Assessment of MUC5AC and MUC2 Immunoexpression in Glandular Odontogenic Cysts, Dentigerous Cysts, and Mucoepidermoid Carcinomas.

Glandular odontogenic cysts (GOCs) and dentigerous cysts may show mucous metaplasia. Central mucoepidermoid carcinoma is very rare and mostly associated with dental cysts. It is hypothesized that odontogenic cysts showing mucus differentiation in their lining, have a propensity to transform into MEC. The present study is the first attempt to explore the relationship between odontogenic cysts [GOCs and dentigerous cysts with mucus metaplasia (DCMM)] and MEC by evaluating immunoexpression of MUC5AC and MUC2. Immunoexpression of MUC5AC and MUC2 was evaluated semiquantitatively in GOCs (20 cases), DCMMs (20 cases), and MECs (20 cases). The percentage of positive cells, intensity, and localization of immunoexpression were assessed for each marker in all cases. Of GOCs, DCMMs, and MECs cases, 85%, 70%, and 80%, respectively, were immunopositive for MUC5AC. Strong cytoplasmic immunoreactivity for MUC5AC was noted, particularly in mucous cells present diffusely within MECs. However, the immunoreactivity was limited to the epithelial lining of GOCs and DCMMs. Most of the MECs (60%) showed more than 25% positivity for MUC5AC, followed by GOCs, and the least in DMMCs. Mild cytoplasmic and nuclear positivity of MUC2 was noted only in epithelial lining cells of 70% GOCs and 45% DCMMs. Whereas, 55% of MECs displayed moderate to strong cytoplasmic and membranous immunopositivity for MUC2 exclusively within mucous cells. As MECs showed strong MUC5AC immunoreactivity in mucous cells, immunoexpression of MUC5AC in odontogenic cysts with mucus cells can possibly explain the pathogenesis of MEC from cysts. However, the variable expression of MUC2 did not give any strong evidence regarding its role as a marker.

Nuclear morphology in breast lesions: refining its assessment to improve diagnostic concordance.

AIMS: Although evaluation of nuclear morphology is important for the diagnosis and categorisation of breast lesions, the criteria used to assess nuclear atypia rely upon the subjective evaluation of several features that may result in inter- and intraobserver variation. This study aims to refine the definitions of cytonuclear features in various breast lesions. METHODS AND RESULTS: ImageJ was used to assess the nuclear morphological features including nuclear diameter, axis length, perimeter, area, circularity and roundness in 160 breast lesions comprising ductal carcinoma in situ (DCIS), invasive breast carcinoma of no special type (IBC-NST), tubular carcinoma, usual ductal hyperplasia (UDH), columnar cell change (CCC) and flat epithelial atypia (FEA). Reference cells included normal epithelial cells, red blood cells (RBCs) and lymphocytes. Reference cells showed size differences not only between normal epithelial cells and RBCs but also between RBCs in varied-sized blood vessels. Nottingham grade nuclear pleomorphism scores 1 and 3 cut-offs in IBC-NST, compared to normal epithelial cells, were < ×1.2 and > ×1.4 that of mean maximum Feret's diameter and < ×1.6 and > ×2.4 that of mean nuclear area, respectively. Nuclear morphometrics were significantly different in low-grade IBC-NST versus tubular carcinoma, low-grade DCIS versus UDH and CCC versus FEA. No differences in the nuclear features between grade-matched DCIS and IBC-NST were identified. CONCLUSION: This study provides a guide for the assessment of nuclear atypia in breast lesions, refines the comparison with reference cells and highlights the potential diagnostic value of image analysis tools in the era of digital pathology.

Assessment of cervical vascularization density in patients with locally advanced squamous cell cervical carcinoma evaluated in colour Doppler and power Doppler functions.

PURPOSE: The aim of the prospective study was to assess changes during treatment and prognostic significance of cervical vascularization density in patients with cervical squamous cell carcinoma (SCC) staged II B and III B and to find relationship of cervical vascularization density with tumour diameter, grading, staging and age of patients. METHODS: The study group included 50 patients who underwent transvaginal Doppler ultrasonography prior to chemoradiotherapy, after external beam radiation therapy (EBRT) and 6 weeks after HDR brachytherapy. The colour Doppler (CD) vascularity index (CDVI) and the power Doppler (PD) vascularity index (PDVI) in cervical tumour were examined. RESULTS: CDVI and PDVI values decreased significantly during radiotherapy (0.13 (95% CI 0.09-0.16); 0.09 (95% CI 0.07-0.11) and 0.05 (95% CI 0.03-0.06) for CDVI (p < 0.001) and 0.26 (95% CI 0.22-0.31); 0.18 (95% CI 0.14-0.22) and 0.08 (95% CI 0.06-0.11) for PDVI (p < 0.001)). No statistically significant associations of CDVI and PDVI with tumour diameter, grading, staging and age of patients were found. The higher (above median) CDVI values before EBRT were associated with better OS (p = 0.041). The higher (above median) PDVI values before EBRT were associated with superior DFS (p = 0.011) and OS (p < 0.001). DFS and OS did not differ significantly regarding CDVI and PDVI values after EBRT and after the treatment. CONCLUSIONS: In the study group, cervical vascularization density evaluated in CD and PD functions did not depend on tumour diameter, grading, staging and age of patients and decreased during radiotherapy. The prognosis for our patients with CDVI and PDVI before the treatment above the median value was better than compared to patients with these parameters below the median value.

Evaluation of PathTezt™, a Liquid-Based Cytology System.

Liquid-based preparation (LBP) cytology is commonly used in most laboratories these days due to its convenience and reliable results for the cervical cancer screening program. The PathTezt™ Liquid-based Pap smear is a second-generation LBP, which uses a filter-based concentration technique in processing the sample. OBJECTIVE: This study was done to evaluate the cellular fixation, morphology, quality of smear in gynae cytology, and diagnostic interpretation of cervical cytological smears produced by the PathTezt liquid-based processor. MATERIALS AND METHODS: A total of 400 pap smear samples were taken and processed using the PathTezt 2000 processor. The slides were evaluated in terms of sample adequacy, percentage of the circle covered by epithelial cells, cellular distribution, obscuring factors, and cell fixation. RESULTS: About 95.25% (381) of the samples were satisfactory for the evaluation. In 19 (4.75%) of the samples, epithelial cells covered less than 50% of the circle. A sample with good cellular distribution was seen in 92% of the cases, while 354 (88.5%) samples showed minimal inflammatory background. Almost all the smears (95.75%) had no erythrocytes in the background. All smears showed good quality fixation features toward nuclear, cytoplasm, and microorganisms. The total performance rate was 99%. CONCLUSION: Although the PathTezt liquid-based processor is still new compared to other first-generation LBP, the smears produced by this method were of high quality and it was cost-effective.

Survey on reporting of epithelial cells in urine sediment as part of external quality assessment programs in Brazilian laboratories.

INTRODUCTION: Epithelial cells (ECs) are structures regularly observed during urine microscopy analysis. The correct identification of EC subtypes can be useful since renal tubular epithelial cells (RTECs) are clinically relevant. We investigate the urinary ECs report and the judgement of its clinical importance by Brazilian laboratories. MATERIALS AND METHODS: A survey with four questions was made available to participants of the Urinalysis External Quality Assessment Program (EQAP) from Controllab. Laboratories composed 3 groups: (1) differentiating ECs subtypes: "squamous", "transitional" and "RTECs"; (2) differentiating ECs subtypes: "squamous" or "non-squamous" cells; (3) without ECs subtype identification. Participants did not necessarily answer to all questions and the answers were evaluated both within the same laboratory's category and within different categories of laboratories. RESULTS: A total of 1336 (94%) laboratories answered the survey; Group 1, 119/140 (85%) reported that ECs differentiation is important to the physician and 62% want to be evaluated by EQAP, while in Group 3, 455/1110 (41%) reported it is useful to them, however only 25% want be evaluated by EQAP. Group 2 laboratories 37/51 (73%) reported that the information is important, but only 13/52 (25%) are interested in an EQAP with differentiation of the 3 ECs subtypes. CONCLUSION: Most of the laboratories do not differentiate ECs in the three subtypes, despite the clinical importance of RTECs. Education of laboratory staff about the clinical significance of urinary particles should be considered a key priority.

Quick assessment of DNA damage in cervical epithelial cells using a chromatin dispersion test.

PURPOSE: This study was aimed to quantify genomic DNA breakages in the cervical epithelium cells of patients diagnosed with different grades of cervical lesions using a quick test based on chromatin dispersion after controlled protein depletion. The association between the progressive stages of cervical dysplasia and the levels of DNA damage, taking into account the presence of papillomavirus human (HPV) infection, was investigated. METHODS: A hospital-based unmatched case-control study was conducted during 2018 with a sample of 78 women grouped according to histological diagnosis as follows: 23 women with low grade-squamous intraepithelial lesion (LG-SIL), 34 women with high grade- squamous intraepithelial lesion (HG-SIL), and three women with cervical carcinoma (CC). In parallel, 15 women without cervical lesions were included as a Control cohort. DNA damage levels in cervical epithelial cells were assessed using the chromatin dispersion test (CDT) and controlled in parallel with DNA breakage detection coupled with florescent in situ hybridization (DBD‒FISH) using whole genomic DNA probes. RESULTS: CDT produces different morphotypes in the cervical epithelium that can be associated with the level of DNA breakage revealed with DBD‒FISH. A significant increase of DNA damage was correlated with the histological progression of the patients and human papillomavirus (HPV) infection. CONCLUSION: The CDT is a simple, accurate and inexpensive morphological bioassay to identify different levels DNA damage that can be associated with the level of abnormal cells present in the cervical epithelium in patients who commonly present HPV infection.

Wide Area Transepithelial Sampling with Computer-Assisted Analysis (WATS3D) Is Cost-Effective in Barrett's Esophagus Screening.

BACKGROUND: Wide area transepithelial sampling with three-dimensional computer-assisted analysis (WATS3D) is an adjunct to the standard random 4-quadrant forceps biopsies (FB, "Seattle protocol") that significantly increases the detection of Barrett's esophagus (BE) and associated neoplasia in patients undergoing screening or surveillance. AIMS: To examine the cost-effectiveness of adding WATS3D to the Seattle protocol in screening patients for BE. METHODS: A decision analytic model was used to compare the effectiveness and cost-effectiveness of two alternative BE screening strategies in chronic gastroesophageal reflux disease patients: FB with and without WATS3D. The reference case was a 60-year-old white male with gastroesophageal reflux disease (GERD). Effectiveness was measured by the number needed to screen to avert one cancer and one cancer-related death, and quality-adjusted life years (QALYs). Cost was measured in 2019 US$, and the incremental cost-effectiveness ratio (ICER) was measured in $/QALY using thresholds for cost-effectiveness of $100,000/QALY and $150,000/QALY. Cost was measured in 2019 US$. Cost and QALYs were discounted at 3% per year. RESULTS: Between 320 and 337 people would need to be screened with WATS3D in addition to FB to avert one additional cancer, and 328-367 people to avert one cancer-related death. Screening with WATS3D costs an additional $1219 and produced an additional 0.017 QALYs, for an ICER of $71,395/QALY. All one-way sensitivity analyses resulted in ICERs under $84,000/QALY. CONCLUSIONS: Screening for BE in 60-year-old white male GERD patients is more cost-effective when WATS3D is used adjunctively to the Seattle protocol than with the Seattle protocol alone.

DNA damage in lens epithelial cells exposed to occupationally-relevant X-ray doses and role in cataract formation.

The current framework of radiological protection of occupational exposed medical workers reduced the eye-lens equivalent dose limit from 150 to 20 mSv per year requiring an accurate dosimetric evaluation and an increase understanding of radiation induced effects on Lens cells considering the typical scenario of occupational exposed medical operators. Indeed, it is widely accepted that genomic damage of Lens epithelial cells (LEC) is a key mechanism of cataractogenesis. However, the relationship between apoptosis and cataractogenesis is still controversial. In this study biological and physical data are combined to improve the understanding of radiation induced effects on LEC. To characterize the occupational exposure of medical workers during angiographic procedures an INNOVA 4100 (General Electric Healthcare) equipment was used (scenario A). Additional experiments were conducted using a research tube (scenario B). For both scenarios, the frequencies of binucleated cells, micronuclei, p21-positive cells were assessed with different doses and dose rates. A Monte-Carlo study was conducted using a model for the photon generation with the X-ray tubes and with the Petri dishes considering the two different scenarios (A and B) to reproduce the experimental conditions and validate the irradiation setups to the cells. The simulation results have been tallied using the Monte Carlo code MCNP6. The spectral characteristics of the different X-ray beams have been estimated. All irradiated samples showed frequencies of micronuclei and p21-positive cells higher than the unirradiated controls. Differences in frequencies increased with the delivered dose measured with Gafchromic films XR-RV3. The spectrum incident on eye lens and Petri, as estimated with MCNP6, was in good agreement in the scenario A (confirming the experimental setup), while the mean energy spectrum was higher in the scenario B. Nevertheless, the response of LEC seemed mainly related to the measured absorbed dose. No effects on viability were detected. Our results support the hypothesis that apoptosis is not responsible for cataract induced by low doses of X-ray (i.e. 25 mGy) while the induction of transient p21 may interfere with the disassembly of the nuclear envelop in differentiating LEC, leading to cataract formation. Further studies are needed to better clarify the relationship we suggested between DNA damage, transient p21 induction and the inability of LEC enucleation.

Use of a common European approach for nanomaterials' testing to support regulation: a case study on titanium and silicon dioxide representative nanomaterials.

The European Union (EU) continuously takes ensuring the safe use of manufactured nanomaterials (MNMs) in consumer products into consideration. The application of a common approach for testing MNMs, including the use of optimized protocols and methods' selection, becomes increasingly important to obtain reliable and comparable results supporting the regulatory framework. In the present study, we tested four representative MNMs, two titanium dioxides (NM100 and NM101) and two silicon dioxides (NM200 and NM203), using the EU FP7-NANoREG approach, starting from suspension and dispersion preparations, through to their characterization and final evaluation of biological effects. MNM dispersions were prepared following a refined NANOGENOTOX protocol and characterized by dynamic light scattering (DLS) in water/bovine serum albumin and in media used for in vitro testing. Potential genotoxic effects were evaluated on human bronchial BEAS-2B cells using micronucleus and Comet assays, and pro-inflammatory effects by cytokines release. Murine macrophages RAW 264.7 were used to detect potential innate immune responses using two functional endpoints (pro-inflammatory cytokines and nitric oxide [NO] production). The interaction of MNMs with RAW 264.7 cells was studied by electron microscopy. No chromosomal damage and slight DNA damage and an oxidative effect, depending on MNMs, were observed in bronchial cells. In murine macrophages, the four MNMs directly induced tumor necrosis factor α or interleukin 6 secretion, although at very low levels; lipopolysaccharide-induced NO production was significantly decreased by the titania and one silica MNM. The application of this approach for the evaluation of MNM biological effects could be useful for both regulators and industries.

Comparative toxicity assessment of nano- and bulk-phase titanium dioxide particles on the human mammary gland in vitro.

There is a major concern that exposure to titanium dioxide (TiO2) nanoparticles (NPs) can have degrading effects on human health as well as mammary gland because of the increased use in numerous sorts of nanotech-based health care and food merchandise. Also, there is a scarcity in NP toxicity studies on the mammary gland; therefore, the aim of the present study was to compare toxicity caused by nano- and bulk-phase TiO2 particles on the human mammary gland in vitro. In comparison to bulk-TiO2 particles, nano-TiO2 cause a significant (p < 0.05) reduction in viability and increased reactive oxygen species generation in the human mammary epithelial cells after a dose- (1, 2, 5, 10, 20, 50, and 100 µg/mL) and time (6, 12, 24, and 48 h)-dependent exposure. Further, an increase in genotoxicity in the mammary epithelial cells was observed as percent tail DNA and comet area was increased significantly (p < 0.05) at 12 h of exposure (10 and 100 µg/mL) with nano-TiO2. The scanning electron microscopic examination showed that a 50 µg/mL dose of both nano-TiO2 and bulk-TiO2 particles cause morphological changes and retarded growth pattern of mammary epithelial cells at 12 h. Moreover, a significant (p < 0.05) increase in apoptosis at 10 µg/mL and necrosis at 50 µg/mL concentrations of nano-TiO2 in comparison to bulk-TiO2 was observed in mammary epithelial cells. Finally, we can conclude that the toxicity caused by nano-TiO2 particles on the human mammary gland cells was comparatively higher than the bulk-TiO2 particles.

Use of a non-hepatic cell line highlights limitations associated with cell-based assessment of metabolically induced toxicity.

Metabolically induced drug-toxicity is a major cause of drug failure late in drug optimization phases. Accordingly, in vitro metabolic profiling of compounds is being introduced at earlier stages of the drug discovery pipeline. An increasingly common method to obtain these profiles is through overexpression of key CYP450 metabolic enzymes in immortalized liver cells, to generate competent hepatocyte surrogates. Enhanced cytotoxicity is presumed to be due to toxic metabolite production via the overexpressed enzyme. However, metabolically induced toxicity is a complex multi-parameter phenomenon and the potential background contribution to metabolism arising from the use of liver cells which endogenously express CYP450 isoforms is consistently overlooked. In this study, we sought to reduce the potential background interference by applying this methodology in kidney-derived HEK293 cells which lack endogenous CYP450 expression. Overexpression of CYP3A4 resulted in increased HEK293 proliferation, while exposure to four compounds with reported metabolically induced cytotoxicity in liver-derived cells overexpressing CYP3A4 resulted in no increase in cytotoxicity. Our results indicate that overexpression of a single CYP450 isoform in hepatic cell lines may not be a reliable method to discriminate which enzymes are responsible for metabolic induced cytotoxicity.

Application of a multi-layer systems toxicology framework for in vitro assessment of the biological effects of Classic Tobacco e-liquid and its corresponding aerosol using an e-cigarette device with MESH™ technology.

We previously proposed a systems toxicology framework for in vitro assessment of e-liquids. The framework starts with the first layer aimed at screening the potential toxicity of e-liquids, followed by the second layer aimed at investigating the toxicity-related mechanism of e-liquids, and finally, the third layer aimed at evaluating the toxicity-related mechanism of the corresponding aerosols. In this work, we applied this framework to assess the impact of the e-liquid MESH Classic Tobacco and its aerosol compared with that of cigarette smoke (CS) from the 3R4F reference cigarette. In the first layer, we evaluated the cytotoxicity profile of the MESH Classic Tobacco e-liquid (containing humectants, nicotine, and flavors) and its Base e-liquid (containing humectant and nicotine only) in comparison with total particulate matter (TPM) of 3R4F CS using primary bronchial epithelial cell cultures. In the second layer, the same culture model was used to explore changes in specific markers using high-content screening assays to identify potential toxicity-related mechanisms induced by the MESH Classic Tobacco and Base e-liquids beyond cell viability in comparison with the 3R4F CS TPM-induced effects. Finally, in the third layer, we compared the impact of exposure to the MESH Classic Tobacco or Base aerosols with 3R4F CS using human organotypic air-liquid interface buccal and small airway epithelial cultures. The results showed that the cytotoxicity of the MESH Classic Tobacco liquid was similar to the Base liquid but lower than 3R4F CS TPM at comparable nicotine concentrations. Relative to 3R4F CS exposure, MESH Classic Tobacco aerosol exposure did not cause tissue damage and elicited lower changes in the mRNA, microRNA, and protein markers. In the context of tobacco harm reduction strategy, the framework is suitable to assess the potential-reduced impact of electronic cigarette aerosol relative to CS.

Assessment of a new arbidol derivative against herpes simplex virus II in human cervical epithelial cells and in BALB/c mice.

As one of the highly contagious forms, herpes simplex virus type 2 (HSV-2) commonly caused severe genital diseases and closely referred to the HIV infection. The lack of effective vaccines and drug-resistance proclaimed the preoccupation for alternative antiviral agents against HSV-2. Molecules bearing indole nucleus presented diverse biological properties involving antiviral and anti-inflammatory activities. In this study, one of the indole molecules, arbidol derivative (ARD) was designed and synthesized prior to the evaluation of its anti-HSV-2 activity. Our data showed that the ARD effectively suppressed HSV-2-induced cytopathic effects and the generation of progeny virus, with 50% effective concentrations of 3.386 and 1.717 µg/mL, respectively. The results of the time-course assay suggested that the ARD operated in a dual antiviral way by interfering virus entry and impairing the earlier period of viral cycle during viral DNA synthesis. The ARD-mediated HSV-2 inhibition was partially attained by blocking NF-κB pathways and down-regulating the expressions of several inflammatory cytokines. Furthermore, in vivo studies showed that oral administration of ARD protected BALB/c mice from intravaginal HSV-2 challenge by alleviating serious vulval lesions and histopathological changes in the target organs. Besides, the treatment with ARD also made the levels of viral protein, NF-κB protein and inflammatory cytokines lower, in consistent with the in-vitro studies. Collectively, ARD unveiled therapeutic potential for the prevention and treatment of HSV-2 infections.

Comparative Study of Micronuclei Count in Patients with Different Tobacco-related Habits using Exfoliated Buccal Epithelial Cells: A Tool for Assessment of Genotoxicity.

AIM: To study and compare the genotoxic effects of tobacco using micronuclei count in individuals with different tobacco-related habits. MATERIALS AND METHODS: A cross-sectional study was done comprising 200 individuals, divided into four groups. Group I: 50 subjects with history of tobacco chewing, group II: 50 subjects with a history of smoking tobacco, group III: 50 subjects with a history of both tobacco chewing and smoking, and group IV: 50 subjects without any habits as controls (age-matched). The study groups were individually further divided into three subgroups which comprised of subjects with history of substance abuse for less than 5, 5 to 10, and greater than 10 years. Exfoliated cells from the buccal mucosa of the subjects were collected and stained using Giemsa stain. A total of 1,000 cells were examined for each case and micronuclei frequency was scored according to the guidelines given by Tolbert et al. Results: The mean number of micronuclei count was 18.28 ± 10.0 in group I (smokeless tobacco users), 11.38 ± 6.3 in group II (subjects with history of tobacco smoking), 22.44 ± 9.8 in group III (subjects with history of using both smokeless and smokable form of tobacco), and 4.86 ± 2.4 in the control group. The statistical difference was found to be highly significant (p < 0.001). Similarly, based on the duration, highly significant difference was notable in the mean number of micronuclei in subjects who had a history of substance abuse for more than 10 years. CONCLUSION: A significantly higher micronucleus frequency was found in smokeless tobacco users than in smokers and controls. Micronuclei assay in the exfoliated buccal cells is a useful and minimally invasive method for monitoring early genotoxic damage. CLINICAL SIGNIFICANCE: Micronuclei assay can be used to detect genotoxic damage at the earliest and, if intervened at this point, may prevent frank malignancy, morbidity, and mortality.

Colonic epithelial miR-31 associates with the development of Crohn's phenotypes.

BACKGROUND: Crohn's disease (CD) is highly heterogeneous, due in large part to variability in cellular processes that underlie the natural history of CD, thereby confounding effective therapy. There is a critical need to advance understanding of the cellular mechanisms that drive CD heterogeneity. METHODS: We performed small RNA sequencing of adult colon tissue from CD and NIBD controls. Colonic epithelial cells and immune cells were isolated from colonic tissues, and microRNA-31 (miR-31) expression was measured. miR-31 expression was measured in colonoid cultures generated from controls and patients with CD. We performed small RNA-sequencing of formalin-fixed paraffin-embedded colon and ileum biopsies from treatment-naive pediatric patients with CD and controls and collected data on disease features and outcomes. RESULTS: Small RNA-sequencing and microRNA profiling in the colon revealed 2 distinct molecular subtypes, each with different clinical associations. Notably, we found that miR-31 expression was a driver of these 2 subtypes and, further, that miR-31 expression was particularly pronounced in epithelial cells. Colonoids revealed that miR-31 expression differences are preserved in this ex vivo system. In adult patients, low colonic miR-31 expression levels at the time of surgery were associated with worse disease outcome as measured by need for an end ileostomy and recurrence of disease in the neoterminal ileum. In pediatric patients, lower miR-31 expression at the time of diagnosis was associated with future development of fibrostenotic ileal CD requiring surgeryCONCLUSIONS. These findings represent an important step forward in designing more effective clinical trials and developing personalized CD therapies. FUNDING: This work was supported by CCF Career Development Award (SZS), R01-ES024983 from NIEHS (SZS and TSF), 1R01DK104828-01A1 from NIDDK (SZS and TSF), P01-DK094779-01A1 from NIDDK (SZS), P30-DK034987 from NIDDK (SZS), 1-16-ACE-47 ADA Pathway Award (PS), UNC Nutrition Obesity Research Center Pilot & Feasibility Grant P30DK056350 (PS), CCF PRO-KIIDS NETWORK (SZS and PS), UNC CGIBD T32 Training Grant from NIDDK (JBB), T32 Training Grant (5T32GM007092-42) from NIGMS (MH), and SHARE from the Helmsley Trust (SZS). The UNC Translational Pathology Laboratory is supported, in part, by grants from the National Cancer Institute (3P30CA016086) and the UNC University Cancer Research Fund (UCRF) (PS).

Clinical follow up and the impact of the Paris system in the assessment of patients with atypical urine cytology.

BACKGROUND: Urinary cytology is routinely used in the diagnosis of urothelial neoplasms, with good sensitivity for high-grade urothelial carcinoma (HGUC) but less so for low-grade urothelial neoplasm (LGUN). There is significant interobserver and interinstitutional variability, especially for the atypical category. The Paris system for reporting urinary cytology (TPS) was introduced to better define the various categories, especially atypical cytology. METHODS: We retrospectively reviewed 630 atypia of undetermined significance (AUS) cases and reclassified them based on TPS. In total, 501 cases previously reported as negative for malignancy had their medical records reviewed to serve as negative controls. RESULTS: Of 630 AUS cases, 299 (47.5%) were reclassified as negative for HGUC (NHGUC), 313 (49.7%) as atypical urothelial cells (AUCs) and 18 (2.9%) as suspicious for HGUC (SHGUC). Based on our institution's previous reporting system, the rate of underlying or subsequent HGUC was 2.8% for AUS, and 0% for negative. When AUS cases were reclassified under TPS, the rates were 1.5% for NHGUC, 4.8% for AUC, and 0% for SHGUC. Review of medical records showed that patients with AUS were more likely to be followed-up compared with those with negative urine cytology (77.8% compared with 54.3%), particularly those under the care of non-urologists. CONCLUSIONS: AUS diagnosis is associated with more patient follow up compared with NEG urine particularly among non-urologists. Reclassifying according to TPS results in significant reduction in the rate of AUS and thus unnecessary testing. This reduction however may be at the expense of slightly decreased detection rate of HGUC.

Establishment of three novel cell lines derived from African American patients with colorectal carcinoma: A unique tool for assessing racial health disparity.

The incidence and mortality rates of colorectal carcinoma (CRC) are higher among African Americans (AAs) compared with Caucasian Americans (CAs). To assess the molecular properties associated with racial health disparity, three cell lines derived from colorectal tumors of three AA subjects were established. Cellular and molecular characterization of the cell lines designated CHTN06, SB501 and SB521 was performed using standard technologies, including immunofluorescence, electron microscopy, karyotyping, reverse transcription-polymerase chain reaction, ELISA and immunoblot analysis. The histology and morphology of CHTN06 xenografts were examined by hematoxylin and eosin staining. A total of three AA CRC cell lines derived from primary tumors were established and characterized. These cell lines were successfully cultured without immortalization and were found to be tumorigenic as mouse xenografts. In the present study, immunoblotting and immunofluorescence confirmed the expression of proteins known to be dysregulated in CRC, such as p53, DNA mismatch repair proteins and villin-1. Oncogenic miRNAs (i.e., miR-17, miR-21, miR-182, miR-210 and miR-222) were overexpressed in the AA CRC lines compared with the CA CRC lines (HT-29, HCT116 and SW480). Additionally, the AA CRC cell lines exhibited a differential inflammatory profile compared with HT-29 (CA CRC cell line); specifically noted was IL-8 secretion in response to inflammatory stimuli. In conclusion, three novel cell lines derived from AA CRC tissues were generated. These cell lines were characterized as epithelial in nature and exhibited differential expression of several miRNAs and inflammatory responses compared with commercially available cell lines of CA origin. The CRC cell lines CHTN06, SB501 and SB521 represent novel tools that may be used to provide diverse in vitro and in vivo models for studying CRC and racial health disparity.

Nephrotoxicity and Kidney Transport Assessment on 3D Perfused Proximal Tubules.

Proximal tubules in the kidney play a crucial role in reabsorbing and eliminating substrates from the body into the urine, leading to high local concentrations of xenobiotics. This makes the proximal tubule a major target for drug toxicity that needs to be evaluated during the drug development process. Here, we describe an advanced in vitro model consisting of fully polarized renal proximal tubular epithelial cells cultured in a microfluidic system. Up to 40 leak-tight tubules were cultured on this platform that provides access to the basolateral as well as the apical side of the epithelial cells. Exposure to the nephrotoxicant cisplatin caused a dose-dependent disruption of the epithelial barrier, a decrease in viability, an increase in effluent LDH activity, and changes in expression of tight-junction marker zona-occludence 1, actin, and DNA-damage marker H2A.X, as detected by immunostaining. Activity and inhibition of the efflux pumps P-glycoprotein (P-gp) and multidrug resistance protein (MRP) were demonstrated using fluorescence-based transporter assays. In addition, the transepithelial transport function from the basolateral to the apical side of the proximal tubule was studied. The apparent permeability of the fluorescent P-gp substrate rhodamine 123 was decreased by 35% by co-incubation with cyclosporin A. Furthermore, the activity of the glucose transporter SGLT2 was demonstrated using the fluorescent glucose analog 6-NBDG which was sensitive to inhibition by phlorizin. Our results demonstrate that we developed a functional 3D perfused proximal tubule model with advanced renal epithelial characteristics that can be used for drug screening studies.

Epithelial-to-mesenchymal transition signature assessment in colorectal cancer quantifies tumour stromal content rather than true transition.

The process of epithelial-to-mesenchymal transition (EMT) in cancer is a well-described process whereby epithelial tumour cells undergo molecular/phenotypic changes and transition to a mesenchymal biology. To aid in the transcriptional characterisation of this process, gene expression signatures have been developed that attribute a relative EMT score to samples in a given cohort. We demonstrate how such EMT signatures can identify epithelial cell line models with high levels of transition but also highlight that, unsurprisingly, fibroblast cell lines, which are inherently mesenchymal, have a higher EMT score relative to any epithelial cell line studied. In line with these data, we demonstrate how increased tumour stromal composition, and reduced epithelial cellularity, significantly correlates with increasing EMT signature score, which is evident using either in silico subtyping analysis (p < 0.00001) or in situ histopathological characterisation (p < 0.001). Considered together, these results reinforce the importance not only of interdisciplinary research to correctly define the nature of EMT biology but also the requirement for a cadre of multidisciplinary researchers who can analyse and interpret the underlying pathological, bioinformatic and molecular data that are essential for advancing our understanding of the malignant process. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Combining Extracellular miRNA Determination with Microfluidic 3D Cell Cultures for the Assessment of Nephrotoxicity: a Proof of Concept Study.

Drug-induced kidney injury is often observed in the clinics and can lead to long-term organ failure. In this work, we evaluated a novel in vitro system that aims at detecting whether compounds can cause renal proximal tubule damage in man. For this, we implemented organotypic cultures of human conditionally immortalized proximal tubule epithelial cells overexpressing the organic anion transporter 1 (ciPTEC-OAT1) in a three-channel OrganoPlate under microfluidic conditions. Cells were exposed to four known nephrotoxicants (cisplatin, tenofovir, cyclosporine A, and tobramycin). The effect on cell viability and NAG release into the medium was determined. A novel panel of four miRNAs (mir-21, mir-29a, mir-34a, and mir-192) was selected as potential biomarkers of proximal tubule damage. After nephrotoxicant treatment, miRNA levels in culture medium were earlier indicators than cell viability (WST-8 assay) and outperformed NAG for proximal tubule damage. In particular, mir-29a, mir-34a, and mir-192 were highly reproducible between experiments and across compounds, whereas mir-21 showed more variability. Moreover, similar data were obtained in two different laboratories, underlining the reproducibility and technical transferability of the results, a key requirement for the implementation of novel biomarkers. In conclusion, the selected miRNAs behaved like sensitive biomarkers of damage to tubular epithelial cells caused by several nephrotoxicity mechanisms. This biomarker panel, in combination with the 3D cultures of ciPTEC-OAT1 in the OrganoPlate, represents a novel tool for in vitro nephrotoxicity detection. These results pave the way for the application of miRNAs in longitudinal, time-course in vitro toxicity studies.