain reaction (PCR). Mixed lymphocyte reaction (MLR) and cytotoxic T-cell assay (CTL) were used to evaluate tolerance vs immunity. Cytokine levels were quantified in MLR supernatants using ELISA assay. The percent of T cells was determined by flow cytometry (FACS) and CD4/CD8 ratio calculated. Postnatal boosts (transplants without conditioning) were performed at 6 months of age to enhance donor chimerism and test the degree of tolerance. RESULTS: When assayed at 4 months of age, donor-type cells were not detected in the spleen or in the peripheral blood of BALB/c mice inoculated with C57BL/6 sca(+)/lin(-) cells. Transplanted but not control animals demonstrated high anti-donor MLR but not CTL responses. The increase of MLR reactivity was correlated with high levels of IL-2. Furthermore, transplanted mice showed higher resistance to postnatal boosts with allogeneic bone marrow (BM) cells, when compared to the control mice. The later resistance was accompanied by the expansion of host-type CD4 cells. CONCLUSION: These data demonstrate that transplantation of cytokine-stimulated sca(+)/lin(-) allogeneic cells at 13 days of fetal development leads to the allosensitization, characterized by an enhancement of MLR alloreactivity and by the rejection of postnatal boosts (transplants without conditioning). Host-type CD4 cells might play a central role in this rejection. These findings indicate that the late injection of allogeneic cells may result in the development of allosensitization with subsequent donor graft rejection. Precise conditions for the development of tolerance must be established before prenatal transplants in humans with conditions other than SCID can be done.

The effect of FTY 720 on engraftment in a model of spontaneous allograft acceptance.

Further development in organ transplantation requires the utilization of new immunosuppressive drugs that-in addition to being effective against rejection-do not block tolerance. We previously reported that FTY 720, a drug that alters lymphocyte trafficking, has marked anti-rejection properties. We now investigate how FTY 720 influences tolerance in a model of graft acceptance by donor-specific blood transfusion (DSBT). Two different transplant models--heart transplantation (Htx) and intestinal transplantation (Itx)--were studied. We performed orthotopic Itx and heterotopic Htx using fully mismatched inbred male RA (RT1(p)) and PVG (RT1(c)) rats as donors and recipients. Tolerance was induced by DSBT on pre-transplant day -12. To test the effect of FTY 720 on DSBT-induced tolerance, we administered FTY 720 orally prior to DSBT. Itx: control rats succumbed to rejection at 18+/-4 days. DSBT alone prolonged survival to 101.9+/-18 days (P<0.05 vs untreated). Long-term survivors were tolerant (acceptance of secondary donor-specific Htx). Adjunction of FTY 720 prior to DSBT reduced survival to 55.9+/-44.7 days (P<0.05). However, long-term survivors still accepted secondary donor-specific Htx. Htx: control rats survived 9+/-0.6 days. DSBT alone prolonged survival indefinitely (>120 days) and induced tolerance (acceptance of secondary donor-specific Htx). Unlike in Itx, adjunction of FTY 720 prior to DSBT did not reduce Htx survival. Acceptance of secondary donor-specific Htx was not influenced by FTY 720. In Itx, FTY 720 counteracts the beneficial effect of pre-transplant DSBT and triggers acute rejection of primary, but not secondary grafts. In Htx, however, FTY 720 allows full development of tolerance. The mechanisms by which FTY 720 causes rejection in primary intestinal but not in heart grafts need to be elucidated.