Farah J. Nassar

Escherichia coli O157:H7—Clinical aspects and novel treatment approaches

Escherichia coli O157:H7 is a notorious pathogen often contracted by intake of contaminated water or food. Infection with this agent is associated with a broad spectrum of illness ranging from mild diarrhea and hemorrhagic colitis to the potentially fatal hemolytic uremic syndrome (HUS). Treating E. coli O157:H7 infection with antimicrobial agents is associated with an increased risk of severe sequelae such as HUS. The difficulty in treating this bacterium using conventional modalities of antimicrobial agent administration has sparked an interest in investigating new therapeutic approaches to this bacterium. These approaches have included the use of probiotic agents and natural products with variable success rates. In addition, novel modalities and regimen of antimicrobial agent administration have been assessed in an attempt at decreasing their association with aggravating infection outcomes.

MicroRNAs as biomarkers for early breast cancer diagnosis, prognosis and therapy prediction

ABSTRACT Breast cancer is a major health problem that affects one in eight women worldwide. As such, detecting breast cancer at an early stage anticipates better disease outcome and prolonged patient survival. Extensive research has shown that microRNA (miRNA) are dysregulated at all stages of breast cancer. miRNA are a class of small noncoding RNA molecules that can modulate gene expression and are easily accessible and quantifiable. This review highlights miRNA as diagnostic, prognostic and therapy predictive biomarkers for early breast cancer with an emphasis on the latter. It also examines the challenges that lie ahead in their use as biomarkers. Noteworthy, this review addresses miRNAs reported in patients with early breast cancer prior to chemotherapy, radiotherapy, surgical procedures or distant metastasis (unless indicated otherwise). In this context, miRNA that are mentioned in this review were significantly modulated using more than one statistical test and/or validated by at least two studies. A standardized protocol for miRNA assessment is proposed starting from sample collection to data analysis that ensures comparative analysis of data and reproducibility of results.

Approaches to treatment of emerging Shiga toxin-producing Escherichia coli infections highlighting the O104:H4 serotype

Shiga toxin-producing Escherichia coli (STEC) are a group of diarrheagenic bacteria associated with foodborne outbreaks. Infection with these agents may result in grave sequelae that include fatality. A large number of STEC serotypes has been identified to date. E. coli serotype O104:H4 is an emerging pathogen responsible for a 2011 outbreak in Europe that resulted in over 4000 infections and 50 deaths. STEC pathogenicity is highly reliant on the production of one or more Shiga toxins that can inhibit protein synthesis in host cells resulting in a cytotoxicity that may affect various organ systems. Antimicrobials are usually avoided in the treatment of STEC infections since they are believed to induce bacterial cell lysis and the release of stored toxins. Some antimicrobials have also been reported to enhance toxin synthesis and production from these organisms. Various groups have attempted alternative treatment approaches including the administration of toxin-directed antibodies, toxin-adsorbing polymers, probiotic agents and natural remedies. The utility of antibiotics in treating STEC infections has also been reconsidered in recent years with certain modalities showing promise.

Effects of subinhibitory concentrations of antimicrobial agents on Escherichia coli O157:H7 Shiga toxin release and role of the SOS response.

Treatment of Escherichia coli O157:H7 by certain antimicrobial agents often exacerbates the patients condition by increasing either the release of preformed Shiga toxins (Stx) upon cell lysis or their production through the SOS response-triggered induction of Stx-producing prophages. Recommended subinhibitory concentrations (sub-MICs) of azithromycin (AZI), gentamicin (GEN), imipenem (IMI), and rifampicin (RIF) were evaluated in comparison to norfloxacin (NOR), an SOS-inducer, to assess the role of the SOS response in Stx release. Relative expression of recA (SOS-inducer), Q (late antitermination gene of Stx-producing prophage), stx1, and stx2 genes was assessed at two sub-MICs of the antimicrobials for two different strains of E. coli O157:H7 using reverse transcription-real-time polymerase chain reaction. Both strains at the two sub-MICs were also subjected to Western blotting for LexA protein expression and to reverse passive latex agglutination for Stx detection. For both strains at both sub-MICs, NOR and AZI caused SOS-induced Stx production (high recA, Q, and stx2 gene expression and high Stx2 production), so they should be avoided in E. coli O157:H7 treatment; however, sub-MICs of RIF and IMI induced Stx2 production in an SOS-independent manner except for one strain at the first twofold dilution below MIC of RIF where Stx2 production decreased. Moreover, GEN caused somewhat increased Stx2 production due to its mode of action rather than any effect on gene expression. The choice of antimicrobial therapy should rely on the antimicrobial mode of action, its concentration, and on the nature of the strain.

miRNA as Potential Biomarkers of Breast Cancer in the Lebanese Population and in Young Women: A Pilot Study

Relative to western populations, the percentage of women diagnosed with breast cancer at a young age in Lebanon is high. While the younger age of the Lebanese population compared to the West certainly contributes to this difference, potential genetic, reproductive and/or biological factors likely play an important role. The objective of this study is to investigate the contribution of miRNAs in this setting through the analysis of the expression of five reported dysregulated miRNAs, miR-148b, miR-10b, miR-21, miR-221, and miR-155 in 20 normal and 57 cancerous breast tissues from Lebanese breast cancer patients. After finding their relative expression by quantitative reverse transcription real time PCR, the results were analyzed with respect to the patients’ clinical and histopathology presentations. Compared to normal breast tissues, significant upregulation of miR-155, miR-21 and miR-148b, notable downregulation of miR-10b and non-significant expression of miR-221 were observed in tumor tissues. Moreover, miR-10b was significantly underexpressed in estrogen/progesterone receptor (ER/PR) negative tumors relative to ER/PR positive tumor tissues. miR-155 was also significantly overexpressed in postmenopausal patients and in those of age at diagnosis greater than 40 years old as well as in PR negative or in human epidermal growth factor 2 (Her2) positive tissues. This study is the first one to report miRNA expression patterns in Lebanese breast cancer patients. We found that differential miRNA expression in breast cancer could be variable between Lebanese and Western populations. miR-10b was positively correlated with the ER and PR status and miR-155 could be a noteworthy biomarker for the menopausal state, age at diagnosis, PR and Her2 status. Hence, miRNA can be used as biomarkers for early breast cancer detection.

Genotypes and serotype distribution of macrolide resistant invasive and non- invasive Streptococcus pneumoniae isolates from Lebanon

BackgroundThis study determined macrolide resistance genotypes in clinical isolates of Streptococcus pneumoniae from multiple medical centers in Lebanon and assessed the serotype distribution in relation to these mechanism(s) of resistance and the source of isolate recovery.MethodsForty four macrolide resistant and 21 macrolide susceptible S. pneumoniae clinical isolates were tested for antimicrobial susceptibility according to CLSI guidelines (2008) and underwent molecular characterization. Serotyping of these isolates was performed by Multiplex PCR-based serotype deduction using CDC protocols. PCR amplification of macrolide resistant erm (encoding methylase) and mef (encoding macrolide efflux pump protein) genes was carried out.ResultsAmong 44 isolates resistant to erythromycin, 35 were resistant to penicillin and 18 to ceftriaxone. Examination of 44 macrolide resistant isolates by PCR showed that 16 isolates harbored the erm(B) gene, 8 isolates harbored the mef gene, and 14 isolates harbored both the erm(B) and mef genes. There was no amplification by PCR of the erm(B) or mef genes in 6 isolates. Seven different capsular serotypes 2, 9V/9A,12F, 14,19A, 19F, and 23, were detected by multiplex PCR serotype deduction in 35 of 44 macrolide resistant isolates, with 19F being the most prevalent serotype. With the exception of serotype 2, all serotypes were invasive. Isolates belonging to the invasive serotypes 14 and 19F harbored both erm(B) and mef genes. Nine of the 44 macrolide resistant isolates were non-serotypable by our protocols.ConclusionMacrolide resistance in S. pneumoniae in Lebanon is mainly through target site modification but is also mediated through efflux pumps, with serotype 19F having dual resistance and being the most prevalent and invasive.

Epidemiologic characteristics, serotypes, and antimicrobial susceptibilities of invasive Streptococcus pneumoniae isolates in a nationwide surveillance study in Lebanon

Invasive pneumococcal disease (IPD) associated with Streptococcus pneumonia is a major public health problem worldwide for all age groups, including in Lebanon. Prevention through vaccination remains the most valuable tool to decrease the burden of disease. Pneumococcal conjugate vaccine 7 (PCV7), marketed internationally including in the Middle East and North Africa region for the prevention of IPD, was introduced in Lebanon in 2006, followed by PCV10 and PCV13 in 2010. However, none of these is currently part of the Extended Program of Immunization schedule and published data on IPD incidence, pneumococcal serotypes and vaccine coverage in the region are lacking. The Lebanese Inter-Hospital Pneumococcal Surveillance Program is a surveillance system set up to determine the burden of IPD and the prevalent serotypes responsible. The aim of this prospective 6-year study carried out in 78 hospitals throughout Lebanon was to obtain such data to help health authorities make informed decisions on the implementation of pneumococcal vaccination at the national level. A total of 257 isolates of culture-confirmed Streptococcus pneumoniae were evaluated. Considering all age groups, vaccine coverage was 41.4%, 53.9%, and 67.2% for PCV7, PCV10, and PCV13 serotypes, respectively; for patients <2, 2-5, and >60 years of age, PCV7 coverage was 50%, 51%, and 35%, respectively; PCV10 coverage was 53%, 74%, 45%, respectively; and PCV13 coverage was 63%, 80%, and 68%, respectively. Overall, 17.4% of these isolates were penicillin-G non-susceptible using the latest established breakpoints and mortality occurred in 23.5% of the patients with non-susceptible isolates. In addition, 10.9% of isolates were multi-drug-resistant. The highest mortality rates were observed in the eldest (>60 years of age) and youngest (<2 years of age) patients. The most prevalent invasive serotypes identified were those found in currently available pneumococcal conjugate vaccines, emphasizing the importance of implementing the vaccine in the routine immunization schedule at the national level. Continuation of current surveillance practices will help assess the impact of vaccine implementation on IPD epidemiology, serotype distribution and antibiotic resistance patterns.

ATL-derived exosomes modulate mesenchymal stem cells: potential role in leukemia progression

BackgroundExosomes are membrane nano-vesicles secreted by a multitude of cells that harbor biological constituents such as proteins, lipids, mRNA and microRNA. Exosomes can potentially transfer their cargo to other cells, implicating them in many patho-physiological processes. Mesenchymal stem cells (MSCs), residents of the bone marrow and metastatic niches, potentially interact with cancer cells and/or their derived exosomes. In this study, we investigated whether exosomes derived from adult T-cell leukemia/lymphoma (ATL) cells act as intercellular messengers delivering leukemia-related genes that modulate the properties of human MSCs in favor of leukemia. We hypothesized that the cargo of ATL-derived exosomes is transferred to MSCs and alter their functional behavior to support the establishment of the appropriate microenvironment for leukemia.ResultsWe showed that both ATL cells (C81 and HuT-102) and patient-derived cells released Tax-containing exosomes. The cargo of HuT-102-derived exosomes consisted of miR-21, miR-155 and vascular endothelial growth factor. We demonstrated that HuT-102-derived exosomes not only deliver Tax to recipient MSCs, but also induce NF-κB activation leading to a change in cellular morphology, increase in proliferation and the induction of gene expression of migration and angiogenic markers.ConclusionsThis study demonstrates that ATL-derived exosomes deliver Tax and other leukemia-related genes to MSCs and alter their properties to presumably create a more conducive milieu for leukemia. These findings highlight the contribution of leukemia-derived exosomes in cellular transformation and their potential value as biomarkers and targets in therapeutic strategies.

MicroRNA Expression in Ethnic Specific Early Stage Breast Cancer: An Integration and Comparative Analysis

Breast cancer (BC) has a higher incidence in young Lebanese woman as compared to the West. We assessed the microRNA (miRNA) microarray profile of tissues derived from Lebanese patients with early BC and performed mRNA-miRNA integration analysis. 173 miRNAs were significantly dysregulated in 45 BC versus 17 normal adjacent breast tissues, including 74 with a fold change more than two of which 17 were never reported before in cancer. Integration analysis of mRNA-miRNA microarray data revealed a potential role of 51 dysregulated miRNA regulating 719 tumor suppressive or oncogenic mRNA associated with increased proliferation and decreased migration and invasion. We then performed a comparative miRNA microarray profile analysis of BC tissue between these 45 Lebanese and 197 matched American BC patients. Notably, Lebanese BC patients had 21 exclusively dysregulated miRNA (e.g. miR-31, 362-3p, and 663) and 4 miRNA with different expression manner compared to American patients (e.g. miR-1288-star and 324-3p). Some of these differences could reflect variation in patient age at diagnosis or ethnic variation affecting miRNA epigenetic regulation or sequence of miRNA precursors. Our data provide a basis for genetic/epigenetic investigations to explore the role of miRNA in early stage BC in young women, including ethnic specific differences.

Ascorbic Acid in Colon Cancer: From the Basic to the Clinical Applications

Given the safety and potential benefits of intravenous ascorbic acid (AA) administration in cancer patients, there is merit in further exploring this therapeutic concept. In this review, we discuss the potential benefits of intravenous AA administration on colorectal cancer and we specifically focus on its effect on glycolysis in mutant and wild type RAS. We perform a PubMed and Ovid MEDLINE search using ascorbic acid, intravenous vitamin C, KRAS mutation, BRAF mutation and colorectal cancer (CRC) as keywords. At the cellular level, colorectal cancer cells undergo a metabolic shift called the Warburg effect to allow for more glucose absorption and utilization of glycolysis. This shift also allows AA to enter which leads to a disruption in the Warburg effect and a shutdown of the downstream KRAS pathway in mutated KRAS colon cancer cells. At the clinical level, AA is associated with tumour regression in advanced disease and improved tolerability and side effects of standard therapy. Based on these findings, we conclude that further clinical trials are needed on a larger scale to examine the therapeutic benefits of AA in colon cancer.