Aaron J. Small

Multiple TLRs Are Expressed in Human Cholangiocytes and Mediate Host Epithelial Defense Responses to Cryptosporidium parvum via Activation of NF-κB

Infection of epithelial cells by Cryptosporidium parvum triggers a variety of host-cell innate and adaptive immune responses including release of cytokines/chemokines and up-regulation of antimicrobial peptides. The mechanisms that trigger these host-cell responses are unclear. Thus, we evaluated the role of TLRs in host-cell responses during C. parvum infection of cultured human biliary epithelia (i.e., cholangiocytes). We found that normal human cholangiocytes express all known TLRs. C. parvum infection of cultured cholangiocytes induces the selective recruitment of TLR2 and TLR4 to the infection sites. Activation of several downstream effectors of TLRs including IL-1R-associated kinase, p-38, and NF-κB was detected in infected cells. Transfection of cholangiocytes with dominant-negative mutants of TLR2 and TLR4, as well as the adaptor molecule myeloid differentiation protein 88 (MyD88), inhibited C. parvum-induced activation of IL-1R-associated kinase, p-38, and NF-κB. Short-interfering RNA to TLR2, TLR4, and MyD88 also blocked C. parvum-induced NF-κB activation. Moreover, C. parvum selectively up-regulated human β-defensin-2 in directly infected cells, and inhibition of TLR2 and TLR4 signals or NF-κB activation were each associated with a reduction of C. parvum-induced human β-defensin-2 expression. A significantly higher number of parasites were detected in cells transfected with a MyD88 dominant-negative mutant than in the control cells at 48–96 h after initial exposure to parasites, suggesting MyD88-deficient cells were more susceptible to infection. These findings demonstrate that cholangiocytes express a variety of TLRs, and suggest that TLR2 and TLR4 mediate cholangiocyte defense responses to C. parvum via activation of NF-κB.

Comparison of Wallstent and Ultraflex stents for palliation of malignant left-sided colon obstruction: a retrospective, case-matched analysis

BACKGROUND Self-expandable metal stents (SEMSs) are accepted palliation for malignant colon obstruction. Outcomes of different stent types is unknown. OBJECTIVE Our purpose was to compare outcomes after palliative placement of the Enteral Wallstent (EW) and the Precision Colonic Ultraflex (PCU) stent. DESIGN Retrospective study of all SEMS placement during a 7-year period. SETTING Tertiary care academic medical center. PATIENTS Malignant left-sided colon obstruction in which through-the-scope (TTS) or non-TTS stent placement was possible. MAIN OUTCOME MEASUREMENTS Technical and clinical success rates, stent-related complications, reintervention. RESULTS Demographics, degree, site, and cause of obstruction were comparable. Technical difficulties were more frequent with EW than PCU (16% vs 9%, P not significant), insufficient stent expansion and stent misplacement being most common. Relief of obstruction occurred in all patients when placement was technically successful. Mean follow-up was 93 days (range 7-691 days). Early (<7 days) stent occlusion (6% vs 0%, P not significant) and migration (4% vs 0%, P not significant) occurred more frequently in the EW group. Self-limited hematochezia was more common with PCU (20% vs 2%, P = .002). Delayed complications (perforation, stent occlusion, migration, and erosion) occurred significantly more often in the EW group (38% vs 20%). Reintervention was needed more frequently for EW, endoscopic (40% vs 17%, P = .01) and operative (46% vs 26%, P = .03). CONCLUSIONS Enteral Wallstents and Precision Ultraflex Colonic stents adequately relieve colonic obstruction. Stent dysfunction, stent-related complications, and need for reintervention are higher after EW placement. Precision Colonic Ultraflex stents appear better suited for palliation of left-sided malignant colon obstruction.

Radiofrequency Ablation Is Associated With Decreased Neoplastic Progression in Patients With Barrett's Esophagus and Confirmed Low-Grade Dysplasia

BACKGROUND & AIMS Barretts esophagus (BE) with low-grade dysplasia (LGD) can progress to high-grade dysplasia (HGD) and esophageal adenocarcinoma (EAC). Radiofrequency ablation (RFA) has been shown to be an effective treatment for LGD in clinical trials, but its effectiveness in clinical practice is unclear. We compared the rate of progression of LGD after RFA with endoscopic surveillance alone in routine clinical practice. METHODS We performed a retrospective study of patients who either underwent RFA (n = 45) or surveillance endoscopy (n = 125) for LGD, confirmed by at least 1 expert pathologist, from October 1992 through December 2013 at 3 medical centers in the United States. Cox regression analysis was used to assess the association between progression and RFA. RESULTS Data were collected over median follow-up periods of 889 days (interquartile range, 264-1623 days) after RFA and 848 days (interquartile range, 322-2355 days) after surveillance endoscopy (P = .32). The annual rates of progression to HGD or EAC were 6.6% in the surveillance group and 0.77% in the RFA group. The risk of progression to HGD or EAC was significantly lower among patients who underwent RFA than those who underwent surveillance (adjusted hazard ratio = 0.06; 95% confidence interval: 0.008-0.48). CONCLUSIONS Among patients with BE and confirmed LGD, rates of progression to a combined end point of HGD and EAC were lower among those treated with RFA than among untreated patients. Although selection bias cannot be excluded, these findings provide additional evidence for the use of endoscopic ablation therapy for LGD.

Cryptosporidium parvum infects human cholangiocytes via sphingolipid-enriched membrane microdomains

Cryptosporidium parvum attaches to intestinal and biliary epithelial cells via specific molecules on host‐cell surface membranes including Gal/GalNAc‐associated glycoproteins. Subsequent cellular entry of this parasite depends on host‐cell membrane alterations to form a parasitophorous vacuole via activation of phosphatidylinositol 3‐kinase (PI‐3K)/Cdc42‐associated actin remodelling. How C. parvum hijacks these host‐cell processes to facilitate its infection of target epithelia is unclear. Using specific probes to known components of sphingolipid‐enriched membrane microdomains (SEMs), we detected aggregation of host‐cell SEM components at infection sites during C. parvum infection of cultured human biliary epithelial cells (i.e. cholangiocytes). Activation and membrane translocation of acid‐sphingomyelinase (ASM), an enzyme involved in SEM membrane aggregation, were also observed in infected cells. Pharmacological disruption of SEMs and knockdown of ASM via a specific small interfering RNA (siRNA) significantly decreased C. parvum attachment (by ∼84%) and cellular invasion (by ∼88%). Importantly, knockdown of ASM and disruption of SEMs significantly blocked C. parvum‐induced accumulation of Gal/GalNAc‐associated glycoproteins at infection sites by ∼90%. Disruption of SEMs and knockdown of ASM also significantly blocked C. parvum‐induced activation of host‐cell PI‐3K and subsequent accumulation of Cdc42 and actin by up to 75%. Our results suggest an important role of SEMs for C. parvum attachment to and entry of host cells, likely via clustering of membrane‐binding molecules and facilitating of C. parvum‐induced actin remodelling at infection sites through activation of the PI‐3K/Cdc42 signalling pathway.

Comparative risk of recurrence of dysplasia and carcinoma after endoluminal eradication therapy of high-grade dysplasia versus intramucosal carcinoma in Barrett's esophagus.

BACKGROUND Endoscopic therapy is the preferred approach for the management of Barretts esophagus (BE) patients with high-grade dysplasia (HGD) and intramucosal carcinoma (IMC). Little is known about outcome differences in patients with HGD versus IMC. OBJECTIVE To determine and compare the rate of recurrent dysplasia or neoplasia in patients with HGD or IMC undergoing endoscopic therapy. DESIGN Retrospective cohort study. PATIENTS A total of 246 BE patients with either HGD or IMC referred for endoscopic therapy. INTERVENTION Patients underwent EMR and/or ablation therapy with the goal of complete eradication of all dysplasia/neoplasia and intestinal metaplasia (CE-IM). Patients were assigned to either the HGD or IMC group based on highest pathology grade at the start of therapy. MAIN OUTCOME MEASUREMENTS Complete eradication and recurrence of IM and/or HGD/neoplasia were assessed among patients with HGD versus IMC. Only patients with CE-IM (documented eradication of all dysplasia/neoplasia and IM on a single endoscopy) were included for analysis of recurrence rates and risk factors. RESULTS CE-IM was achieved in 113 of 135 patients (83.7%) with HGD and in 84 of 111 patients (75.7%) with IMC (P = .16). Overall recurrence rates of dysplasia or neoplasia after CE-IM were similar in both groups (HGD, 8.0% vs IMC, 9.5%; P = .44; relative risk, 1.2; 95% confidence interval, 0.5-3.0) and remained similar in patients with 5 years of surveillance after CE-IM (HGD, 13.5% vs IMC, 11.4%; P = .53; relative risk, 0.85; 95% confidence interval, 0.3-2.7). LIMITATIONS Retrospective, observational study and evolution of endoscopic modalities and experience. CONCLUSION Endoluminal therapy can successfully achieve eradication of IM and dysplasia or neoplasia in BE patients with HGD and IMC at comparable rates. There were no differences in the rates of recurrent HGD/IMC in the 2 groups.

The Human Immunodeficiency Virus Type 1 Tat Protein Enhances Cryptosporidium parvum-Induced Apoptosis in Cholangiocytes via a Fas Ligand-Dependent Mechanism

ABSTRACT While Cryptosporidium parvum infection of the intestine has been reported in both immunocompetent and immunocompromised individuals, biliary infection is seen primarily in adult AIDS patients and is associated with development of AIDS cholangiopathy. However, the mechanisms of pathogen-induced AIDS cholangiopathy remain unclear. Since we previously demonstrated that the Fas/Fas ligand (FasL) system is involved in paracrine-mediated C. parvum cytopathicity in cholangiocytes, we also tested the potential synergistic effects of human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (Tat)-mediated FasL regulation on C. parvum-induced apoptosis in cholangiocytes by semiquantitative reverse transcription-PCR, immunoblotting, immunofluorescence analysis, and immunogold electron microscopy. H69 cells do not express CXCR4 and CCR5, which are receptors required for direct HIV-1 viral infection. However, recombinant biologically active HIV-1-associated Tat protein increased FasL expression in the cytoplasm of cholangiocytes without a significant increase in apoptosis. We found that C. parvum-induced apoptosis was associated with translocation of intracellular FasL to the cell membrane surface and release of full-length FasL from infected H69 cells. Tat significantly (P < 0.05) increased C. parvum-induced apoptosis in bystander cells in a dose-dependent manner. Moreover, Tat enhanced both C. parvum-induced FasL membrane translocation and release of full-length FasL. In addition, the FasL neutralizing antibody NOK-1 and the caspase-8 inhibitor Z-IETD-fmk both blocked C. parvum-induced apoptosis in cholangiocytes. The data demonstrated that HIV-1 Tat enhances C. parvum-induced cholangiocyte apoptosis via a paracrine-mediated, FasL-dependent mechanism. Our results suggest that concurrent active HIV replication, with associated production of Tat protein, and C. parvum infection synergistically increase cholangiocyte apoptosis and thus jointly contribute to AIDS-related cholangiopathies.

Host Cell Actin Remodeling in Response to Cryptosporidium

Cryptosporidium exhibits a complex strategy to invade and establish productive infection sites, involving complimentary parasite and host cell processes. While the work regarding host cell actin remodeling has greatly enhanced our understanding of the molecular pathways involved in the parasite induced actin reorganization, the specific function of host cell actin remodeling is still equivocal. We contend that host cell actin polymerization contributes to the development of productive C. parvum infection sites by generating membrane protrusion events, which may assist in the retention of the parasite at the apical surface within the unique extracytoplasmic niche. With our current understanding of the molecular pathways initiating actin remodeling upon C. parvum interactions with host cells, the next logical step is to determine the upstream events resulting in PI3K activation and the specific role of actin remodeling in parasite development, a process that may have implications beyond host-pathogen interactions.

Novel endoscopic approaches for assessing biliary tract diseases.

Purpose of review Biliary endoscopy offers both diagnostic and therapeutic value in complex clinical situations. This review addresses the latest advances over the past year in endoscopic approaches of biliary tract diseases. Recent findings Specifically, we focus on the latest findings on endoscopic retrograde cholangiopancreatography for the evaluation of biliary strictures. In addition, key studies have demonstrated the enhanced role of cholangioscopy, photodynamic therapy in cholangiocarcinoma, and biliary stent technology. Summary The following review focuses on the latest advancements in the field of biliary endoscopy. Pivotal studies were selected to highlight some of the current investigations in therapeutic endoscopic retrograde cholangiopancreatography as well as knowledge gaps for future research.

A Lower Cyst Fluid CEA Cut-Off Increases Diagnostic Accuracy in Identifying Mucinous Pancreatic Cystic Lesions

Context Carcinoembryonic antigen analysis of pancreatic cyst fluid is the tumor marker of choice for preoperatively differentiating mucinous from non-mucinous cystic lesions. Objective We aim to determine the most accurate cyst carcinoembryonic antigen cut-off value for distinguishing mucinous cysts from non-mucinous cysts with a focus on discriminating intraductal papillary mucinous neoplasms. Methods The results of pancreatic cyst aspiration carcinoembryonic antigen levels from a single center were retrospectively collected and evaluated for a diagnosis of a mucinous cyst and an assessment of malignancy using surgical histology as the diagnostic standard in 86 patients. Results The median cyst carcinoembryonic antigen level (ng/mL) was significantly higher in mucinous cysts compared with non-mucinous cysts (218 vs. 4.4; P=0.0006) and in intraductal papillary mucinous neoplasms compared with non-mucinous cysts (135 vs. 4.4; P=0.0027). A cyst carcinoembryonic antigen cut-off of 30.7 ng/mL was most accurate (87.2%) for differentiating mucinous from non-mucinous cysts and specifically for differentiating intraductal papillary mucinous neoplasms from non-mucinous cysts (82.7%). Cyst carcinoembryonic antigen levels were not significantly different between malignant and non-malignant mucinous cysts (68.5 vs. 238.1; P=0.51). Conclusions Pancreatic cyst fluid carcinoembryonic antigen can accurately differentiate histologically verified mucinous lesions, including intraductal papillary mucinous neoplasms, from non-mucinous lesions with an optimal cut-off that is much lower than previously reported values. Cyst carcinoembryonic antigen levels are not a reliable predictor of malignancy. Image: Sensitivity and specificity curves of cyst fluid CEA levels for differentiating mucinous from non-mucinous cysts.

How safe and effective is a nitinol self-expanding metallic stent for palliation of malignant colonic obstruction?

How safe and effective is a nitinol self-expanding metallic stent for palliation of malignant colonic obstruction?