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Rationale: The exploration of the gut microbiome and related metabolites holds an exciting future in health science. The challenges associated with fecal sample testing are proper sample collection, sterile transportation, optimal transport conditions, and processing as all these factors could potentially change the microbiome composition, further exacerbated by the patient's customary discomfort regarding feces samples. The study aimed to compare the usage of rectal swabs and stool samples for short-chain fatty acid estimation using gas chromatography-mass spectrometry (GC-MS) and indole estimation using spectrophotometry., Method: From May 2022 to June 2022, three women were recruited from the Department of Obstetrics and Gynecology (OBG) in a secondary care hospital in coastal Karnataka. During their clinical visit, a rectal swab was collected, and the stool sample was transported to the hospital from the patient's home in sterile containers provided. After the extraction, short-chain fatty acids (acetate, propionate, and butyrate) were quantified using GC-MS. The fecal indole concentration was determined using a hydroxylamine-based assay., Results: The GC-MS analysis failed to detect the concentrations of short-chain fatty acids in rectal swab samples. Indole concentrations in stool and swab samples were significantly different., Conclusion: The study's findings do not support the use of rectal swabs to analyze gut metabolites., (© 2024 John Wiley & Sons Ltd.)

Background: Hirschsprung's Disease (HD) is characterized by intestinal sub-occlusion and the absence of enteric ganglion cells. A rectal biopsy examination is performed to confirm the diagnosis. In a recent study, we demonstrated that the analysis of 60 sections of rectal mucosa and submucosa stained by H&E may ensure a 90% diagnostic accuracy. Although the need to analyze so many sections makes the process of reading the slides more time-consuming, this encouraged us to study their distribution in the healthy rectal submucosa, to simplify the diagnosis., Objectives: To develop a method that facilitates HD diagnosis by studying the distribution of ganglion cells in the submucosal plexus., Methods: Using the calretinin technique, we studied the distribution of plexuses in 60 fragments of rectal submucosa from 19 cadavers. After the study, the reading method created was used for diagnosis in 47 cases of suspected HD, using H&E staining. The accuracy was verified by comparing the results obtained with H&E to those obtained with the acetylcholinesterase technique, the golden standard in our laboratory., Results: The study of submucosal plexus distribution showed that just by examining the submucosal region every 20 µm, approximately, it is possible to locate a ganglionic plexus, and we have already been able to diagnose HD with 93% accuracy., Conclusion: The study of ganglion cell distribution enabled the creation of a simplified method for reading the slides. The method applied achieved good accuracy and it can be used as an alternative method in HD diagnosis., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (Copyright © 2023 HCFMUSP. Published by Elsevier España, S.L.U. All rights reserved.)

Introduction: The majority of new HIV infections occur through mucosal transmission. The availability of readily applicable and accessible platforms for anti-retroviral (ARV) delivery is critical for the prevention of HIV acquisition through sexual transmission in both women and men. There is a compelling need for developing new topical delivery systems that have advantages over the pills, gels and rings, which currently fail to guarantee protection against mucosal viral transmission in vulnerable populations due to lack of user compliance. The silk fibroin (SF) platform offers another option that may be better suited to individual circumstances and preferences to increase efficacy through user compliance. The objective of this study was to test safety and efficacy of SF for anti-HIV drug delivery to mucosal sites and for viral prevention., Methods: We formulated a potent HIV inhibitor Griffithsin (Grft) in a mucoadhesive silk fibroin (SF) drug delivery platform and tested the application in a non-human primate model in vivo and a pre-clinical human cervical and colorectal tissue explant model. Both vaginal and rectal compartments were assessed in rhesus macaques (Mucaca mulatta) that received SF (n = 4), no SF (n = 7) and SF-Grft (n = 11). In this study, we evaluated the composition of local microbiota, inflammatory cytokine production, histopathological changes in the vaginal and rectal compartments and mucosal protection after ex vivo SHIV challenge., Results: Effective Grft release and retention in mucosal tissues from the SF-Grft platform resulted in protection against HIV in human cervical and colorectal tissue as well as against SHIV challenge in both rhesus macaque vaginal and rectal tissues. Mucoadhesion of SF-Grft inserts did not cause any inflammatory responses or changes in local microbiota., Conclusions: We demonstrated that in vivo delivery of SF-Grft in rhesus macaques fully protects against SHIV challenge ex vivo after two hours of application and is safe to use in both the vaginal and rectal compartments. Our study provides support for the development of silk fibroin as a highly promising, user-friendly HIV prevention modality to address the global disparity in HIV infection., (© 2020 The Authors. Journal of the International AIDS Society published by John Wiley & Sons Ltd on behalf of the International AIDS Society.)

Background: Daily oral pre- or post-exposure prophylaxis (PrEP or PEP) is highly effective in preventing HIV infection. However, many people find it challenging to adhere to a daily oral regimen. Chemoprophylaxis with single oral doses of antiretroviral drugs taken before or after sex may better adapt to changing or unanticipated sexual practices and be a desirable alternative to daily PrEP or PEP. We investigated willingness to use a single oral pill before or after sex among men who have sex with men (MSM) and assessed the biological efficacy of a potent antiretroviral combination containing elvitegravir (EVG), emtricitabine (FTC), and tenofovir alafenamide (TAF)., Methods: Data on willingness to use single-dose PrEP or PEP were obtained from the 2017 cycle of the American Men's Internet Survey (AMIS), an annual online behavioral surveillance survey of MSM in the United States. Antiretroviral drug levels were measured in humans and macaques to define drug distribution in rectal tissue and identify clinically relevant doses for macaque modeling studies. The biological efficacy of a single dose of FTC/TAF/EVG as PrEP or PEP was investigated using a repeat-challenge macaque model of rectal HIV infection., Findings: Through pharmacokinetic assessment in humans and macaques we found that EVG penetrates and concentrates in rectal tissues supporting its addition to FTC/TAF to boost and extend chemoprophylactic activity. Efficacy estimates for a single oral dose given to macaques 4h before or 2h after SHIV exposure was 91•7%[35•7%-98•9%] and 100%, respectively, compared to 80•1%[13•9%-95•4%] and 64•6%[-19•4%-89•5%] when single doses were given 6 and 24h post challenge, respectively. A two-dose regimen at 24h and 48h after exposure was also protective [77•1%[1•7%-94•7%]., Interpretation: Informed by user willingness, human and macaque pharmacokinetic data, and preclinical efficacy we show that single-dose prophylaxis before or after sex is a promising HIV prevention strategy. Carefully designed clinical trials are needed to determine if any of these strategies will be effective in humans., Funding: Funded by CDC intramural funds, CDC contract HCVJCG2-2016-03948 (to CFK), and a grant from the MAC AIDS Fund and by the National Institutes of Health [P30AI050409] - the Emory Center for AIDS Research (to MZ and TS)., (Published by Elsevier B.V.)

Diagnosis or exclusion of Hirschsprung disease (HSCR) is a frequent exercise in any pediatric hospital. Although HSCR may present at different ages and with varied clinical findings, the most common presentation is a neonate with severe constipation or signs of intestinal obstruction. A variety of diagnostic tests including contrast enema and anorectal manometry may be used as diagnostic screens, but diagnosis ultimately rests upon histopathological evaluation of a rectal biopsy. For the experienced pathologist, conventional hematoxylin-and-eosin-stained sections often suffice to exclude HSCR or establish the diagnosis. However, ancillary diagnostic tests such as acetylcholinesterase histochemistry or calretinin immunohistochemistry are complementary and extremely helpful in some cases. In this Perspectives article, we review the clinical and pathological features of HSCR, highlight those that are found in most patients, and discuss how to address particularly challenging aspects of the diagnostic workup.

The scientific interest in cadmium (Cd) as a human health damaging agent has significantly increased over the past decades. However, particularly the histological distribution of Cd in human tissues is still scarcely defined. Using inductively coupled plasma-mass spectrometry (ICP-MS), we determined the concentration of Cd in 40 different human tissues of four body donors and provided spatial information by elemental imaging on the microscopic distribution of Cd in 8 selected tissues by laser ablation (LA)-ICP-MS. ICP-MS results show that Cd concentrations differ by a factor of 20 000 between different tissues. Apart from the well know deposits in kidney, bone, and liver, our study provides evidence that muscle and adipose tissue are underestimated Cd pools. For the first time, we present spatially resolved Cd distributions in a broad panel of human soft tissues. The defined histological structures are mirrored by sharp cut differences in Cd concentrations between neighboring tissue types, particularly in the rectum, testis, and kidneys. The spatial resolution of the Cd distribution at microscopic level visualized intratissue hot spots of Cd accumulation and is suggested as a powerful tool to elucidate metal based toxicity at histological level.

Herbivores exhibit specializations at the intestinal level that facilitate the bacterial fermentation. The available information on the digestive physiology of Lagostomus maximus makes this rodent an interesting model to evaluate morpho-functional adaptations to herbivory. The general objective of this work was centered on the study of the morphology and histochemistry of the descending colon and rectum of L. maximus. To do so, a comparative analysis of the morphology, ultrastructure and glycosylation pattern of both anatomical regions was carried out. Histochemical results revealed that in both sectors of the large intestine, there are goblet cells with different glycosylation pattern within a morphologically homogeneous cell population. The main difference between both intestinal segments lay in the fact that the most distal region of the large intestine showed a greater proportion of sialomucins, characterized by being slightly O-acetylated. Further specific differences were revealed by lectin histochemistry. These data allowed to perform a functional interpretation of the cell types and secreted substances, thus contributing to a better understanding of the role of mucins in the intestinal tract functioning., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Objective: HIV Prevention Trials Network 069/AIDS Clinical Trials Group A5305 was a study of 48-week oral pre-exposure prophylaxis (PrEP) regimens in MSM and transgender women. A rectal substudy was included to evaluate drug concentrations in rectal compartment vs. blood, gut-associated lymphoid tissue (GALT) responses to four antiretroviral PrEP regimens [maraviroc (MVC), MVC + emtricitabine (FTC), MVC + tenofovir (TFV) disoproxil fumarate, and TFV disoproxil fumarate + FTC], and to determine whether ARV exposure was associated with ex-vivo suppression of HIV infection in colorectal explants., Methods: C-C chemokine receptor type 5 (CCR5) genotype was characterized using PCR. At baseline and at Weeks 24, 48, and 49, GALT phenotype was characterized by flow cytometry, rectal biopsies were challenged with HIV-1BaL, and tissue and plasma pharmacokinetics were measured via mass spectrometry., Results: Exposure to MVC was not associated with increased expression of CD4+/CCR5+ HIV target T cells. Significant ex-vivo viral suppression compared with baseline was seen at Weeks 24 and 48, ranging from 1.4 to 1.8 log10 for all study regimens except the MVC-alone arm which did not show statistically significant viral suppression at Week 48. Tissue concentrations of TFV, TFV-diphosphate, and FTC were correlated with viral suppression., Conclusion: MVC-containing HIV PrEP regimens did not increase GALT CD4+ T-cell activation or the CD4+/CCR5+ phenotype. No virologic suppression was seen with MVC-alone at Week 48 compared with combination regimens, suggesting MVC monotherapy might be less effective than combination antiretroviral PrEP regimens.

Objectives: Despite plasma virologic suppression with antiretroviral therapy (ART), HIV persists in gut tissue. The objectives of this study were to compare plasma and rectal tissue HIV RNA dynamics and to assess relationships with dolutegravir (DTG) plasma and tissue concentrations., Design: A longitudinal cohort study of HIV-infected treatment-naïve individuals initiating DTG-based ART was conducted over 12 weeks with plasma and rectal tissue sampling (Clinicaltrials.gov:NCT02924389)., Methods: HIV RNA and DTG concentrations were quantified in plasma and rectal tissue samples collected pre-ART (baseline) and post-ART at weeks 2, 6, and 12 using Abbott Real-Time HIV-1 assays and high-performance liquid chromatography tandem mass spectroscopy, respectively. Relationships between rectal tissue RNA and DTG concentrations were modeled using binary logistic regression, controlling for repeated measures., Results: Twelve participants were enrolled: six (50.0%) women, nine (75.0%) black, median age 42.0 years (Q1 31.2, Q3 52.0). All attained plasma virologic suppression by week 6. 11 of 12 (91.7%) had detectable rectal tissue HIV RNA at baseline, and only three of 11 (27.3%) achieved rectal tissue virologic suppression at any time-point. Compared with rectal tissue nonsuppressors, three of three (100.0%) of rectal tissue suppressors were women, had higher BMI, 35.9 kg/m (range 24.9-38.5) versus 20.6 (17.7-29.9), P = 0.05, and lower baseline log plasma HIV RNA: 3.7 copies/ml (range 3.6-4.4) versus 4.7 (3.8-5.4), P = 0.02. No significant relationships between rectal tissue RNA suppression and DTG concentrations were seen., Conclusion: Rectal tissue HIV RNA persisted in most participants and was not predicted by DTG concentrations. Impact of host factors, particularly sex, on tissue HIV viral dynamics warrants further exploration.

The objective of the study is the quantitative analysis of the dose-time dependences of changes occurring in collagen of bladder and rectum after gamma-irradiation using optical methods [nonlinear microscopy in a second harmonic generation (SHG) detection regime and cross-polarization optical coherence tomography (CP OCT)]. For quantitative assessment of the collagen structure, regions of interest on the SHG-images of two-dimensional (2-D) distribution of SHG signal intensity of collagen were chosen in the submucosa. The mean SHG signal intensity and its standard deviation were calculated by ImageJ 1.39p (NIH). For quantitative analysis of CP OCT data, an integral depolarization factor (IDF) was calculated. Quantitative calculation of the SHG signal intensity and the IDF can provide additional information about the processes of the collagen radiation-induced degradation and subsequent remodeling. High positive correlation between the mean SHG signal intensity and the mean IDF of bladder and rectum demonstrates that CP OCT can be used as an "optical biopsy" in the grading of collagen radiation damage., ((2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).)

Background: Although the pelvic autonomic plexus branches are considered to be a mixture of sympathetic and parasympathetic nerves, little is known regarding the composite fibers of the pelvic plexus branches. This study aimed to investigate the immunohistochemical features of sympathetic and parasympathetic nerves in the pelvic autonomic plexus branches., Methods: Using 10 donated elderly male cadavers, the detailed topohistology of nerve fibers at and around the bladder, seminal vesicle, prostate, and rectum was examined. Neuronal nitric oxide synthase (nNOS) and vasoactive intestinal polypeptide (VIP) were used as parasympathetic nerve markers; tyrosine hydroxylase (TH) was used as a sympathetic nerve marker. The myenteric plexus of the colon was utilized as a positive control., Results: Most nerve fibers in the bladder, seminal vesicle, prostate, and rectum were both nNOS- and TH-positive. Thus, pelvic plexus branches were classified into two types: 1) triple-positive mixed nerves (nNOS+, VIP+, TH+, thick myelinated fibers + or -) and 2) double-positive mixed nerves (nNOS+, VIP-, TH+, thick myelinated fibers + or -). Notably, triple-positive nerves were localized within the posterosuperior part of the plexus (near the rectum) and travelled anteroinferiorly toward the posterolateral corner of the prostate. The posteriorly and inferiorly located nerves were predominantly composed of parasympathetic, rather than sympathetic, fibers. In contrast, nerve fibers within and along the bladder and seminal vesicle contained either no or few VIP-positive nerves. These superiorly located nerves were characterized by clear sympathetic nerve dominance., Conclusions: The nerves of the pelvic plexus branches were clearly classified into nerves around the bladder and seminal vesicle (VIP-negative) and nerves around the prostate (VIP-positive). Although nNOS- and VIP-positive nerve fibers are candidate cavernous nerves, cavernous nerve identity cannot be definitively concluded for these nerves in the periprostatic region.

Background: Cell-free circulating tumour-derived DNA (ctDNA) can be detected by testing for methylated BCAT1 and IKZF1 DNA, which has proven sensitivity for colorectal cancer (CRC). A prospective correlative biomarker study between presence of methylated BCAT1 and IKZF1 in tissue and blood was conducted in cases with CRC to explore how detection of such ctDNA biomarkers relates to cancer characteristics, methylation in tissue and surgical resection of the primary cancer., Methods: Enrolled patients with invasive CRC had blood collected at diagnosis, prior to any treatment or surgery (peri-diagnostic sample). A subgroup of patients also had cancer and adjacent non-neoplastic tissue collected at surgical resection, as well as a second blood sample collected within 12 months of surgery (post-surgery sample). DNA was extracted from all samples and assayed for methylated BCAT1 and IKZF1 to determine the degree of methylation in tissue and the presence of ctDNA in blood., Results: Of 187 cases providing peri-diagnostic blood samples, tissue was available in 91, and 93 provided at least one post-surgery blood sample for marker analysis. Significant methylation of either BCAT1 or IKZF1 was seen in 86/91 (94.5%) cancer tissues, with levels independent of stage and higher than that observed in adjacent non-neoplastic specimens ( P  < 0.001). ctDNA methylated in BCAT1 or IKZF1 was detected in 116 (62.0%) cases at diagnosis and was significantly more likely to be detected with later stage ( P  < 0.001) and distal tumour location ( P  = 0.004). Of the 91 patients who provided pre-and post-surgery blood samples, 47 patients were ctDNA-positive at diagnosis and 35 (74.5%) became negative after tumour resection., Conclusion: This study has shown that BCAT1 and IKZF1 methylation are common events in CRC with almost all cancer tissues showing significant levels of methylation in the two genes. The presence of ctDNA in blood is stage-related and show rapid reversion to negative following surgical resection. Monitoring methylated BCAT1 and IKZF1 levels could therefore inform adequacy of surgical resection., Trial Registration: Australian New Zealand Clinical Trial Registry number 12611000318987. Registered 25 March 2011., Competing Interests: The study was approved by the Southern Adelaide Clinical Human Research Ethics Committee (134.045). Written informed consent was obtained from all participants.GPY is a paid consultant to Clinical Genomics. SKP, DHM and RTB are paid employees of Clinical Genomics. The other authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Background: Colorectal cancer (CRC) screening might be improved by using a measure of prior risk to modulate screening intensity or the faecal immunochemical test threshold. Intermediate molecular biomarkers could aid risk prediction by capturing both known and unknown risk factors., Methods: We sampled normal bowel mucosa from the proximal colon, distal colon and rectum of 317 individuals undergoing colonoscopy. We defined cases as having a personal history of colorectal polyp(s)/cancer, and controls as having no history of colorectal neoplasia. Molecular analyses were performed for: telomere length (TL); global methylation; and the expression of genes in molecular pathways associated with colorectal tumourigenesis. We also calculated a polygenic risk score (PRS) based on CRC susceptibility polymorphisms., Results: Bowel TL was significantly longer in cases than controls, but was not associated with blood TL. PRS was significantly and independently higher in cases. Hypermethylation showed a suggestive association with case:control status. No gene or pathway was differentially expressed between cases and controls. Gene expression often varied considerably between bowel locations., Conclusions: PRS and bowel TL (but not blood TL) may be clinically-useful predictors of CRC risk. Sample collection to assess these biomarkers is feasible in clinical practice, especially where population screening uses flexible sigmoidoscopy or colonoscopy.

Background: It remains unclear whether regional variation exists in the human enteric nervous system (ENS) ie, whether intrinsic innervation varies along the gut. Recent classification of gastrointestinal neuropathies has highlighted inadequacies in the quantification of the human ENS. This study used paired wholemounts to accurately quantify and neurochemically code the hindgut myenteric plexus, comparing human distal colon and rectum., Methods: Paired human descending colonic/rectal specimens were procured from 15 patients undergoing anterior resection. Wholemounts of myenteric plexi were triple-immunostained with anti-Hu/NOS/ChAT antibodies. Images were acquired by motorized epifluorescence microscopy, allowing assessment of ganglionic density/size, ganglionic area density, and neuronal density. 'Stretch-corrected' values were calculated using stretched/relaxed tissue dimensions., Key Results: Tile-stitching created a collage with average area 99 300 000 μm 2 . Stretch-corrected ganglionic densities were similar (colon: median 510 ganglia/100 mm 2 [range 386-1170], rectum: 585 [307-923]; P = .99), as were average ganglionic sizes (colon: 57 593 μm 2 [40 301-126 579], rectum: 54 901 [38 701-90 211], P = .36). Ganglionic area density (colon: 11.92 mm 2 per 100 mm 2 [7.53-18.64], rectum: 9.84 [5.80-17.19], P = .10) and stretch-corrected neuronal densities (colon: 189 neurons/mm 2 [117-388], rectum: 182 [89-361], P = .31) were also similar, as were the neurochemical profiles of myenteric ganglia, with comparable proportions of NOS+ and ChAT+ neurons (P > .10)., Conclusions and Inferences: This study has revealed similar neuronal and ganglionic densities and neurochemical profiles in human distal colon and rectum. Further investigation of other components of the ENS, incorporating additional immunohistochemical markers are required to confirm that there is no regional variation in the human hindgut ENS., (© 2017 John Wiley & Sons Ltd.)

Introduction: This study investigates the use of rectal suction biopsy (RSB) with calretinin immunohistochemical staining (CIS) in patients suspected of having abnormally innervated bowel after pull through operation for Hirschsprung disease (HD)., Method: This study was conducted in Children's Hospital 2, Ho Chi Minh City from January 2015 through June 2016. Patients suspected with abnormally innervated bowel after pull through operation for HD were submitted for a RSB with CIS. Following histological results, the patients underwent an appropriate treatment (redo pull through operation or non-operative medical management) and followed up at least 6 months to evaluate their outcomes., Results: Ten patients were enrolled in our study. The median age was 5.6 (range 1.4-20) years. The diagnosis of normally innervated bowel was made in five patients by showing positive reactivity of CIS on RSB, whereas five others were diagnosed with abnormally innervated bowel. Final diagnosis of the five latter patients was confirmed by analyses of the resected bowel after a redo pull through operation (including 4 cases with residual aganglionosis and one case with transition zone bowel)., Conclusions: RSB with CIS is a reliable and simple method for diagnosis of abnormally innervated bowel after pull through operation for HD., Level of Evidence: III TYPE OF STUDY: Study of diagnostic test (evaluate the effectiveness of a diagnostic test or outcome assessment)., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Some case reports of neuroendocrine tumors and neuroendocrine carcinoma associated with ulcerative colitis (UC) have been published. Most neuroendocrine tumor cases are small lesions corresponding to microcarcinoids (MCs). However, published case reports have presented findings of MCs as single-case reports. Thus, the frequency of MCs is still unclear. In this study, we described the clinical and morphological features of 14 cases of UC-associated MCs and estimated the frequency of MCs. Consecutive patients with UC who underwent complete removal of the large intestine were assessed, and 135 patients were selected. Of the 135 cases, 14 cases (10.4%) in which MC lesions were observed histologically were classified as the MC group, and the remaining 121 cases were classified as the control group. Seven cases in the MC group (50%) exhibited colitic cancer. No cases in either group had distinct carcinoid tumors. All MC lesions were located in the rectum, and the sizes ranged from 0.1 to 5.5 mm. Eight cases (57%) had multiple MC lesions. The frequency of MCs in UC was estimated to be 10.4%. Most cases of MC were quite unlikely to develop into clinically distinct carcinoid tumors. Thus, when MC lesions remain microscopic, they may not represent true neoplasms, which require immediate surgical resection. Because MC often arose in cases with UC complicated by dysplasia or cancer, patients with UC whose rectal biopsies reveal MC may be at high risk of colitic cancer., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objectives: Tenofovir alafenamide, a prodrug of tenofovir, produces higher PBMC concentrations of tenofovir diphosphate (tenofovir-dp) than tenofovir disoproxil fumarate. To understand tenofovir alafenamide's mucosal tissue distribution and its implications for pre-exposure prophylaxis, we characterized tenofovir-dp in female genital tract (FGT) and lower gastrointestinal (GI) tissues., Methods: Healthy seronegative women were given 5, 10 or 25 mg of tenofovir alafenamide ( n  =   8/group). Each participant provided plasma, PBMC and cervical, vaginal and rectal tissue samples over 14 days. Plasma, cell lysate and tissue homogenate concentrations were analysed by LC-MS/MS. Dose proportionality was declared in plasma and PBMCs if the natural log AUC versus natural log dose regression line 90% CI was within 0.57-1.43. In vitro tenofovir-dp formation was assessed in PBMCs and ectocervical (Ect1/E6E7) and vaginal (VK2/E6E7) cells incubated in 0.5 and 10 μM tenofovir alafenamide or tenofovir. clinicaltrials.gov: NCT02357602., Results: Following single doses of 5, 10 and 25 mg, median (IQR) tenofovir plasma AUC 0-14 days was 52.8 (49.5-59.6), 78.1 (68.2-86.9) and 169.7 (131.2-211.4) ng·h/mL and tenofovir-dp PBMC AUC 0-14 days was 2268 (1519-4090), 4584 (3113-5734) and 9306 (6891-10785) fmol·h/10 6 cells, respectively. Tenofovir was quantifiable in 52% and 92% of FGT and GI tissues, whereas tenofovir-dp was quantifiable in only 5% and 19% of FGT and GI tissues, respectively. Plasma tenofovir and PBMC tenofovir-dp were dose proportional (90% CI = 0.87-1.15 and 0.62-1.02, respectively). In vitro tenofovir-dp was 1.7-17-fold higher in epithelial cells than PBMCs., Conclusions: After tenofovir alafenamide dosing in vivo , tenofovir-dp was unquantifiable in most tissues (91%) although cervical and vaginal epithelial cells efficiently formed tenofovir-dp from tenofovir alafenamide in vitro . These findings warrant further investigation of tenofovir alafenamide's pharmacology., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

This paper details the case of a 26-year-old woman with depletion syndrome and the effectiveness of her treatment with indomethacin. Villous adenomas are benign neoplasms with a high incidence of becoming malignant. A small percentage of villous adenomas are known to cause depletion syndrome, also referred to as the McKittrick-Wheelock syndrome, a condition characterised by secretory diarrhoea, dehydration, hyponatremia, hypokalaemia, hypochloraemia, metabolic acidosis and acute renal failure. Prostaglandin-E2 mediates the hypersecretion mechanism observed in depletion syndrome, and can be inhibited by cyclo-oxygenase inhibitors. This case study measured the effectiveness of prostaglandin inhibition on a patient with oral and parenteral electrolyte replacement refractory depletion syndrome. Fluid loss and prostaglandin levels were measured before and after pharmacological treatment. This case demonstrates a 49% decrease in rectal effluent and a marked commensurate decrease in daily replenishment requirements within 48 hours of indomethacin treatment initiation, resulting in subsequent electrolyte stabilisation., Competing Interests: Conflicts of Interest: None declared., (2017 BMJ Publishing Group Ltd.)

Background: Long-acting injectable antiretroviral agents are being developed for HIV-1 prevention. The MWRI-01 study was done to characterise the safety, acceptability, and pharmacokinetic and pharmacodynamic profile of long-acting rilpivirine., Methods: We did a phase 1 open-label study at the University of Pittsburgh. We enrolled healthy individuals (aged 18-45 years) who were seronegative for HIV-1. Participants were assigned alternately one intramuscular dose of either 1200 mg or 600 mg long-acting rilpivirine, beginning with the 1200 mg dose. We obtained plasma specimens, genital and rectal fluids, and tissue samples (rectal, cervical, and vaginal) before and after exposure to long-acting rilpivirine for assessment of pharmacokinetics and ex-vivo biopsy challenge with HIV-1. Our primary objective was to characterise product safety, and the analysis included all enrolled participants. This trial is registered with ClinicalTrials.gov, number NCT01656018., Findings: 36 participants were enrolled into the study, of whom 24 were women and 12 men. 12 women and six men received each dose. 204 adverse events were reported among the 36 participants, of which 200 (98%) were grade 1-2. The most common adverse event was injection site reaction. All grade 3 and 4 adverse events were deemed not related to rilpivirine. Geometric mean (90% CI) concentrations in plasma of rilpivirine at day 28 post dose were 53 ng/mL (38-67) in women and 43 ng/mL (23-63) in men for the 1200 mg dose and 28 ng/mL (19-37) in women and 17 ng/mL (9-24) in men for the 600 mg dose. The tissue-to-plasma ratio for rilpivirine in rectal tissue was about two-fold higher than in vaginal and cervical tissue (1·10-1·53 vs 0·61-0·72 and 0·50-0·71, respectively). Exposure to long-acting rilpivirine suppressed viral replication significantly in rectal tissue (p<0·0001), and this suppression persisted for up to 4 months. By contrast, no viral suppression was seen in cervical or vaginal tissue., Interpretation: Ongoing research will characterise longer term safety and acceptability of multiple injections and help ascertain whether long-acting rilpivirine should advance to assessment of efficacy in preventing HIV-1 infection., Funding: Bill & Melinda Gates Foundation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The pharmacokinetics (PK) of tenofovir-diphosphate (TFV-DP) and emtricitabine-triphosphate (FTC-TP), the active anabolites of tenofovir disoproxil fumarate (TDF), and emtricitabine (FTC) in blood, genital, and rectal compartments was determined in HIV-positive and seronegative adults who undertook a 60-day intensive PK study of daily TDF/FTC (plus efavirenz in HIV positives). Lymphocyte cell sorting, genital, and rectal sampling occurred once per subject, at staggered visits. Among 19 HIV-positive (3 female) and 21 seronegative (10 female) adults, TFV-DP in peripheral blood mononuclear cells (PBMC) accumulated 8.6-fold [95% confidence interval (CI): 7.2-10] from first-dose to steady-state concentration (Css) versus 1.7-fold (95% CI: 1.5-1.9) for FTC-TP. Css was reached in ∼11 and 3 days, respectively. Css values were similar between HIV-negative and HIV-positive individuals. Css TFV-DP in rectal mononuclear cells (1,450 fmol/10 6 cells, 898-2,340) was achieved in 5 days and was >10 times higher than PBMC (95 fmol/10 6 cells, 85-106), seminal cells (22 fmol/10 6 cells, 6-79), and cervical cells (111 fmol/10 6 cells, 64-194). FTC-TP Css was highest in PBMC (5.7 pmol/10 6 cells, 5.2-6.1) and cervical cells (7 pmol/10 6 cells, 2-19) versus rectal (0.8 pmol/10 6 cells, 0.6-1.1) and seminal cells (0.3 pmol/10 6 cells, 0.2-0.5). Genital drug concentrations on days 1-7 overlapped with estimated Css, but accumulation characteristics were based on limited data. TFV-DP and FTC-TP in cell sorted samples were highest and achieved most rapidly in CD14 + compared with CD4 + , CD8 + , and CD19 + cells. Together, these findings demonstrate cell-type and tissue-dependent cellular pharmacology, preferential accumulation of TFV-DP in rectal mononuclear cells, and rapid distribution into rectal and genital compartments., Competing Interests: Author Disclosure Statement Peter Anderson receives contract work and grant support from Gilead Sciences, paid to his institution. All other authors report no competing financial interests exit.

Objective: Colorectal cancer remains the fourth most common cause of cancer-related mortality worldwide. Here we investigate the role of nuclear factor-κB (NF-κB) co-factor B-cell CLL/lymphoma 3 (BCL-3) in promoting colorectal tumour cell survival., Design: Immunohistochemistry was carried out on 47 tumour samples and normal tissue from resection margins. The role of BCL-3/NF-κB complexes on cell growth was studied in vivo and in vitro using an siRNA approach and exogenous BCL-3 expression in colorectal adenoma and carcinoma cells. The question whether BCL-3 activated the AKT/protein kinase B (PKB) pathway in colorectal tumour cells was addressed by western blotting and confocal microscopy, and the ability of 5-aminosalicylic acid (5-ASA) to suppress BCL-3 expression was also investigated., Results: We report increased BCL-3 expression in human colorectal cancers and demonstrate that BCL-3 expression promotes tumour cell survival in vitro and tumour growth in mouse xenografts in vivo, dependent on interaction with NF-κB p50 or p52 homodimers. We show that BCL-3 promotes cell survival under conditions relevant to the tumour microenvironment, protecting both colorectal adenoma and carcinoma cells from apoptosis via activation of the AKT survival pathway: AKT activation is mediated via both PI3K and mammalian target of rapamycin (mTOR) pathways, leading to phosphorylation of downstream targets GSK-3β and FoxO1/3a. Treatment with 5-ASA suppressed BCL-3 expression in colorectal cancer cells., Conclusions: Our study helps to unravel the mechanism by which BCL-3 is linked to poor prognosis in colorectal cancer; we suggest that targeting BCL-3 activity represents an exciting therapeutic opportunity potentially increasing the sensitivity of tumour cells to conventional therapy., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Proximal and distal colorectal cancers (CRCs) are regarded as distinct disease entities, evolving through different genetic pathways and showing multiple clinicopathological and molecular differences. Segmental distribution of some common markers (e.g., KRAS, EGFR, Ki-67, Bcl-2, COX-2) is clinically important, potentially affecting their prognostic or predictive value. However, this distribution is influenced by a variety of factors such as the anatomical overlap of tumorigenic molecular events, associations of some markers with other clinicopathological features (stage and/or grade), and wide methodological variability in markers' assessment. All these factors represent principal influences followed by intratumoral heterogeneity and geographic variation in the frequency of detection of particular markers, whereas the role of other potential influences (e.g., pre-adjuvant treatment, interaction between markers) remains rather unclear. Better understanding and elucidation of the various influences may provide a more accurate picture of the segmental distribution of molecular markers in CRC, potentially allowing the application of a novel patient stratification for treatment, based on particular molecular profiles in combination with tumor location.

Colorectal cancer arises in part from the cumulative effects of multiple gene lesions. Recent studies in selected cancer types have revealed significant intra-tumor genetic heterogeneity and highlighted its potential role in disease progression and resistance to therapy. We hypothesized the existence of significant intra-tumor genetic heterogeneity in rectal cancers involving variations in localized somatic mutations and copy number abnormalities. Two or three spatially disparate regions from each of six rectal tumors were dissected and subjected to the next-generation whole-exome DNA sequencing, Oncoscan SNP arrays, and targeted confirmatory sequencing and analysis. The resulting data were integrated to define subclones using SciClone. Mutant-allele tumor heterogeneity (MATH) scores, mutant allele frequency correlation, and mutation percent concordance were calculated, and copy number analysis including measurement of correlation between samples was performed. Somatic mutations profiles in individual cancers were similar to prior studies, with some variants found in previously reported significantly mutated genes and many patient-specific mutations in each tumor. Significant intra-tumor heterogeneity was identified in the spatially disparate regions of individual cancers. All tumors had some heterogeneity but the degree of heterogeneity was quite variable in the samples studied. We found that 67-97% of exonic somatic mutations were shared among all regions of an individual's tumor. The SciClone computational method identified 2-8 shared and unshared subclones in the spatially disparate areas in each tumor. MATH scores ranged from 7 to 41. Allele frequency correlation scores ranged from R(2)=0.69-0.96. Measurements of correlation between samples for copy number changes varied from R(2)=0.74-0.93. All tumors had some heterogeneity, but the degree was highly variable in the samples studied. The occurrence of significant intra-tumor heterogeneity may allow selected tumors to have a genetic reservoir to draw from in their evolutionary response to therapy and other challenges.

There is an increasing recognition of the role that cancer cell derived exosomes play in intercellular signaling upon fusion or uptake with a target cell, including immune system evasion, tumor growth and metastasis. To date, however, although exosomal membrane and cargo lipids are expected to play a pivotal role in exosome biogenesis and secretion, as well as in fusion or uptake and target cell functional response, the detailed characterization of cancer cell derived exosome lipids across a range of different cancers has not yet been broadly explored. Here, a simple and straightforward lipidome analysis strategy consisting of optimized sample extraction and novel sample derivatization techniques, coupled with high-resolution 'shotgun' mass spectrometry and 'targeted' tandem mass spectrometry methods, is demonstrated for the rapid identification of >520 individual lipids in 36 lipid classes and sub classes from exosomes secreted by the colorectal cancer cell line, LIM1215. Relative quantification and comparison of exosome versus cellular lipid profiles reveals significant enrichment of certain lipid classes, as well as substantial lipid subclass remodeling and changes in abundance of individual lipids, including sphingolipids, sterol lipids, glycerolipids and glycerophospholipids, and particularly plasmalogen- and alkyl ether-containing glycerophospholipids. This analysis strategy therefore provides a platform for comprehensive lipidome profiling across a wide range of cancer cell or tissue derived exosomes, that will facilitate subsequent functional studies aimed at elucidating the role of specific cellular or exosome lipids in the onset and progression of colorectal cancer, or to identify specific lipid(s) that could serve as effective diagnostic or prognostic disease biomarkers., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Some studies have linked colorectal cancer to metal exposure., Aims: Our objective was to evaluate the element distribution in colorectal adenocarcinoma biopsies, adjacent non-tumour tissues, and healthy controls., Methods: The study is a case-control study which compared the element distribution in colon biopsies from two groups of patients: with colorectal cancer (2 types of samples: colorectal cancer biopsies and adjacent non-tumour tissues) and healthy controls. Fifteen metal concentrations (Aluminium, Boron, Cadmium, Chromium, Copper, Iron, Magnesium, Manganese, Nickel, Lead, Selenium, Silicon, Titanium, Vanadium, and Zinc) were quantified by using inductively coupled plasma atomic emission spectrometry., Results: 104 patients were included: 76 in the colorectal cancer group, 28 in the healthy control group. Among the 15 elements analyzed, only boron, chromium, zinc, silicon and magnesium were found at clearly detectable concentrations. Colorectal tumour biopsies had significantly higher concentrations of magnesium as compared to adjacent non-tumour or healthy tissues. Zinc concentration followed the same trend but differences were not statistically significant. In addition, concentration of silicon was higher in colorectal cancer tissue than in healthy non-cancer tissue, while chromium was mostly found in adjacent non-tumour tissue., Conclusion: Magnesium, chromium, zinc and silicon were found in noteworthy concentrations in colorectal tumour. Their potential role in colorectal carcinogenesis should be explored., (Copyright © 2015 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Objectives: Pharmacokinetic studies in animal models are important for assessing the prophylactic potential of antiretroviral drugs for HIV prevention. This study sought to identify clinically relevant doses of the marketed integrase inhibitors raltegravir, elvitegravir and dolutegravir in macaques and investigate drug penetration and antiviral activity in mucosal secretions., Methods: Macaques received one oral dose of raltegravir, elvitegravir or dolutegravir alone or in combination with emtricitabine and tenofovir disoproxil fumarate followed by drug level measurements in blood and rectal and vaginal secretions. Antiviral activity was investigated in TZM-bl cells exposed to SHIV162p3 in the presence of rectal secretions collected from treated animals., Results: Plasma drug concentrations with 50 mg/kg raltegravir or elvitegravir were within the range seen in humans receiving 400-800 mg of raltegravir or 800 mg of unboosted elvitegravir but lower than with 150 mg of elvitegravir boosted with cobicistat. AUC0-24 values for dolutegravir increased proportionally with the dose, with a calculated human-equivalent dose of 20 mg/kg. Elvitegravir showed the highest penetration in rectal and vaginal fluids despite the absence of pharmacological boosting, followed by raltegravir and dolutegravir. Rectal secretions collected at 24 h from treated macaques blocked infection of TZM-bl cells by 50% at dilutions of 1/1000 (raltegravir), 1/800 (dolutegravir) and >1/30 000 (elvitegravir)., Conclusions: We defined macaque doses of HIV integrase inhibitors that recapitulate human clinical doses, which will facilitate efficacy and dose escalation studies in macaques. High and sustained drug concentrations and activity in mucosal secretions suggest that integrase inhibitors are promising candidates for HIV prevention., (Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Endometriosis involving intestinal mucosa is relatively uncommon. It poses a diagnostic challenge for clinicians and pathologists. We herein report a case of colonoscopic specimen revealing rectal mucosal endometriosis. A 39-year-old woman complained of red rectal bleeding and intermittent abdominal pain. Colonoscopic examination showed a rectal mass with ulceration and circum wall involvement. Biopsy was processed in the suspicious of carcinoma. Morphologically, irregular glands replaced residual colorectal ones, displayed mucin depletion, nuclear stratification and subtile subnuclear vacuoles. The stroma was full of spindle cells with abundant pink cytoplasm and unclear boundary. Due to subjectively interpreting as dysplastic glands in desmoplastic setting, primary rectal adenocarcinoma was firstly raised. Immunohistochemically, CK7, ER and CD10 identified the essence of ectopic endometrium. CK20 and CDX2 highlighted residual glands. In case of misdiagnosis, any pathologists should be aware of intestinal endometriosis for each female's colorectal biopsy, especially for that morphology not typical for primary adenocarcinoma or endometriosis. Reading slides carefully combined with a panel of immunomarkers would solve the pitfall.

Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae is a pathogen causing an epidemic of carbapenem-resistant Enterobacteriaceae (CRE) in healthcare settings in developed countries. The spread is primarily by patient-to-patient transmission. Understanding the mode of spread is important for designing effective control measures. We studied CRE dissemination by quantifying environmental contamination from the vicinity of 34 carriers using selective contact plates. We examined rectal CRE concentrations and clinical characteristics and correlated these with environmental contamination. Eight (24%) carriers were non-spreaders: no CRE was detected in their vicinity. Faecal continence was the only independent predictor of being a non-spreader. Among the 26 spreaders, we identified a distinct group of six (18%) super-spreaders who accounted for 79% of environmental colonies detected. Super-spreaders were likely to have high rectal CRE concentrations and to have been admitted with respiratory disease. CRE spread to the environment follows the 20/80 rule: 20% of carriers are responsible for 80% of shedding and may play a central role in CRE transmission., (Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Purpose: Unexpected focal colonic or rectal radiotracer activity is an usual finding in patients subjected to a PET study. The aim of this work has been to evaluate the clinical significance of this finding in the prediction of an existing colorectal malignancy., Material and Methods: During the last three years, all patients studied with (18)F-FDG PET/CT and PET for oncologic work-up purposes were prospectively surveyed for focal colorectal radiotracer activity. Colonoscopy was performed in all patients with this incidental finding in order to exclude colonic malignancy. CEA level, maximum standardized uptake value (SUVmax), CT findings, colonoscopy findings and histopathological results were prospectively analyzed in all patients., Results: A total of 2290 patients were evaluated, 158 of whom were studied with PET and the remainder with a hybrid PET/CT. Focal FDG colorectal activity was incidentally detected in 27 patients with no previous history of colorectal cancer. Colorectal adenocarcinoma was diagnosed in seven (25.9%) patients. A pre-cancerous lesion was found in eleven patients (40.7%). Eight patients (29.6%) had no macroscopic lesions. One patient was diagnosed with a benign lesion. Any focal activity found in the colon by (18)F-FDG PET/CT examination predicts a probability greater than 50% of an underlying malignant or premalignant lesion in the histopathological analysis (logistic regression, p=0.01), independently of the calculated SUVmax., Conclusion: According to the results of the present study, we recommend the performance of a colonoscopy and biopsy of any suspicious lesions, in all patients with unexpected focal FDG activity found in colon or rectum during a (18)F-FDG PET/CT examination., (Copyright © 2014 Elsevier España, S.L.U. and SEMNIM. All rights reserved.)

Background: This study estimated the number of daily tenofovir disoproxil fumarate/emtricitabine (TDF/FTC) doses required to achieve and maintain (after discontinuation) intracellular drug concentrations that protect against human immunodeficiency virus (HIV) infection for men who have sex with men (MSM)., Methods: Tenofovir diphosphate (TFV-DP) concentrations in peripheral blood mononuclear cells (PBMCs) and rectal mononuclear cells from an intensive pharmacokinetic study ("Cell-PrEP" [preexposure prophylaxis]) of 30 days of daily TDF/FTC followed by 30 days off drug were evaluated. A regression formula for HIV risk reduction derived from PBMCs collected in the preexposure prophylaxis initiative study was used to calculate inferred risk reduction. The time required to reach steady state for TFV-DP in rectal mononuclear cells was also determined., Results: Twenty-one HIV-uninfected adults participated in Cell-PrEP. The inferred HIV risk reduction, based on PBMC TFV-DP concentration, reached 99% (95% confidence interval [CI], 69%-100%) after 5 daily doses, and remained >90% for 7 days after stopping drug from steady-state conditions. The proportion of participants reaching the 90% effective concentration (EC90) was 77% after 5 doses and 89% after 7 doses. The percentage of steady state for natural log [TFV-DP] in rectal mononuclear cells was 88% (95% CI, 66%-94%) after 5 doses and 94% (95% CI, 78%-98%) after 7 doses., Conclusions: High PrEP activity for MSM was achieved by approximately 1 week of daily dosing. Although effective intracellular drug concentrations persist for several days after stopping PrEP, a reasonable recommendation is to continue PrEP dosing for 4 weeks after the last potential HIV exposure, similar to recommendations for postexposure prophylaxis., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Biogalvanic characterisation is a promising method for obtaining health-specific tissue information. However, there is a dearth of understanding in the literature regarding the underlying galvanic cell, electrode reactions and their controlling factors which limits the application of the technique. This work presents a parametric electrochemical investigation into a zinc–copper galvanic system using salt (NaCl) solution analogues at physiologically-relevant concentrations (1.71, 17.1 & 154 mM). The potential difference at open cell, closed cell maximum current and the internal resistance (based on published characterisation methods) were measured. Additionally, independent and relative polarisation scans of the electrodes were performed to improve understanding of the system. Our findings suggest that the prominent reaction at the cathode is that of oxygen-reduction, not hydrogen-evolution. Results indicate that cell potentials are influenced by the concentration of dissolved oxygen at low currents and maximum closed cell currents are limited by the rate of oxygen diffusion to the cathode. Characterised internal resistance values for the salt solutions did not correspond to theoretical values at the extremes of concentration (1.71 and 154 mM) due to electrode resistance and current limitation. Existing biogalvanic models do not consider these phenomena and should be improved to advance the technique and its practical application.

It has recently been shown that disease associated gene signatures can be identified by profiling tissue other than the disease related tissue. In this paper, we investigate gene signatures for Irritable Bowel Syndrome (IBS) using gene expression profiling of both disease related tissue (colon) and surrogate tissue (rectum). Gene specific joint ANOVA models were used to investigate differentially expressed genes between the IBS patients and the healthy controls taken into account both intra and inter tissue dependencies among expression levels of the same gene. Classification algorithms in combination with feature selection methods were used to investigate the predictive power of gene expression levels from the surrogate and the target tissues. We conclude based on the analyses that expression profiles of the colon and the rectum tissue could result in better predictive accuracy if the disease associated genes are known.

Purpose: Recent studies indicated that the scaffolding adaptor protein GAB2 (GRB2-associated binding protein 2) plays a critical role in the proliferation and migration of various cancers. This study aimed to determine the role of aberrant GAB2 expression in human colorectal cancer (CRC)., Methods: Quantitative real-time reverse transcription polymerase chain reaction was used to evaluate GAB2 mRNA expression in 152 CRC tissues samples to determine the clinicopathological significance of GAB2 expression. We also performed in vitro proliferation assays using siGAB2-transfected CRC cells., Results: GAB2 expression in tumor colorectal tissues was significantly higher than in normal colorectal tissues (p = 0.0212). High GAB2 expression levels were associated with malignant clinicopathologic potential factors, including lymphatic invasion (p = 0.0003), venous invasion (p = 0.0170), and liver metastasis (p = 0.0144). The survival rate of patients with high GAB2 expression levels was significantly lower than that of patients with low GAB2 expression (p = 0.0074). Multivariate analysis indicated that GAB2 expression was a factor affecting lymph node metastasis. Cell proliferation was significantly suppressed by siGAB2 expression in CRC cells in vitro., Conclusions: GAB2 expression was associated with lymph node metastasis and may play a role in the growth and metastasis of CRC. These results suggest that GAB2 is a potential therapeutic target in CRC.

Background: Although colorectal cancer is a disease characterized by sequential accumulation of mutations in epithelial cells, mechanisms leading to genomic vulnerability contributing to tumor initiation remain undefined. GUCY2C has emerged as an intestine-specific tumor suppressor controlling epithelial homeostasis through circuits canonically disrupted in cancer. Surprisingly, the GUCY2C tumor suppressor is universally overexpressed by human colorectal cancer cells. This apparent paradox likely reflects silencing of GUCY2C through loss of its paracrine hormone guanylin. Here, we quantified expression of guanylin mRNA and protein in tumors and normal epithelia from patients with colorectal cancer., Methods: Guanylin mRNA was quantified in tumors and normal adjacent epithelia from 281 patients by the reverse transcriptase-polymerase chain reaction. Separately, the guanylin protein was quantified by immunohistochemistry in 54 colorectal tumors and 30 specimens of normal intestinal epithelium., Results: Guanylin mRNA in colorectum varied more than a 100-fold across the population. Guanylin mRNA was reduced 100- to 1,000-fold in >85% of tumors compared with matched normal adjacent mucosa (P < 0.001). Loss of guanylin mRNA was greatest in tumors from patients <50 years old (P < 0.005) and with the highest expression in normal adjacent mucosa (Spearman correlation coefficient = 0.61; P < 0.001). In a separate validation cohort, guanylin protein was detected in all 30 normal colorectal mucosa specimens, but in none of 54 colorectal tumors., Conclusions: Colorectal cancer may initiate as a disease of paracrine hormone insufficiency through loss of guanylin expression, silencing the GUCY2C tumor suppressor and disrupting homeostatic mechanisms regulating colorectal epithelia cells., Impact: Intestinal tumorigenesis may be prevented by oral GUCY2C hormone replacement therapy., (©2014 American Association for Cancer Research.)

Objectives: Combination microbicide vaginal rings may be more effective than single microbicide rings at reducing/preventing sexual transmission of HIV. Here, we report the pre-clinical development and macaque pharmacokinetics of matrix-type silicone elastomer vaginal rings containing dapivirine and darunavir., Methods: Macaque rings containing 25 mg dapivirine, 100 mg dapivirine, 300 mg darunavir or 100 mg dapivirine+300 mg darunavir were manufactured and characterized by differential scanning calorimetry. In vitro release was assessed into isopropanol/water and simulated vaginal fluid. Macaque vaginal fluid and blood serum concentrations for both antiretrovirals were measured during 28 day ring use. Tissue levels were measured on day 28. Ex vivo challenge studies were performed on vaginal fluid samples and IC50 values were calculated., Results: Darunavir caused a concentration-dependent reduction in the dapivirine melting temperature in both solid drug mixes and in the combination ring. In vitro release from rings was dependent on drug loading, the number of drugs present and the release medium. In macaques, serum concentrations of both microbicides were maintained between 10(1) and 10(2) pg/mL. Vaginal fluid levels ranged between 10(3) and 10(4) ng/g and between 10(4) and 10(5) ng/g for dapivirine and darunavir, respectively. Both dapivirine and darunavir showed very similar concentrations in each tissue type; the range of drug tissue concentrations followed the general rank order: vagina (1.8 × 10(3)-3.8 × 10(3) ng/g)  > cervix (9.4 × 10(1)-3.9 × 10(2) ng/g)  > uterus (0-108 ng/g)  > rectum (0-40 ng/g). Measured IC50 values were >2 ng/mL for both compounds., Conclusions: Based on these results, and in light of recent clinical progress of the 25 mg dapivirine ring, a combination vaginal ring containing dapivirine and darunavir is a viable second-generation HIV microbicide candidate., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The main cause of death in colorectal carcinoma (CRC) patients is tumor metastasis; however, the underlying molecular mechanisms are largely unknown. In the present study, a novel metastasis-related gene, fibrinogen-like protein 2 (FGL2), was characterized for its role in CRC metastasis and underlying molecular mechanisms. The clinical significance of FGL2 was investigated using tissue microarray analysis of samples from 82 patients with CRC. The molecular effects of FGL2 in CRC cells were determined using RNA interference and ectopic expression of FGL2. The overexpression of FGL2 was examined by immunohistochemistry in 82 CRC patients, and it was determined to be an independent predictor of overall survival (P < 0.05). The depletion of FGL2 expression inhibited tumor progression and epithelial-to-mesenchymal transition (EMT) in vitro and in vivo, while ectopic overexpression of FGL2 enhanced cell invasion and induced EMT in vitro. Our results suggest that FGL2 plays an important oncogenic role in CRC aggressiveness by inducing EMT, and FGL2 could be employed as a novel prognostic marker and effective therapeutic target for CRC.

Objective: Enteric glia activation has been reported to amplify intestinal inflammation via the enteroglial-specific S100B protein. This neurotrophin promotes macrophage recruitment in the mucosa, amplify colonic inflammation and interacts with toll-like receptors (TLR). Molecules inhibiting S100B-driven enteric activation might mitigate the course of ulcerative colitis (UC). This study aims to investigate the effects of palmitoylethanolammide (PEA), a drug able to counteract astroglial activation in the central nervous system, on intestinal inflammation, in humans and mice., Design: Mouse models of dextran sodium sulphate (DSS)-induced colitis, colonic biopsies deriving from UC patients and primary cultures of mouse and human enteric glial cells (EGC), have been used to assess the effects of PEA, alone or in the presence of specific PPARα or PPARγ antagonists, on: macroscopic signs of UC (DAI score, colon length, spleen weight, macrophages/neutrophils infiltration); the expression and release of proinflammatory markers typical of UC; TLR pathway in EGCs., Results: PEA treatment improves all macroscopic signs of UC and decreases the expression and release of all the proinflammatory markers tested. PEA anti-inflammatory effects are mediated by the selective targeting of the S100B/TLR4 axis on ECG, causing a downstream inhibition of nuclear factor kappa B (NF-kB)-dependent inflammation. Antagonists at PPARα, but not PPARγ, abolished PEA effects, in mice and in humans., Conclusions: Because of its lack of toxicity, its ability in reducing inflammation and its selective PPARα action, PEA might be an innovative molecule to broaden pharmacological strategies against UC., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

The purpose of this study was to set the optimal preanalytical fixation protocol to enhance analytical and postanalytical phase accuracy and consistency. Twenty-five normal colorectal tissues were fixed using various formalin concentrations, pHs, and fixation periods. All specimens were embedded in paraffin and 4 μm sections were used for immunohistochemistry of Ki-67, and extraction and amplification of DNA and RNA. The Ki-67 labeling index and the successful gene amplification rate for DNA and mRNA were evaluated and compared among variously fixed tissue samples. Ki-67 positivity was enhanced by low pH and short fixation time, and was influenced by the type of antibody, but not by the staining (with or without using an autostainer) method. DNA amplification by PCR was strongly influenced by pH of formalin. cDNA amplification could be accomplished only with the shortest PCR fragment of 142 bp, and longer fixation times impaired the amplification. These data suggest that multiple different factors influence immunohistochemical results and gene amplification using DNA and mRNA. We recommend, based on data from this comprehensive analysis, a 10% neutral buffered formalin and fixation times of no longer than 1 week to produce consistent immunohistochemical slides and DNA amplification within 500 bp in pathology laboratories., (© 2014 The Authors. Pathology International © 2014 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.)

Matrix-assisted laser desorption ionisation imaging mass spectrometry (MALDI-MSI) is a rapidly advancing technique for intact tissue analysis that allows simultaneous localisation and quantification of biomolecules in different histological regions of interest. This approach can potentially offer novel insights into tumour microenvironmental (TME) biochemistry. In this study we employed MALDI-MSI to evaluate fresh frozen sections of colorectal cancer (CRC) tissue and adjacent healthy mucosa obtained from 12 consenting patients undergoing surgery for confirmed CRC. Specifically, we sought to address three objectives: (1) To identify biochemical differences between different morphological regions within the CRC TME; (2) To characterise the biochemical differences between cancerous and healthy colorectal tissue using MALDI-MSI; (3) To determine whether MALDI-MSI profiling of tumour-adjacent tissue can identify novel metabolic 'field effects' associated with cancer. Our results demonstrate that CRC tissue harbours characteristic phospholipid signatures compared with healthy tissue and additionally, different tissue regions within the CRC TME reveal distinct biochemical profiles. Furthermore we observed biochemical differences between tumour-adjacent and tumour-remote healthy mucosa. We have referred to this 'field effect', exhibited by the tumour locale, as cancer-adjacent metaboplasia (CAM) and this finding builds on the established concept of field cancerisation., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Background: The aim of this study was to investigate differences of miRNA-34a expression in benign and malignant colorectal lesions., Materials and Methods: Samples of cancer, paraneoplastic tissues and polyps were selected and total RNA was extracted by conventional methods for real-time PCR to detect the miRNA- 34a expression. In addition, the LOVO colorectal cancer cell line was cultured, treated with the demethylating agent 5-azacytidine and screened for differentially expressed miRNA-34a., Results: After the drug treatment, the miRNA-34a expression of colorectal cancer cell line LOVO was increased and real-time PCR showed that levels of expression in both cell line and colorectal cancer tissues were low, as compared to paraneoplastic tissue (p<0.05). Polyps tissues had significantly higher expression than paraneoplastic and colorectal cancer samples (p<0.05)., Conclusions: miRNA-34a-5p may play a role as a tumor suppressor gene in colorectal cancer, with involvement of DNA methylation.

Background and Aims: To prevent bacterial adherence and translocation, the colonic mucosa is covered by a protecting mucus layer and the epithelium synthesizes antimicrobial peptides. The present qualitative study investigated the contents and interaction of these peptides in and with rectal mucus., Methods: Rectal mucus extracts were analyzed for antimicrobial activity and screened with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Dot blot and immunohistochemistry for antimicrobial peptides. In addition, binding of AMPs to mucins was investigated by Western blot and enzyme-linked lectin assays., Results: In functional tests the mucus layer exhibited a strong antimicrobial activity. We detected 11 antimicrobial peptides in mucus extracts from healthy persons including the defensins HBD-1 and -3, the cathelicidin LL-37, ubiquitin, lysozyme, histones, high mobility group nucleosome-binding domain-containing protein 2, ubiquicidin and other ribosomal proteins. AMPs were bound by mucins but this was demonstrated to be reversible and inhibition of antibacterial activity was limited., Conclusion: These findings indicate that epithelial antimicrobial peptides are retained in the intestinal mucus layer without losing their efficacy. Thus, the mucus layer and its composition provide an attractive drug target to restore antimicrobial barrier function in intestinal diseases., (Copyright © 2013 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.)

Background: The use of calretinin immunostain (IHC) in the evaluation of rectal suction biopsies for Hirschsprung disease (HD) has been reported by Kapur et al. and others. The first goal of this article is to report our institutional experience with the use of calretinin in specimens for evaluation of HD. The second goal is to describe the pattern of expression of calretinin in the junction of ganglionic-to-aganglionic segment of pull through specimens of patients with a previous diagnosis of HD on suction rectal biopsy., Material and Methods: Three pathologists at University of Texas at Houston evaluated 28 rectal biopsy specimens from 2010-2011. The patients' age ranged from 15 days to 8 years. Twenty-three cases were suction biopsies, and five were rectal full thickness biopsies. Hematoxylin-eosin (H&E) stain was performed on at least 80 levels for the suction biopsy specimens. Calretinin immunohistochemical stain was performed on levels 40-42 in all cases, with adequate controls. The H&E slides of nine pull through specimens with a diagnosis of HD on a suction rectal biopsy that was evaluated in this study, were evaluated. Calretinin IHC was performed on the slide(s) showing the junction of aganglionic-to-normal rectum, along with adequate controls., Results: The presence of ganglion cells consistently correlated with calretinin-positive thin nerve fibrils in the lamina propria, muscularis mucosae and superficial submucosa. These nerve fibrils were absent in the aganglionic segments of bowel and in the areas without ganglion cells from the junction of normal with diseased rectum. Calretinin was strongly expressed in the submucosal and subserosal nerve trunks in the ganglionic segment. It had faint expression in the thick nerve trunks from the areas without ganglion cells 1.6-2.5 cm proximal to the normal rectum. No calretinin expression was seen in the nerve trunks in the rest of the aganglionic segment., Conclusion: The pattern of expression of calretinin in rectal suction biopsies in HD and normal rectum coincide with the ones previously described in the literature. Calretinin IHC offered additional diagnostic value in the specimens with inadequate amount of submucosa and rarely seen ganglion cells. The pattern of expression of calretinin in HD pull-through specimens correlates with the rectal biopsy ones. Faint positivity of the thick submucosal and subserosal nerves in the absence of ganglion cells and calretinin positive nerve fibrils, is characteristic of the junction of the aganglionic-to-normal rectum. We are the first ones to document this finding.

Objective: The goal of this work was to evaluate whether the volume reduction related to removal of gas in the rectum could be translated in lower doses to organs at risk (OAR) during vaginal cuff brachytherapy (VBT)., Material and Methods: Fourteen pairs of brachytherapy planning CT scans derived from 11 patients were re-segmented and re-planned using the same parameters. The only difference between pairs of CTs was the presence or lack of gas in the rectum. The first CT showed the basal status and the second was carried out after gas removal with a tube. A set of values derived from bladder and rectum dose-volume histograms (DVH) and dose-surface histograms (DSH) were extracted. Moreover the cylinder position related to the patient craniocaudal axis was recorded., Results: Rectum volume decreased significantly from 77.8 ± 45 to 55.43 ± 17.6 ml (p = 0.0052) after gas removal. Such volume diminution represented a significant reduction on all rectal DVH parameters analyzed except D25 % and D50 %. DSH parameter results were similar to previous ones. A nonsignificant increase of the bladder volume was observed and was associated with an increase of the DVH metrics analyzed., Conclusion: Removal of gas pockets is a simple and inexpensive maneuver that decreases rectal dose parameters on VBT, which can be translated as a better therapeutic ratio. It also suggests that other actions directed to empty the rectum could have a similar effect.

Maraviroc (MVC) is a potent CCR5 coreceptor antagonist that is in clinical testing for daily oral pre-exposure prophylaxis (PrEP) for HIV prevention. We used a macaque model consisting of weekly SHIV162p3 exposures to evaluate the efficacy of oral MVC in preventing rectal SHIV transmission. MVC dosing was informed by the pharmacokinetic profile seen in blood and rectal tissues and consisted of a human-equivalent dose given 24 h before virus exposure, followed by a booster postexposure dose. In rectal secretions, MVC peaked at 24 h (10,242 ng/ml) with concentrations at 48 h that were about 40 times those required to block SHIV infection of peripheral blood mononuclear cells (PBMCs) in vitro. Median MVC concentrations in rectal tissues at 24 h (1,404 ng/g) were 30 and 10 times those achieved in vaginal or lymphoid tissues, respectively. MVC significantly reduced macrophage inflammatory protein 1β-induced CCR5 internalization in rectal mononuclear cells, an indication of efficient binding to CCR5 in rectal lymphocytes. The half-life of CCR5-bound MVC in PBMCs was 2.6 days. Despite this favorable profile, 5/6 treated macaques were infected during five rectal SHIV exposures as were 3/4 controls. MVC treatment was associated with a significant increase in the percentage of CD3(+)/CCR5(+) cells in blood. We show that high and durable MVC concentrations in rectal tissues are not sufficient to prevent SHIV infection in macaques. The increases in CD3(+)/CCR5(+) cells seen during MVC treatment point to unique immunological effects of CCR5 inhibition by MVC. The implications of these immunological effects on PrEP with MVC require further evaluation.

Aim: to evaluate the expression of NF-kB and COX2 in native Indonesians with sporadic colorectal cancer (CRC)., Methods: we conducted a matched-pair case-control study by acquiring both CRC and tumor-adjacent normal tissues from the same subjects. CRC patients who underwent surgery at Cipto Mangunkusumo Hospital, Jakarta, or Hasan Sadikin Hospital, Bandung, were enrolled in the study. The specimens were immunohistologically stained with antibody directed against p65 (RelA) to assess NF-kB expression and against human COX2 protein to assess COX2 expression., Results: sixty-seven specimens consisting of both CRC and tumor-adjacent normal tissues were analyzed. COX2 expression was positive in 39 CRC tissues (58.2%), but in only 19 tumor-adjacent normal tissues (28.4%; p=0.0002). NF-kB expression was positive in 47 CRC tissues (70.1%), but in only 27 tumor-adjacent normal tissues (40.3%; p<0.0001)., Conclusion: inflammation plays a role in the carcinogenesis of sporadic CRC in native Indonesians. This support potential use of nonsteroidal anti-inflammatory drugs as chemopreventive agents for CRC.

Aim: To study an assessment of the number of enterochromaffin cells and expression of hydroxyindole-O-methyltransferase in colonic mucosa and urine excretion of 6-sulfatoxymelatonin in patients with ulcerative colitis., Methods: The study included 30 healthy subjects (group I-C), 30 patients with ulcerative proctitis [group II-ulcerative proctitis (UP)] and 30 patients with ulcerative colitis [group III-ulcerative colitis (UC)] in acute phases of these diseases. The number of enterochromaffin cells (EC) was estimated in rectal and colonic mucosa. Bioptates were assembled from many different parts of the large intestine. Immunorective cells collected from various parts of the colon were counted according to the Eurovision DAKO (Dako A/S, Copenhagen, Denmark) System in the range of 10 fields in each bioptate at × 200 magnification. The level of mRNA expression of hydroxyindole-O-methyltransferase (HIOMT) in colonic mucosa was estimated with RT-PCR. Urine 6-sulfatoxymelatonin (6-HMS) excretion was determined immunoenzymatically using an IBL (IBL International GmbH, Hamburg, Germany) kit (RE 54031)., Results: The number of EC cells in healthy subjects (C) was 132.40 ± 31.26. In patients of group II (UP) and group III (UC) the number of these cells was higher--225.40 ± 37.35 (P < 0.001) and--225.24 ± 40.50 (P < 0.001) respectively. Similar differences were related to HIOMT expression, which was 1.04 ± 0.36 in group C, 1.56 ± 0.56 (P < 0.01) in group UP and 2.00 ± 0.35 (P < 0.001) in group UC. Twenty-four hour 6-HMS urinary excretion was as follows: C--6.32 ± 4.95 μg/24 h, UP - 26.30 ± 7.29 μg/24 h (P < 0.01), UC--2.30 ± 12.56 μg/24 h (P < 0.001). A correlation between number of EC cells and 6-HMS excretion was noted in all groups: r = 0.766 in patients with UP, r = 0.703 with UC and r = 0.8551 in the control group; the correlation between the results is statistically significant., Conclusion: In the acute phases of both UP and UC, proliferation of EC cells and high expression of HIOMT and urine excretion of 6-HMS is noted. These changes may represent a beneficial response in the anti-inflammatory and defense mechanism.

Purpose: Preoperative chemoradiation therapy (CRT) significantly decreases local recurrence in locally advanced rectal cancer. Various biomarkers in biopsy specimens obtained before CRT have been proposed as predictors of response. However, reliable biomarkers remain to be established., Methods and Materials: The study group comprised 101 consecutive patients with locally advanced rectal cancer who received preoperative CRT with oral uracil/tegafur (UFT) or S-1. We evaluated histologic findings on hematoxylin and eosin (H&E) staining and immunohistochemical expressions of Ki67, p53, p21, and apoptosis in biopsy specimens obtained before CRT and 7 days after starting CRT. These findings were contrasted with the histologic response and the degree of tumor shrinkage., Results: In biopsy specimens obtained before CRT, histologic marked regression according to the Japanese Classification of Colorectal Carcinoma (JCCC) criteria and the degree of tumor shrinkage on barium enema examination (BE) were significantly greater in patients with p21-positive tumors than in those with p21-negative tumors (P=.04 and P<.01, respectively). In biopsy specimens obtained 7 days after starting CRT, pathologic complete response, histologic marked regression according to both the tumor regression criteria and JCCC criteria, and T downstaging were significantly greater in patients with apoptosis-positive and p21-positive tumors than in those with apoptosis-negative (P<.01, P=.02, P=.01, and P<.01, respectively) or p21-negative tumors (P=.03, P<.01, P<.01, and P=.02, respectively). The degree of tumor shrinkage on both BE as well as MRI was significantly greater in patients with apoptosis-positive and with p21-positive tumors than in those with apoptosis-negative or p21-negative tumors, respectively. Histologic changes in H&E-stained biopsy specimens 7 days after starting CRT significantly correlated with pathologic complete response and marked regression on both JCCC and tumor regression criteria, as well as with tumor shrinkage on BE and MRI (P<.01, P<.01, P<.01, P<.01, and P=.03, respectively)., Conclusions: Immunohistochemical expressions of p21 and apoptosis together with histologic changes on H&E-stained biopsy specimens obtained 7 days after starting CRT are strong predictors of the response to CRT., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS) method has been developed for the determination of 5,10-methylenetetrahydrofolate (methyleneTHF), tetrahydrofolate (THF) and 5-methyltetrahydrofolate (methylTHF) in colorectal mucosa and tumor tissues. The folate extraction method includes homogenization, heat and folate conjugase treatment to hydrolyze polyglutamyl folate to monoglutamyl folate. Before analysis on LC-MS/MS, simple and fast sample purification with ultrafiltration (molecular weight cut-off membrane, 10 kDa) was performed. Folates were detected and quantified using positive electrospray. The method described in the present paper was successfully applied to determine the level of three folate monoglutamates in mucosa and tumor samples from 77 colorectal cancer patients, starting from a limited amount of tissue. The results showed that the LC-MS/MS method has a great advantage over other previously used methods because of its high sensitivity and selectivity. Significantly higher levels of methyleneTHF and THF were found in tumor compared with matched mucosa tissues. Folate levels in adjacent mucosa were associated with tumor location, age and gender. The correlation between folate levels and tumor site further strengthens the fact that development of right- and left-sided tumors follows different pathways., (Copyright © 2012 John Wiley & Sons, Ltd.)

A reliable antemortem test is needed to understand the ecology of chronic wasting disease (CWD) in elk (Cervus elaphus nelsoni). We measured the ability of antemortem biopsy samples from the rectal mucosa to detect the abnormal prion protein associated with CWD (PrP(CWD)), the relationship between test results from the obex and rectal biopsies at varying stages of CWD progression, and the prevalence of CWD in free-ranging elk from Rocky Mountain National Park, Colorado, USA. We sampled and placed radio collars on 136 adult female elk in the winter of 2007-08. Elk with biopsy samples found positive for PrP(CWD) by immunohistochemistry (IHC) were euthanized and the obex and retropharyngeal lymph nodes were examined with IHC. We resampled, euthanized, and necropsied 20, 25, and 34 of the remaining study elk in each of the three following winters, respectively. Sensitivity of rectal biopsy samples increased in an asymptotic fashion with follicle count and was maximized at 85% (95% credible limits [CL]=60, 98) in the beginning of the study, when a greater proportion of elk were in a detectable stage of prion infection. However, maximum sensitivity was reduced to 72% (CL=46, 94) when we included resampled elk, which included recently infected elk that were initially negative using rectal biopsies and IHC. Test results were similar between rectal biopsies and the obex, but the earliest stages of prion infection were only detected by using retropharyngeal lymph nodes. Minimum CWD prevalence was estimated to be 9.9% (CL=5.7, 15.7) using rectal biopsies, but this rose to 12.9% (CL=8.0, 19.1) when we included four elk that were likely misdiagnosed at initial capture. Our results indicate rectal biopsies can provide a useful research tool for CWD in elk populations, but should be used with caution because they can miss individuals in early stages of infection and underestimate prevalence. Prevalence estimates from this population are the highest reported to date in elk and indicate that under appropriate conditions, CWD may be able to affect the dynamics of high-density elk populations.