Increased T-helper 2 cytokines in bile from patients with IgG4-related cholangitis disrupt the tight junction-associated biliary epithelial cell barrier.

BACKGROUND & AIMS: IgG4-related cholangitis is a chronic inflammatory biliary disease that involves different parts of the pancreatobiliary system, but little is known about its mechanisms of pathogenesis. A T-helper (Th) 2 cell cytokine profile predominates in liver tissues from these patients. We investigated whether Th2 cytokines disrupt the barrier function of biliary epithelial cells (BECs) in patients with IgG4-related cholangitis. METHODS: We assessed the Th2 cytokine profile in bile samples and brush cytology samples from 16 patients with IgG4-related cholangitis and respective controls, and evaluated transcription of tight junction (TJ)-associated proteins in primary BECs from these patients. The effect of Th2 cytokines on TJ-mediated BEC barrier function and wound closure was examined by immunoblot, transepithelial resistance, charge-selective Na(+)/Cl(-) permeability, and 4-kDa dextran flux analyses. RESULTS: Bile samples from patients with IgG4-related cholangitis had significant increases in levels of Th2 cytokines, interleukin (IL)-4, and IL-5. IL-13 was not detected in bile samples, but polymerase chain reaction analysis of whole-brush cytology samples from patients with IgG4-related cholangitis revealed increased levels of IL-13 mRNA, compared with controls. BECs isolated from the brush cytology samples revealed decreased levels of claudin-1 and increased levels of claudin-2 mRNAs. In vitro, IL-4 and IL-13 significantly reduced TJ-associated BEC barrier function by activating claudin-2-mediated paracellular pore pathways. Th2 cytokines also impaired wound closure in BEC monolayers. CONCLUSIONS: Th2 cytokines predominate in bile samples from patients with IgG4-related cholangitis and disrupt the TJ-mediated BEC barrier in vitro. Subsequent increases in biliary leaks might contribute to the pathogenesis of chronic biliary inflammation in these patients.

Enhanced innate immune responsiveness and intolerance to intestinal endotoxins in human biliary epithelial cells contributes to chronic cholangitis.

BACKGROUND: Pattern recognition receptors (PRRs) orchestrate the innate immune defence in human biliary epithelial cells (BECs). Tight control of PRR signalling provides tolerance to physiological amounts of intestinal endotoxins in human bile to avoid constant innate immune activation in BECs. AIMS: We wanted to determine whether inappropriate innate immune responses to intestinal endotoxins contribute to the development and perpetuation of chronic biliary inflammation. METHODS: We examined PRR-mediated innate immune responses and protective endotoxin tolerance in primary BECs isolated from patients with primary sclerosing cholangitis (PSC), alcoholic liver disease and patients without chronic liver disease. Expression studies comprised northern blots, RT-PCR, Western blots and immunocytochemistry. Functional studies comprised immuno-precipitation Western blots, FACS for endotoxin uptake, and NF-κB activation assays and ELISA for secreted IL-8 and tumour necrosis factor (TNF)-α. RESULTS: Primary BECs from explanted PSC livers showed reversibly increased TLR and NOD protein expression and activation of the MyD88/IRAK signalling complex. Consecutively, PSC BECs exhibited inappropriate innate immune responses to endotoxins and did not develop immune tolerance after repeated endotoxin exposures. This endotoxin hyper-responsiveness was probably because of the stimulatory effect of abundantly expressed IFN-γ and TNF-α in PSC livers, which stimulated TLR4-mediated endotoxin signalling in BECs, leading to increased TLR4-mediated endotoxin incorporation and impaired inactivation of the TLR4 signalling cascade. As TNF-α inhibition partly restored protective innate immune tolerance, endogenous TNF-α secretion probably contributed to inappropriate endotoxin responses in BECs. CONCLUSION: Inappropriate innate immune responses to intestinal endotoxins and subsequent endotoxin intolerance because of enhanced PRR signalling in BECs probably contribute to chronic cholangitis.

Efficient and selective tumor cell lysis and induction of apoptosis in melanoma cells by a conditional replication-competent CD95L adenovirus.

The high mortality of melanoma demands the development of new strategies, and gene therapy may be considered provided improvements in efficacy and selectivity. Overexpression of the death ligand CD95L/FasL has been shown in previous studies as highly effective for apoptosis induction in melanoma cells. For efficient and selective targeting of melanoma, a conditional replication-competent adenoviral vector was constructed (Ad5-FFE-02), which drives CD95L expression by a tetracycline-inducible promoter. For restricting its replication to melanoma cells, the adenoviral E1A gene is controlled by a tyrosinase-derived promoter. Furthermore, adenoviral E1B was deleted and a mutated E1A was used to preferentially support replication in tumor cells. Proving its high selectivity and efficiency, strong expression of E1A and doxycycline-dependent induction of CD95L were characteristic for tyrosinase-positive melanoma cells after Ad5-FFE-02 transduction, whereas absent in non-melanoma cell lines. Importantly, Ad5-FFE-02-mediated cell lysis was restricted to melanoma cells, and induction of apoptosis was found only in tyrosinase and CD95 expressing cells. Finally, the combination of adenoviral replication and CD95L-mediated apoptosis resulted in an enhanced repression of melanoma cell growth. This new adenoviral vector may provide a basis for an efficient targeting of melanoma.

A bidirectional Tet-dependent promotor construct regulating the expression of E1A for tight control of oncolytic adenovirus replication.

Tight regulation of oncolytic adenoviruses (oAdV) represents an important requirement for their safe application. Here we describe a new doxycycline (Dox)-dependent oAdV with a bidirectional expression cassette, which drives the expression of the reverse tetracycline-controlled transactivator (rtTA(s)-M2) from a lung tumor-specific promoter and, in the opposite direction, the expression of the adenoviral E1A gene from a second generation TetO(7) sequence linked to an isolated TATA box. In H441 lung cancer cells, this oAdV showed a strictly Dox-dependent E1A expression, adenoviral replication, cell killing activity and a 450-fold induction of progeny virus production. The virus could be shut off again by withdrawal of Dox and, in contrast to a control oAdV expressing E1A directly from the SP-B promoter, did not replicate in non-target cells. However, the absolute values of virus production and the cell killing activity in the presence of the inducer were still reduced as compared to the control oAdV. The results demonstrate, for the first time, Dox-dependent oAdV replication from a single adenoviral vector genome. Future improvement of the Dox-dependent E1A regulation cassette should lead to the generation of an oAdV well suited to meet the demands for a highly regulated and efficient oncolytic virus for in vivo applications.