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

BACKGROUND & AIMS: Barrett's 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.

Natural history and management of refractory benign esophageal strictures.

BACKGROUND AND AIMS: The natural history of refractory benign esophageal strictures (RBES) is unclear, and surgery or percutaneous endoscopic gastrostomy (PEG) may be the only viable long-term options. The aim of the present study was to assess the long-term outcomes of patients with RBES. METHODS: Clinical data of consecutive patients with RBES treated in the previous 15 years in 2 tertiary-care referral academic centers with specialized interest in esophageal stricture management were retrospectively analyzed. RBES was defined as the persistence and/or recurrence of dysphagia despite at least 5 dilation sessions and/or cycles with dilation to at least 14 mm. Information regarding the use of dilation or stents and the dysphagia-free period between subsequent interventions and adverse events was collected. Clinical success was defined as no need for endoscopic interventions for at least 6 months; unfavorable outcomes were defined as the need for endoscopic treatment at the end of follow-up, surgery, or percutaneous endoscopic gastrostomy (PEG). Predictors of unfavorable outcomes were assessed by multivariate analysis. A linear mixed-effect model was used to measure dysphagia-free period changes over time. RESULTS: Overall, 70 patients with RBES (46 male; mean age 60 years) were followed for a mean of 43.9 months (range 3.7-157 months). Caustic, postradiotherapy, surgical, mixed, and postinflammatory etiology accounted for 10%, 14.3%, 31.4%, 40%, and 4.3% of causes, respectively. All patients underwent sequential sessions of pneumatic or bougie dilation, with a median of 15.5 dilation sessions per patient. Self-expandable metal stents (SEMSs) and biodegradable stents were placed in 18 (25.7%) and 14 (20%) patients, respectively. RBES resolution was achieved in only 22 of 70 (31.4%) patients. Two deaths (3%) were related to RBES. The success rate was lower in those who also were treated with endoprosthetics (odds ratio [OR] 3.7; 95% confidence interval [CI], 1.01-18.0). The mean dysphagia-free period was 3.3 months (95% CI, 2.4-4.1) for patients treated with dilation and 2.4 months (95% CI, 1.2-3.6) for those treated with stents (P = .062). Over time, the total dysphagia-free period increased at a rate of 4.1 days (95% CI, 1.7-6.4) per dilation. There was no difference in the rate of change across groups defined by sex (P = .976), age (P = .633), or endoscopic treatment (P = .267). CONCLUSIONS: Our multicenter series showed a disappointing long-term outcome for RBES, with only 1 of 3 achieving clinical resolution. The dysphagia-free period was relatively short, affecting the quality of life. Endoprosthetics did not appear to affect the natural history of RBES.

Esophageal anastomotic strictures: outcomes of endoscopic dilation, risk of recurrence and refractory stenosis, and effect of foreign body removal.

BACKGROUND & AIMS: Esophageal anastomotic strictures often require repeat dilation to relieve dysphagia. Little is known about factors that affect their remediation. We investigated long-term success and rates of recurrence or refractoriness after dilation and factors associated with refractory stenosis. METHODS: We performed a retrospective study of 74 patients with an anastomotic stricture that had been dilated during a 5-year period (564 dilations; median follow-up period, 8 months). A stricture was refractory if luminal patency could not be maintained after ≥5 dilation sessions during 10 weeks. RESULTS: Of the 74 patients, 93% had initial relief of dysphagia. The stricture recurred in 43% of patients, and 69% were considered refractory. Removal of sutures/staples protruding into the lumen did not accelerate time to initial patency (median, 37 days; interquartile range [IQR], 20-82 days) or lengthen the dysphagia-free interval (37.4 days; IQR, 8-41 weeks), compared with patients who did not undergo removal (initial patency, median 55 days; IQR, 14-109 days; P = .66 and median dysphagia-free interval, 21.7 days; IQR, 9-64 weeks; P = .8). Use of fluoroscopy during dilation (odds ratio, 8.92; 95% confidence interval, 1.98-40.14) was positively associated with development of refractory strictures, whereas neoadjuvant chemotherapy (odds ratio, 0.28; 95% confidence interval, 0.07-0.97) was inversely associated. Female sex and distal location of strictures increased risk of refractoriness as effect modifiers in multivariate analysis. CONCLUSIONS: Endoscopic dilation is highly successful in achieving luminal remediation, yet anastomotic strictures are often refractory and frequently recur. Removal of sutures/staples within the lumen does not help achieve patency. Need for fluoroscopic guidance indicates a high likelihood of refractoriness to dilation, whereas prior neoadjuvant chemotherapy indicates a lower risk.

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 Barrett's 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.

Risk of recurrent or refractory strictures and outcome of endoscopic dilation for radiation-induced esophageal strictures.

BACKGROUND: Radiation therapy for head, neck, and esophageal cancer can result in esophageal strictures that may be difficult to manage. Radiation-induced esophageal strictures often require repeat dilation to obtain relief of dysphagia. This study aimed to determine the long-term clinical success and rates of recurrent and refractory stenosis in patients with radiation-induced strictures undergoing dilation. METHODS: Retrospective cohort study of patients with radiation-induced strictures who underwent endoscopic dilation by a single provider from October 2007-October 2012. Outcomes measured included long-term clinical efficacy, interval between sessions, number of dilations, and proportion of radiation strictures that were recurrent or refractory. Risk factors for refractory strictures were assessed. RESULTS: 63 patients underwent 303 dilations. All presented with a stricture >30 days after last radiation session. Clinical success to target diameter was achieved in 52 patients (83%). A mean of 3.3 (±2.6) dilations over a median period of 4 weeks was needed to achieve initial patency. Recurrence occurred in 17 (33%) at a median of 22 weeks. Twenty-seven strictures (43%) were refractory to dilation therapy. Fluoroscopy during dilation (OR 22.88; 95% CI 3.19-164.07), severe esophageal stenosis (lumen <9 mm) (OR 10.51; 95% CI 1.94-56.88), and proximal location with prior malignancy extrinsic to the lumen (OR 6.96; 95% CI 1.33-36.29) were independent predictors of refractory strictures in multivariate analysis. CONCLUSIONS: (1) Radiation-induced strictures have a delayed onset (>30 days) from time of radiation injury. (2) Endoscopic dilation can achieve medium-term luminal remediation but the strictures have a high long-term recurrence rate of up to 33%. (3) Remediation of radiation strictures following laryngectomy can be achieved but require frequent dilations. (4) Clinical and procedural predictors may identify patients at high risk of refractory strictures. (5) The optimal strategy in highly selected refractory patients is not clear.

Perforation in colorectal stenting: a meta-analysis and a search for risk factors.

BACKGROUND: Recent studies suggest that there is a substantial risk of perforation after colorectal stent placement. OBJECTIVE: To identify risk factors for perforation from colonic stenting. DESIGN: A meta-analysis of 86 studies published between 2005 and 2011. SETTING: Multicenter review. PATIENTS: All patients who underwent colorectal stent placement. INTERVENTION: Colorectal stent placement. MAIN OUTCOME MEASUREMENTS: The occurrence of perforation with subgroup analyses for stent design, stricture etiology, stricture dilation, and concomitant chemotherapy, including the use of bevacizumab. RESULTS: A total of 4086 patients underwent colorectal stent placement; perforation occurred in 207. Meta-analysis revealed an overall perforation rate of 7.4%. Of the 9 most frequently used stent types, the WallFlex, the Comvi, and the Niti-S D-type had a higher perforation rate (>10%). A lower perforation rate (<5%) was found for the Hanarostent and the Niti-S covered stent. Stenting benign strictures was associated with a significantly increased perforation rate of 18.4% compared with 7.5% for malignant strictures. Dilation did not increase the risk of perforation: 8.5% versus 8.5% without dilation. The subgroup of post-stent placement dilation had a significantly increased perforation risk of 20.4%. With a perforation rate of 12.5%, bevacizumab-based therapy was identified as a risk factor for perforation, whereas the risk for chemotherapy without bevacizumab was 7.0% and not increased compared with the group without concomitant therapies during stent therapy (9.0%). LIMITATIONS: Heterogeneity; a considerable proportion of data is unavailable for subgroup analysis. CONCLUSIONS: The perforation rate of colonic stenting is 7.4%. Stent design, benign etiology, and bevacizumab were identified as risk factors for perforation. Intraprocedural stricture dilation and concomitant chemotherapy were not associated with an increased risk of perforation.

Endoscopic placement of self-expandable metal stents for malignant colonic obstruction: long-term outcomes and complication factors.

BACKGROUND: Self-expandable metal stents (SEMSs) are used for colonic obstruction palliatively and preoperatively. OBJECTIVE: Determine long-term efficacy, incidence of complications, and risk factors of SEMS placement for colonic obstruction. DESIGN: Retrospective review of SEMSs placed for malignant colorectal obstruction from 1999 to 2008. SETTING: Tertiary-care center. PATIENTS: This study involved 168 patients who underwent SEMS placement for palliation and 65 patients who underwent SEMS placement as a "bridge to surgery." INTERVENTION: Colonic SEMS placement. MAIN OUTCOME MEASUREMENTS: Stricture location, stent-induced complications, time to adverse events, need for reintervention. RESULTS: Technical and immediate clinical success rates were 96% and 99% in the palliative group and 95% and 98% in the preoperative group. Forty-one patients (24.4%) in the palliative group had complications including perforation (9%), occlusion (9%), migration (5%), and erosion/ulcer (2%). Mean stent patency was 145 days in the palliative group. One hundred eight of 122 patients (88.5%) were free of obstruction from implantation until death. Preoperatively placed stents remained in situ for a mean of 25.4 days and remained patent until surgery in 73.8% of patients. Complications were present preoperatively in 23.1% of patients; 94% underwent elective colectomy. Univariate analysis identified males, complete obstruction, stent diameter < or = 22 mm, stricture dilation during SEMS insertion, and operator experience as significant risk factors for complication. In the palliative group, intraluminal lesions (27% vs 19%), bevacizumab (35% vs 23%), and distal colon placement of the stent (27% vs 13%) were also associated with higher complication rates as compared to extraluminal lesions, patients not treated with bevacizumab, and stents in the proximal colon, respectively. Bevacizumab therapy nearly tripled the risk of perforation. LIMITATIONS: Retrospective analysis, single institution. CONCLUSION: Colorectal SEMS placement is relatively safe and effective but with a complication rate of nearly 25%. Patient characteristics and technical variables appear to affect the outcome of SEMS therapy.

Retrograde endoscopic cystgastrostomy for pancreatic pseudocyst drainage after a Prior Roux-en-Y gastric bypass.

A 47-year-old woman with a history of Roux-en-Y gastric bypass developed a pancreatic pseudocyst after an episode of acute necrotizing pancreatitis. She presented with intractable abdominal pain and weight loss. Computed tomography scan revealed an enlarging pancreatic fluid collection abutting the gastric antrum. The patient underwent exploratory laparotomy, at which a Whipple procedure was aborted due to severe fibrosis and necrosis of her pancreas. Retrograde peroral endoscopic pancreatic pseudocyst drainage was successfully performed through the defunctionalized stomach.

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.

Helicobacter pylori interstrain restriction-modification diversity prevents genome subversion by chromosomal DNA from competing strains.

Helicobacter pylori, bacteria that colonize the human gastric mucosa, possess a large number of genes for restriction-modification (R-M) systems, and essentially, every strain possesses a unique complement of functional and partial R-M systems. Nearly half of the H.pylori strains studied possess an active type IIs R-M system, HpyII, with the recognition sequence GAAGA. Recombination between direct repeats that flank the R-M cassette allows for its deletion whereas strains lacking hpyIIRM can acquire this cassette through natural transformation. We asked whether strains lacking HpyII R-M activity can acquire an active hpyIIRM cassette [containing a 1.4 kb kanamycin resistance (aphA) marker], whether such acquisition is DNase sensitive or resistant and whether restriction barriers limit acquisition of chromosomal DNA. Our results indicate that natural transformation and conjugation-like mechanisms may contribute to the transfer of large (4.8 kb) insertions of chromosomal DNA between H.pylori strains, that inactive or partial R-M systems can be reactivated upon recombination with a functional allele, consistent with their being contingency genes, and that H.pylori R-M diversity limits acquisition of chromosomal DNA fragments of >/=1 kb.

EUS-guided biliary drainage or enteroscopy-assisted ERCP in patients with surgical anatomy and biliary obstruction: an international comparative study.

Background and study aims: How enteroscopy-assisted ERCP (e-ERCP) and endoscopic ultrasound-guided biliary drainage (EUS-BD) compare in patients with surgically altered upper gastrointestinal anatomy is currently unknown. The aims of this study were to compare efficacy and safety of both techniques and study predictors of these outcomes. Patients and methods: This was an international, multicenter comparative cohort study at 10 tertiary centers. Outcomes data included technical success (biliary access with cholangiography and stent placement [when indicated]), clinical success (resolution of biliary obstruction) and adverse events (AEs) (graded according to the ASGE lexicon). Results: A total of 98 patients underwent EUS-BD (n = 49) or e-ERCP (n = 49). Technical success was achieved in 48 (98 %) patients in the EUS-BD group as compared to 32 (65.3 %) patients in the e-ERCP group (OR 12.48, P = 0.001). Clinical success was attained in 88 % of patients in EUS-BD group as compared to 59.1 % in the e-ERCP group (OR 2.83, P = 0.03). Procedural time was significantly shorter in the EUS-BD group (55 min vs 95 min, P < 0.0001). AEs occurred more commonly in the EUS-BD group (20 % vs. 4 %, P = 0.01). However, the majority (90 %) of AEs were mild/moderate. Length of stay was significantly longer in the EUS-BD group (6.6 d vs. 2.4 d, P < 0.0001). Conclusions: EUS-BD can be performed with a higher degree of clinical efficacy and shorter procedure time than e-ERCP in patients with surgically-altered upper gastrointestinal anatomy. Whether or not this approach should be first-line therapy in this patient population is highly dependent on the indication for the procedure, the patient's anatomy, and local practice and expertise.

Sphincter of Oddi Dysfunction.

This article reviews the diagnosis and management of sphincter of Oddi dysfunction (SOD), including the various factors to consider before embarking on endoscopic therapy for SOD. Selection starts with patient education to include possible patient misconceptions related to symptoms caused by the pancreaticobiliary sphincter as well as reinforcing the risks associated with the diagnosis and therapy. The likelihood of relief of recurrent abdominal pain attributed to SOD is related to the classification of SOD type and a crucial consideration before considering endoscopic therapy in light of recent evidence.

HIV-1 Tat protein suppresses cholangiocyte toll-like receptor 4 expression and defense against Cryptosporidium parvum.

Biliary cryptosporidiosis is associated with acquired immunodeficiency syndrome (AIDS) cholangiopathy and occurs almost exclusively in adult patients with AIDS. Infection of biliary epithelial cells (cholangiocytes) with Cryptosporidium parvum induces Toll-like receptor (TLR) 4 expression and stimulates a TLR-dependent response against infection. Here, we tested whether human immunodeficiency virus type 1 (HIV-1) Tat affects TLR expression and, hence, anti-C. parvum defense responses. Using an in vitro model of human biliary cryptosporidiosis, we found that recombinant Tat protein increased TLR4 mRNA expression in both uninfected and C. parvum-infected cholangiocytes. Conversely, Tat decreased TLR4 protein levels and suppressed C. parvum-induced TLR4 protein expression. Using actinomycin to inhibit transcription, we found that Tat increased the half-life of TLR4 mRNA from approximately 25 to 60 min, and RNA gel-shift assays demonstrated direct binding of Tat to TLR4 mRNA. In vitro transcription/translation studies suggested that Tat does not affect transcription but does decrease TLR4 translation. Importantly, more parasites were found in Tat-treated cells than in control cells 48 h after infection. These findings suggest that Tat inhibits cholangiocyte TLR4 protein expression through translational inhibition. These events appear to diminish the ability of cholangiocytes to initiate an innate immune response to C. parvum. We suggest that these findings may contribute to the unusual susceptibility of HIV-infected individuals to biliary cryptosporidiosis.

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.

A case of IgG4-associated cholangitis and autoimmune pancreatitis responsive to corticosteroids.

BACKGROUND: A 72-year-old male presented to the emergency department with epigastric pain, anorexia and progressive jaundice of 1 week's duration. He had no prior history of gastrointestinal illness, diabetes or cancer. He did not smoke or consume alcohol. He did have a family history of colon and bone cancer. INVESTIGATIONS: Biochemical and serologic studies, CT scan, abdominal ultrasound, endoscopic ultrasound, endoscopic retrograde cholangiopancreatography, biliary cytology, pancreas needle biopsies and immunohistochemical stainings. DIAGNOSIS: Autoimmune pancreatitis with IgG(4)-associated sclerosing cholangitis affecting the extrahepatic biliary ducts and mimicking primary sclerosing cholangitis and cholangiocarcinoma. MANAGEMENT: Corticosteroids and immunomodulatory therapy.

The human immunodeficiency virus type 1 tat protein enhances Cryptosporidium parvum-induced apoptosis in cholangiocytes via a Fas ligand-dependent mechanism.

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.

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 approximately 84%) and cellular invasion (by approximately 88%). Importantly, knockdown of ASM and disruption of SEMs significantly blocked C. parvum-induced accumulation of Gal/GalNAc-associated glycoproteins at infection sites by approximately 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.

Multiple TLRs are expressed in human cholangiocytes and mediate host epithelial defense responses to Cryptosporidium parvum via activation of NF-kappaB.

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-kappaB 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-kappaB. Short-interfering RNA to TLR2, TLR4, and MyD88 also blocked C. parvum-induced NF-kappaB activation. Moreover, C. parvum selectively up-regulated human beta-defensin-2 in directly infected cells, and inhibition of TLR2 and TLR4 signals or NF-kappaB activation were each associated with a reduction of C. parvum-induced human beta-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-kappaB.