Katia Cheaito

Modeling Human Neurological and Neurodegenerative Diseases: From Induced Pluripotent Stem Cells to Neuronal Differentiation and Its Applications in Neurotrauma

With the help of several inducing factors, somatic cells can be reprogrammed to become induced pluripotent stem cell (iPSCs) lines. The success is in obtaining iPSCs almost identical to embryonic stem cells (ESCs), therefore various approaches have been tested and ultimately several ones have succeeded. The importance of these cells is in how they serve as models to unveil the molecular pathways and mechanisms underlying several human diseases, and also in its potential roles in the development of regenerative medicine. They further aid in the development of regenerative medicine, autologous cell therapy and drug or toxicity screening. Here, we provide a comprehensive overview of the recent development in the field of iPSCs research, specifically for modeling human neurological and neurodegenerative diseases, and its applications in neurotrauma. These are mainly characterized by progressive functional or structural neuronal loss rendering them extremely challenging to manage. Many of these diseases, including Parkinsons disease (PD), Huntingtons disease (HD), Amyotrophic lateral sclerosis (ALS) and Alzheimers disease (AD) have been explored in vitro. The main purpose is to generate patient-specific iPS cell lines from the somatic cells that carry mutations or genetic instabilities for the aim of studying their differentiation potential and behavior. This new technology will pave the way for future development in the field of stem cell research anticipating its use in clinical settings and in regenerative medicine in order to treat various human diseases, including neurological and neurodegenerative diseases.

Escherichia coli Isolated from Urinary Tract Infections of Lebanese Patients between 2005 and 2012: Epidemiology and Profiles of Resistance

The early treatment of urinary tract infections (UTIs) is directly related to decrease in morbidity, which makes the empirical treatment of great importance. Recently, beta lactamases of several types have emerged as significant mechanisms of resistance in Gram-negative bacilli, especially Escherichia coli. Our aim was to study the urinary E. coli isolated from Lebanese patients and to characterize their mechanisms of resistance. The study analyzed data between 2005 and 2012 of UTIs caused by E. coli. The mechanisms of resistance were characterized by phenotypic and genotypic methods and the pulsed-field gel electrophoresis (PFGE) was used to determine the different bacterial clusters. As expected, the highest incidence was observed with E. coli (60.53–73.98%) followed by K. pneumoniae (5.32–8.33%). ICU isolates were constantly associated with the lowest rates of susceptibility to extended-spectrum cephalosporins, ciprofloxacin, as well as most of the tested antibiotics. A 100% occurrence of CTX-M in extended-spectrum β-lactamase (ESBL)-producing isolates was recorded, followed by TEM, SHV, and OXA. In addition, 15.9% harbored 4 different ESBL enzymes and only 13 isolates (14.8%) harbored only one enzyme (CTX-M). Over the years, the simultaneous susceptibility of E. coli to ceftazidime and ciprofloxacin decreased from 62.5% in 2006 to 48.7% in 2012. PFGE results demonstrated that 10 clusters were 32 generated, denoting diversity among detected isolates. Understanding the epidemiology of resistance is 33 instrumental for the implementation of recommendations for the management of antimicrobials, infection 34 control measures, as well as active surveillance and antimicrobial stewardship.

Assessment of combination therapy in BALB/c mice injected with carbapenem-resistant Enterobacteriaceae strains.

Monotherapeutic options for carbapenem resistant infections are limited. Studies suggest that combination therapy may be associated with better outcomes than monotherapies. However, this is still controversial. This study assessed, the efficacy of combination therapy against carbapenem resistant Enterobacteriaceae harboring singly various extended spectrum beta lactamase or carbapenemase encoding genes. Thus, four isolates harboring either blaCTXM-15, blaCTXM-15 and blaOXA-48, blaNDM-1, or blaKPC-2 genes were selected for testing. Minimal inhibitory concentration was determined by broth dilution method. Gene transcript levels on single and combined treatments were done in vitro and in vivo by qRT-PCR. Assessment of treatments was done in BALB/c mice according to a specific protocol. As such, the qRT-PCR revealed a significant decrease of transcript levels in all isolates upon using rifampicin or tigecycline, singly or in combination with colistin. However, variable levels were obtained using colistin singly or in combination with meropenem or fosfomycin. In vivo assessment showed that all combinations used were effective against isolates harboring blaCTXM-15, blaOXA-48, and blaNDM-1. Conversely, the most significant combination against the isolate harboring blaKPC-2 gene was colistin with either carbapenem, fosfomycin, or kanamycin. As a conclusion, combination therapy selected based on the type of carbapenemase produced, appeared to be non-toxic and might be effective in BALB/c mice. Therefore, the use of a rationally optimized combination therapy might lead to better results than monotherapy, however, clinical trials are needed for human consumption.

The Mediterranean Region: A Reservoir for CTX-M-ESBL- Producing Enterobacteriacae

The incidence of ESBL-producing bacteria is increasing worldwide, which represents a challenge for the healthcare systems. More recently, the emergence of the CTX-M group has been frequently reported as being associated with both nosocomial and community acquired infections. CTX-M-15 was identified as being the most prevalent in a large geographic area including Europe and the Middle East, suggesting the presence of a community reservoir of CTX-M enzymes disseminating in the Mediterranean area. Thus, this review will focus on the Mediterranean region to highlight the increasing prevalence of CTX-M-ESBL producing Enterobacteriacae.

Sphere-Formation Assay: Three-Dimensional in vitro Culturing of Prostate Cancer Stem/Progenitor Sphere-Forming Cells

Cancer Stem Cells (CSCs) are a sub-population of cells, identified in most tumors, responsible for the initiation, recurrence, metastatic potential, and resistance of different malignancies. In prostate cancer (PCa), CSCs were identified and thought to be responsible for the generation of the lethal subtype, commonly known as Castration-Resistant Prostate Cancer (CRPC). In vitro models to investigate the properties of CSCs in PCa are highly required. Sphere-formation assay is an in vitro method commonly used to identify CSCs and study their properties. Here, we report the detailed methodology on how to generate and propagate spheres from PCa cell lines and from murine prostate tissue. This model is based on the ability of stem cells to grow in non-adherent serum-free gel matrix. We also describe how to use these spheres in histological and immuno-fluorescent staining assays to assess the differentiation potential of the CSCs. Our results show the sphere-formation Assay (SFA) as a reliable in vitro assay to assess the presence and self-renewal ability of CSCs in different PCa models. This platform presents a useful tool to evaluate the effect of conventional or novel agents on the initiation and self-renewing properties of different tumors. The effects can be directly evaluated through assessment of the sphere-forming efficiency (SFE) over five generations or other downstream assays such as immuno-histochemical analysis of the generated spheres.

Abstract B090: Personalized research: Establishment and characterization of prostate cancer patient-derived organoids and cells

Several attempts have been made to understand the exact mechanisms underlying the pathogenesis of prostate cancer (PC); however, the currently available cancer models fail to recapitulate the heterogeneity of this tumor, its metastasis, and progression to castration-resistant states. Moreover, many drug candidates that succeed in preclinical models fail to deliver a good outcome in clinical trials, resulting in ineffective patient treatment and misused resources. In this aspect, the development of three-dimensional (3D) organoid culture systems has rendered it feasible to recap the convolution of organogenesis in vitro, promoting the generation of novel and more representative cancer models. Thus, the aim of this study is to generate patient-specific 3D organoids and cell lines, then characterize these models to identify potential prognostic biomarkers and treatments for PC while correlating the outcome with the collected clinical parameters. In our study, we are employing the R-spondin-1-based organoids technology to generate PC organoids and primary cell lines derived from fresh normal and tumor tissues of patients undergoing radical prostatectomy. Consequently, molecular characterization of the different patient-derived PC organoids and cell lines will be performed, followed by using this model to assess different classical and in-clinical trials drugs. We succeeded in establishing 21 normal and 18 tumor patient-derived organoids, out of a total of 23 patients, and then propagated the established organoids for up to 6 generations. Interestingly, 2D cells were derived from these organoids using the same culture media and were continuously passaged for up to 20 passages so far (more than 3 months). Our newly patient-derived organoids and cells show a typical epithelial phenotype and express CK8 and CK14 (prostate epithelial markers). To the best of our knowledge, this is the first study to address the establishment of primary PC cell lines from organoids, which will likely lead to a comprehensive understanding of mechanisms exploited in PC. Moreover, molecular characterization of this model when combined with pharmacologic profiles can aid in predicting a patient’s drug response; thus, our study represents an attempt to inaugurate what can possibly lead to personalized treatment of PC. Citation Format: Katia Anis Cheaito, Ola Hadadeh, Hisham Bahmad, Marwan El-Sabban, Albert El-Hajj, Deborah Mukherji, Wassim Abou-Kheir. Personalized research: Establishment and characterization of prostate cancer patient-derived organoids and cells [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B090.

Corrigendum: Escherichia coli Isolated from Urinary Tract Infections of Lebanese Patients between 2005 and 2012: Epidemiology and Profiles of Resistance.

[This corrects the article on p. 26 in vol. 2, PMID: 4415468.].

184Prevalence and Molecular Epidemiology of ESBL producing E.coli and K.pneumoniae in Lebanese Medical Centers; Strong Correlation between Antibiotic Consumption and Resistance to Cephalosporins and Ciprofloxacin

analyzed. 29.9% of E.coli strains and 31.1% of K.penumoniae were ESBL producers. Correlation data showed strong significance between the use of 3rd and/or 4th generation cephalosporins and resistance to these antibiotics. In addition, the correlation factor of cephalosporin consumption versus susceptibility to ciprofloxacin in E.coli (Hospital 1) was as low as -0.899 and -0.886 in K. pneumoniae (Hospital 3). blaCTX-M was produced by 97% of E.coli and 96.7% of K.pneumoniae. Specifically, blaCTX-M15 was commonly found. 22.3% of E.coli and 37.4% of K.pneumoniae co-produced the 4 studied beta-lactamases. Pulsed-field gel electrophoresis analysis demonstrated that there was no major clonal relationship among these ESBL producers.