A new systematization of histological analysis for the diagnosis of Hirschsprung's disease.

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.

Efficacy of silk fibroin biomaterial vehicle for in vivo mucosal delivery of Griffithsin and protection against HIV and SHIV infection ex vivo.

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.

Single oral dose for HIV pre or post-exposure prophylaxis: user desirability and biological efficacy in macaques.

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).

Diagnosis of Hirschsprung Disease.

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.

Chemical imaging and assessment of cadmium distribution in the human body.

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.

Analysis of glycoconjugates and morphological characterization of the descending colon and rectum of the plains viscacha, Lagostomus maximus.

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.

The pharmacokinetics, pharmacodynamics, and mucosal responses to maraviroc-containing pre-exposure prophylaxis regimens in MSM.

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.

Quantitative assessment of radiation-induced changes of bladder and rectum collagen structure using optical methods.

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.

HIV RNA persists in rectal tissue despite rapid plasma virologic suppression with dolutegravir-based therapy.

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.

Circulating tumour DNA for monitoring colorectal cancer-a prospective cohort study to assess relationship to tissue methylation, cancer characteristics and surgical resection.

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.

Site-dependent differences in the composite fibers of male pelvic plexus branches: an immunohistochemical analysis of donated elderly cadavers.

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.

Telomere length and genetics are independent colorectal tumour risk factors in an evaluation of biomarkers in normal bowel.

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.

Quantification and neurochemical coding of the myenteric plexus in humans: No regional variation between the distal colon and rectum.

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 µm2 . Stretch-corrected ganglionic densities were similar (colon: median 510 ganglia/100 mm2 [range 386-1170], rectum: 585 [307-923]; P = .99), as were average ganglionic sizes (colon: 57 593 µm2 [40 301-126 579], rectum: 54 901 [38 701-90 211], P = .36). Ganglionic area density (colon: 11.92 mm2 per 100 mm2 [7.53-18.64], rectum: 9.84 [5.80-17.19], P = .10) and stretch-corrected neuronal densities (colon: 189 neurons/mm2 [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.

Microcarcinoid arising in patients with long-standing ulcerative colitis: histological analysis.

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.

Single-dose pharmacokinetics of tenofovir alafenamide and its active metabolite in the mucosal tissues.

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.

Rectal suction biopsy with calretinin immunohistochemistry in patients suspected with residual aganglionosis after operation for Hirschsprung disease.

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).

Indomethacin therapy effective in a patient with depletion syndrome from secretory villous adenoma.

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.

Long-acting rilpivirine as potential pre-exposure prophylaxis for HIV-1 prevention (the MWRI-01 study): an open-label, phase 1, compartmental, pharmacokinetic and pharmacodynamic assessment.

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.

Intracellular Tenofovir and Emtricitabine Anabolites in Genital, Rectal, and Blood Compartments from First Dose to Steady State.

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/106 cells, 898-2,340) was achieved in 5 days and was >10 times higher than PBMC (95 fmol/106 cells, 85-106), seminal cells (22 fmol/106 cells, 6-79), and cervical cells (111 fmol/106 cells, 64-194). FTC-TP Css was highest in PBMC (5.7 pmol/106 cells, 5.2-6.1) and cervical cells (7 pmol/106 cells, 2-19) versus rectal (0.8 pmol/106 cells, 0.6-1.1) and seminal cells (0.3 pmol/106 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.