Muhammad W. Shahid

The learning curve of in vivo probe-based confocal laser endomicroscopy for prediction of colorectal neoplasia.

BACKGROUND Probe-based confocal laser endomicroscopy (pCLE) is an emerging tool for in vivo imaging of the GI tract that requires the endoscopist to interpret microscopic images. The learning curve for interpretation of pCLE images is unknown. OBJECTIVE To examine the learning curve of correctly identifying benign and neoplastic colorectal lesions by using pCLE and to evaluate the learning curve of obtaining high-quality images. DESIGN Prospective, double-blind review of pCLE images of 76 colorectal lesions by using corresponding polypectomies as the reference standard. A training set of 20 images with known histology was first reviewed to standardize image interpretation, followed by blinded review of 76 unknown images. SETTING Eleven endoscopists from 3 different endoscopy centers evaluated the images obtained by 1 endoscopist using the high-definition confocal probe. PATIENTS Patients undergoing screening and surveillance colonoscopies. INTERVENTION Intravenous fluorescein pCLE imaging of colorectal lesions followed by polypectomies. MAIN OUTCOME MEASUREMENTS Accuracy of image interpretation with constructing learning curve for pCLE image interpretation and acquisition. RESULTS Of the 76 colorectal lesions, 51 (67%) were neoplastic and 25 (33%) were benign, based on histopathology. Accuracy for the overall group was 63% for lesions 1 to 20, 64% for lesions 21 to 40, 79% for lesions 41 to 60, and 86% for lesions 61 to 76. The ability to obtain high-quality images was stable over the 76 cases. LIMITATIONS Small sample size and use of offline video sequences. CONCLUSIONS Accurate interpretation of pCLE images for predicting neoplastic lesions can be learned rapidly by a wide range of GI specialists. Furthermore, the ability to acquire high-quality pCLE images is also quickly learned.

Cytoprotective effect of chlorogenic acid against hydrogen peroxide-induced oxidative stress in MC3T3-E1 cells through PI3K/Akt-mediated Nrf2/HO-1 signaling pathway

Osteoporosis is a disorder of bone and its development is closely associated with oxidative stress and reactive oxygen species (ROS). Chlorogenic acid (CGA) has potential antioxidant effects and its pharmacological action in osteoblasts is not clearly understood. The present study aimed to clarify the protective effects and mechanisms of CGA on hydrogen peroxide (H2O2)-induced oxidative stress in osteoblast cells. MC3T3-E1 cells were treated with H2O2 to induce oxidative stress model in vitro. Cells were treated with CGA prior to H2O2 exposure, the intracellular ROS production, malondialdehyde content, nitric oxide release and glutathione level were measured. We also investigated the protein levels of heme oxygenase-1 (HO-1), the nuclear translocation of transcription factor NF-erythroid 2-related factor (Nrf2) and the phosphorylation levels of Akt in CGA-treated cells. The results showed that pretreatment of CGA could reverse the inhibition of cell viability and suppress the induced apoptosis and caspase-3 activity. Additionally, it significantly reduced H2O2-induced oxidative damage in a dose-dependent manner. Furthermore, it induced the protein expression of HO-1 together with its upstream mediator Nrf2, and activated the phosphorylation of Akt in MC3T3-E1 cells. LY294002, a PI3K/Akt inhibitor, significantly suppressed the CGA-induced Nrf2 nuclear translocation and HO-1 expression. Reduction of cell death mediated by CGA in presence of H2O2 was significantly inhibited by Zinc protoporphyrin IX (a HO-1 inhibitor) and LY294002. These data demonstrated that CGA protected MC3T3-E1 cells against oxidative damage via PI3K/Akt-mediated activation of Nrf2/HO-1 pathway, which may be an effective drug in treatment of osteoporosis.

Characteristics and genetic diversity of multi-drug resistant extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolated from bovine mastitis

A characterization of the drug resistance profiles, identification of PCR-based replicon typing, and multilocus sequence typing (MLST) and analysis of 46 ESBL-producing Escherichia coli from cows with mastitis are described. All multidrug-resistant isolates of various phylogenetic groups (A = 31, B1= 3, B2 = 2, D = 10) were ESBL-producers of genotypes CTX-M-15 (29), CTX-M-55 (4), CTX-M-14 (4), CTX-M-3 (1), CTX-M-1 (1), TEM (22) and SHV (8) that were found on conjugative plasmids of diverse incompatibility groups (primarily IncF). Transconjugation experiments indicated successful (100%) trans-conjugation, which was verified phenotypically and genotypically. A total of 28 sequence types (ST) were identified, with 10% of isolates being ST410, and 9 other ST that were assigned arbitrary numbers, reflecting the degree of diversity. Multilocus sequence analysis revealed two lineages, a dominant and a small lineage. Split-decomposition showed intraspecies recombination clearly contributed in genetic recombination generating genotypic diversity among the isolates, and a lack of interspecies recombination. This coherent analysis on genetic structure of multidrug-resistant pathogenic E. coli population isolated from mastitic-milk weaponized with resistance elements from a large, rapidly developing country will be a helpful contribution for epidemiology and surveillance of drug resistance patterns, and understanding their global diversity.

T1166 Interobserver Agreement and Accuracy Among International Experts of Probe-Based Confocal Laser Microscopy (pCLE) in Predicting Colorectal Neoplasia

G A A b st ra ct s infliximab. We describe the first case of disseminated mucocutaneous granulomas in a patient with UC receiving infliximab. Case: 19-year-old female with UC in clinical remission, who had been treated with infliximab (10mg/kg/every 8 weeks) for 2 years, presented with multiple skin, nasal and oral lesions developing over the course of three months. The skin lesions were erythematous annular plaques on the right hip, inner thighs and forearms. Multiple ulcers were found in the nasal turbinates and one on the hard palate. Repeat esophagogastroduodenoscopy and colonoscopy at the time of evaluation for these lesions showed mild chronic esophagitis andmild chronic proctitis. She had a normal MRI enterography. Her IBD serology (pANCA positive; ASCA IgA, IgG negative) was also supportive of a diagnosis of UC. Histological examination of the skin biopsy revealed a diffuse interstitial granulomatous infiltrate with lymphocytes and histiocytes. Biopsy of the nasal and oral mucosa revealed a marked submucosal inflammatory infiltrate composed of lymphocytes, plasma cells and eosinophils with granulomas and foci of necrosis. Special stains and cultures for bacteria, fungus and mycobacteria were negative. Evaluation was negative for Cryptococcus, Bordetella henselae, Blastomyces and Histoplasma. Angiotensin I converting enzyme was normal. CT scan of the sinuses and chest radiograph were normal as well. Discussion: Metastatic Crohns disease, opportunistic infections and granulomatous reaction to infliximab were considered in the differential diagnosis. IGD was the most likely cause of these lesions in our patient. Metastatic Crohns disease was unlikely given the quiescence of her intestinal disease compared to the activity of the mucocutaneous involvement. Opportunistic infections, which can be fatal in the face of anti-TNF therapy were ruled out by negative cultures. Furthermore, in our patient, her skin and mucosal lesions had significantly improved with no other intervention except for the discontinuation of her anti-TNF therapy. Conclusion: Granulomatous diseases, including IGD, should be considered in the differential diagnosis of annular or ulcerated lesions in the setting of anti TNFtherapy in a patient with IBD.

Reprogrammed lipid metabolism in bladder cancer with cisplatin resistance

Due to its tendency to recur and acquire chemoresistance quickly, bladder cancer (BC) remains to be an elusive and difficult disease. Patients with recurrent and chemoresistant BC have an extremely poor prognosis. One possible approach that may provide insightful and valuable information regarding resistance mechanisms is looking into the lipid metabolism of BC cells. Metabolism of lipids is essential for cancer cells and is associated with the regulation of a variety of key cellular processes and functions. This study conducted a comparative lipidomic profiling of two isogenic human T24 bladder cancer cell lines, one of which is clinically characterized as cisplatin-sensitive (T24S) and the other as cisplatin-resistant (T24R). Immunohistochemistry analysis revealed that expression of cytosolic acetyl-CoA synthetase 2 (ACSS2) is positively correlated with aggressive BC. Ultra performance liquid chromatography-mass spectrometry (UPLC-MS) analysis profiled a total of 1,864 lipids and levels of differentially expressed lipids suspected of being associated with cisplatin resistance were determined. In addition, we found that ACSS2 inhibition greatly perturbed levels of metabolites, including CE(18:1), CE(22:6), TG(49:1), and TG(53:2). This study broadens our current knowledge on the links between cisplatin resistance and lipid metabolism in aggressive BC and suggests potential biomarkers for identifying higher-risk patients.

The Prognostic 97 Chemoresponse Gene Signature in Ovarian Cancer

Patient diagnosis and care would be significantly improved by understanding the mechanisms underlying platinum and taxane resistance in ovarian cancer. Here, we aim to establish a gene signature that can identify molecular pathways/transcription factors involved in ovarian cancer progression, poor clinical outcome, and chemotherapy resistance. To validate the robustness of the gene signature, a meta-analysis approach was applied to 1,020 patients from 7 datasets. A 97-gene signature was identified as an independent predictor of patient survival in association with other clinicopathological factors in univariate [hazard ratio (HR): 3.0, 95% Confidence Interval (CI) 1.66–5.44, p = 2.7E-4] and multivariate [HR: 2.88, 95% CI 1.57–5.2, p = 0.001] analyses. Subset analyses demonstrated that the signature could predict patients who would attain complete or partial remission or no-response to first-line chemotherapy. Pathway analyses revealed that the signature was regulated by HIF1α and TP53 and included nine HIF1α-regulated genes, which were highly expressed in non-responders and partial remission patients than in complete remission patients. We present the 97-gene signature as an accurate prognostic predictor of overall survival and chemoresponse. Our signature also provides information on potential candidate target genes for future treatment efforts in ovarian cancer.

An 8-gene signature for prediction of prognosis and chemoresponse in non-small cell lung cancer

Identification of a potential gene signature for improved diagnosis in non-small cell lung cancer (NSCLC) patient is necessary. Here, we aim to establish and validate the prognostic efficacy of a gene set that can predict prognosis and benefits of adjuvant chemotherapy (ACT) in NSCLC patients from various ethnicities. An 8-gene signature was calculated from the gene expression of 181 patients using univariate Cox proportional hazard regression analysis. The prognostic value of the signature was robustly validated in 1,477 patients from five microarray independent data sets and one RNA-seq data set. The 8-gene signature was identified as an independent predictor of patient survival in the presence of clinical parameters in univariate and multivariate analyses [hazard ratio (HR): 2.84, 95% confidence interval CI (1.74-4.65), p=3.06e-05, [HR] 2.62, 95% CI (1.51-4.53), p=0.001], respectively. Subset analysis demonstrated that the 8-gene signature could identify high-risk patients in stage II-III with improved survival from ACT [(HR) 1.47, 95% CI (1.01-2.14), p=0.044]. The 8-gene signature also stratified risk groups in EGFR-mutated and wild-type patients. In conclusion, the 8-gene signature is a strong and independent predictor that can significantly stratify patients into low- and high-risk groups. Our gene signature also has the potential to predict patients in stage II-III that are likely to benefit from ACT.

Alpha-oxoglutarate inhibits the proliferation of immortalized normal bladder epithelial cells via an epigenetic switch involving ARID1A

Interstitial cystitis (IC) is a chronic urinary tract disease that is characterized by unpleasant sensations, such as persistent pelvic pain, in the absence of infection or other identifiable causes. We previously performed comprehensive metabolomics profiling of urine samples from IC patients using nuclear magnetic resonance and gas-chromatography/mass spectrometry and found that urinary α-oxoglutarate (α-OG), was significantly elevated. α-OG, a tricarboxylic acid (TCA) cycle intermediate, reportedly functions to suppress the proliferation of immortalized normal human bladder epithelial cells. Here, we identified AT-rich interactive domain 1 A (ARID1A), a key chromatin remodeler, as being hypomethylated and upregulated by α-OG treatment. This was done through EPIC DNA methylation profiling and subsequent biochemical approaches, including quantitative RT-PCR and western blot analyses. Furthermore, we found that α-OG almost completely suppresses ten-eleven translocation (TET) activity, but does not affect DNA methyltransferase (DNMT) activity. Altogether, our studies reveal the potential role of α-OG in epigenetic remodeling through its effects on ARID1A and TET expression in the bladder. This may provide a new possible therapeutic strategy in treating IC.

Menthol, a unique urinary volatile compound, is associated with chronic inflammation in interstitial cystitis.

Chronic inflammation is a potential systemic risk factor for many bladder dysfunctions, including interstitial cystitis (IC). However, the underlying mechanism through which a healthy bladder protects itself from inflammatory triggers remains unknown. In this study, we identified odor compounds in urine obtained from IC patients and healthy controls. Using comprehensive solid-phase microextraction-gas chromatography-time-of-flight-mass spectrometry (SPME-GC-TOF-MS) profiling and bioinformatics, we found that levels of urinary volatile metabolites, such as menthol, were significantly reduced in IC patients, compared to healthy controls. In an attempt to understand the mechanistic meaning of our volatile metabolites data and the role of menthol in the immune system, we performed two independent experiments: (a) cytokine profiling, and (b) DNA microarray. Our findings suggest that lipopolysaccharide (LPS)-stimulated inflammatory events, such as the production and secretion of inflammatory cytokines (e.g., TNF-α, IL-6, and IL-1β) and the activation of NF-κB and associated proteins within a large signaling network (e.g., Akt, TLR1, TNFAIP3, and NF-κB), are suppressed by the presence of menthol. These findings broaden our knowledge on the role of urinary menthol in suppressing inflammatory events and provide potential new strategies for alleviating both the odor and inflammation associated with IC.

Quantitative Proteomic Analysis Reveals Caffeine-Perturbed Proteomic Profiles in Normal Bladder Epithelial Cells

Lower urinary tract symptoms (LUTSs) are highly prevalent among the elderly and negatively impact quality of life. Since caffeinated beverages are enjoyed worldwide and the relationship between LUTS and caffeine is still not fully understood, it would be of particular interest to examine the underlying mechanisms that drive caffeines influence on LUTS development and progression. The aim of this study is to characterize the effects of caffeine on hTert‐immortalized normal bladder epithelial cells by investigating whether exposure to caffeine can cause potential changes in the bladder proteome and/or biological pathways. In labeled LC–MS/MS proteomic analysis, 57 proteins are found as being differentially expressed in caffeine‐treated bladder epithelial cells, compared to controls; this included 32 upregulated and 25 downregulated proteins. Further functional gene enrichment analysis reveals that caffeine affects major biological pathways, including those for “muscle contraction” and “chromatin assembly.” These findings provide new scientific insights that may be useful in future studies investigating the role of caffeine in bladder dysfunctions.