Prevention of Rebleeding From Esophageal Varices in Patients With Cirrhosis Receiving Small-Diameter Stents Versus Hemodynamically Controlled Medical Therapy.

BACKGROUND & AIMS: Patients with cirrhosis and variceal hemorrhage have a high risk of rebleeding. We performed a prospective randomized trial to compare the prevention of rebleeding in patients given a small-diameter covered stent vs those given hepatic venous pressure gradient (HVPG)-based medical therapy prophylaxis. METHODS: We performed an open-label study of patients with cirrhosis (92% Child class A or B, 70% alcoholic) treated at 10 medical centers in Germany. Patients were assigned randomly more than 5 days after variceal hemorrhage to groups given a small covered transjugular intrahepatic portosystemic stent-shunt (TIPS) (8 mm; n = 90), or medical reduction of portal pressure (propranolol and isosorbide-5-mononitrate; n = 95). HVPG was determined at the time patients were assigned to groups (baseline) and 2 weeks later. In the medical group, patients with an adequate reduction in HVPG (responders) remained on the drugs whereas nonresponders underwent only variceal band ligation. The study was closed 10 months after the last patient was assigned to a group. The primary end point was variceal rebleeding. Survival, safety (adverse events), and quality of life (based on the Short Form-36 health survey) were secondary outcome measures. RESULTS: A significantly smaller proportion of patients in the TIPS group had rebleeding within 2 years (7%) than in the medical group (26%) (P = .002). A slightly higher proportion of patients in the TIPS group experienced adverse events, including encephalopathy (18% vs 8% for medical treatment; P = .05). Rebleeding occurred in 6 of 23 patients (26%) receiving medical treatment before hemodynamic control was possible. Per-protocol analysis showed that rebleeding occurred in a smaller proportion of the 32 responders (18%) than in nonresponders who received variceal band ligation (31%) (P = .06). Fifteen patients from the medical group (16%) underwent TIPS placement during follow-up evaluation, mainly for refractory ascites. Survival time and quality of life did not differ between both randomized groups. CONCLUSIONS: Placement of a small-diameter, covered TIPS was straightforward and prevented variceal rebleeding in patients with Child A or B cirrhosis more effectively than drugs, which often required step-by-step therapy. However, TIPS did not increase survival time or quality of life and produced slightly more adverse events. Clinical Trial no: ISRCTN 16334693.

AMP affects intracellular Ca2+ signaling, migration, cytokine secretion and T cell priming capacity of dendritic cells.

The nucleotide adenosine-5'-monophosphate (AMP) can be released by various cell types and has been shown to elicit different cellular responses. In the extracellular space AMP is dephosphorylated to the nucleoside adenosine which can then bind to adenosine receptors. However, it has been shown that AMP can also activate A(1) and A(2a) receptors directly. Here we show that AMP is a potent modulator of mouse and human dendritic cell (DC) function. AMP increased intracellular Ca(2+) concentration in a time and dose dependent manner. Furthermore, AMP stimulated actin-polymerization in human DCs and induced migration of immature human and bone marrow derived mouse DCs, both via direct activation of A(1) receptors. AMP strongly inhibited secretion of TNF-α and IL-12p70, while it enhanced production of IL-10 both via activation of A(2a) receptors. Consequently, DCs matured in the presence of AMP and co-cultivated with naive CD4(+)CD45RA(+) T cells inhibited IFN-γ production whereas secretion of IL-5 and IL-13 was up-regulated. An enhancement of Th2-driven immune response could also be observed when OVA-pulsed murine DCs were pretreated with AMP prior to co-culture with OVA-transgenic naïve OTII T cells. An effect due to the enzymatic degradation of AMP to adenosine could be ruled out, as AMP still elicited migration and changes in cytokine secretion in bone-marrow derived DCs generated from CD73-deficient animals and in human DCs pretreated with the ecto-nucleotidase inhibitor 5'-(alpha,beta-methylene) diphosphate (APCP). Finally, the influence of contaminating adenosine could be excluded, as AMP admixed with adenosine desaminase (ADA) was still able to influence DC function. In summary our data show that AMP when present during maturation is a potent regulator of dendritic cell function and point out the role for AMP in the pathogenesis of inflammatory disorders.

MR-based visualization and quantification of three-dimensional flow characteristics in the portal venous system.

PURPOSE: To evaluate the feasibility of time-resolved flow-sensitive MRI for the three-dimensional (3D) visualization and quantification of normal and pathological portal venous (PV) hemodynamics. MATERIALS AND METHODS: Portal venous hemodynamics were evaluated in 18 healthy volunteers and 5 patients with liver cirrhosis. ECG- and adaptive respiratory navigator gated flow-sensitive 4D MRI (time-resolved 3D MRI with three-directional velocity encoding) was performed on a 3 Tesla MR system (TRIO, Siemens, Germany). Qualitative flow analysis was achieved using 3D streamlines and time-resolved particle traces originating from seven emitter planes precisely placed at anatomical landmarks in the PV system. Quantitative analysis included retrospective extraction of regional peak and mean velocities and vessel area. Results were compared with standard 2D flow-sensitive MRI and to the reference standard Doppler ultrasound. RESULTS: Qualitative flow analysis was successfully used in the entire PV system. Venous hemodynamics in all major branches in 17 of 18 volunteers and 3 of 5 patients were reliably depicted with good interobserver agreement (kappa = 0.62). Quantitative analysis revealed no significant differences and moderate agreement for peak velocities between 3D MR and 2D MRI (r = 0.46) and Doppler ultrasound (US) (r = 0.35) and for mean velocities between 3D and 2D MRI (r = 0.41). The PV area was significantly (P < 0.01) higher in 3D and 2D MRI compared with US. CONCLUSION: We successfully applied 3D MR velocity mapping in the PV system, providing a detailed qualitative and quantitative analysis of normal and pathological hemodynamics.

5-hydroxytryptamine modulates migration, cytokine and chemokine release and T-cell priming capacity of dendritic cells in vitro and in vivo.

Beside its well described role in the central and peripheral nervous system 5-hydroxytryptamine (5-HT), commonly known as serotonin, is also a potent immuno-modulator. Serotoninergic receptors (5-HTR) are expressed by a broad range of inflammatory cell types, including dendritic cells (DCs). In this study, we aimed to further characterize the immuno-biological properties of serotoninergic receptors on human monocyte-derived DCs. 5-HT was able to induce oriented migration in immature but not in LPS-matured DCs via activation of 5-HTR(1) and 5-HTR(2) receptor subtypes. Accordingly, 5-HT also increased migration of pulmonary DCs to draining lymph nodes in vivo. By binding to 5-HTR(3), 5-HTR(4) and 5-HTR(7) receptors, 5-HT up-regulated production of the pro-inflammatory cytokine IL-6. Additionally, 5-HT influenced chemokine release by human monocyte-derived DCs: production of the potent Th1 chemoattractant IP-10/CXCL10 was inhibited in mature DCs, whereas CCL22/MDC secretion was up-regulated in both immature and mature DCs. Furthermore, DCs matured in the presence of 5-HT switched to a high IL-10 and low IL-12p70 secreting phenotype. Consistently, 5-HT favoured the outcome of a Th2 immune response both in vitro and in vivo. In summary, our study shows that 5-HT is a potent regulator of human dendritic cell function, and that targeting serotoninergic receptors might be a promising approach for the treatment of inflammatory disorders.

Analysis of CD127 and KLRG1 expression on hepatitis C virus-specific CD8+ T cells reveals the existence of different memory T-cell subsets in the peripheral blood and liver.

The differentiation and functional status of virus-specific CD8+ T cells is significantly influenced by specific and ongoing antigen recognition. Importantly, the expression profiles of the interleukin-7 receptor alpha chain (CD127) and the killer cell lectin-like receptor G1 (KLRG1) have been shown to be differentially influenced by repetitive T-cell receptor interactions. Indeed, antigen-specific CD8+ T cells targeting persistent viruses (e.g., human immunodeficiency virus and Epstein-Barr virus) have been shown to have low CD127 and high KLRG1 expressions, while CD8+ T cells targeting resolved viral antigens (e.g., FLU) typically display high CD127 and low KLRG1 expressions. Here, we analyzed the surface phenotype and function of hepatitis C virus (HCV)-specific CD8+ T cells. Surprisingly, despite viral persistence, we found that a large fraction of peripheral HCV-specific CD8+ T cells were CD127+ and KLRG1- and had good proliferative capacities, thus resembling memory cells that usually develop following acute resolving infection. Intrahepatic virus-specific CD8+ T cells displayed significantly reduced levels of CD127 expression but similar levels of KLRG1 expression compared to the peripheral blood. These results extend previous studies that demonstrated central memory (CCR7+) and early-differentiated phenotypes of HCV-specific CD8+ T cells and suggest that insufficient stimulation of virus-specific CD8+ T cells by viral antigen may be responsible for this alteration in HCV-specific CD8+ T-cell differentiation during chronic HCV infection.

Expression of the interleukin-7 receptor alpha chain (CD127) on virus-specific CD8+ T cells identifies functionally and phenotypically defined memory T cells during acute resolving hepatitis B virus infection.

Virus-specific CD8+ T cells play a central role in the outcome of several viral infections, including hepatitis B virus (HBV) infection. A key feature of virus-specific CD8+ T cells is the development of memory. The mechanisms resulting in the establishment of T-cell memory are still only poorly understood. It has been suggested that T-cell memory may depend on the survival of virus-specific CD8+ T cells in the contraction phase. Indeed, a population of effector cells that express high levels of the interleukin-7 receptor alpha chain (CD127) as the precursors of memory CD8+ T cells has recently been identified in mice. However, very little information is currently available about the kinetics of CD127 expression in an acute resolving viral infection in humans and its association with disease pathogenesis, viral load, and functional and phenotypical T-cell characteristics. To address these important issues, we analyzed the HBV-specific CD8+ T-cell response longitudinally in a cohort of six patients with acute HBV infection who spontaneously cleared the virus. We observed the emergence of CD127 expression on antigen-specific CD8+ memory T cells during the course of infection. Importantly, the up-regulation of CD127 correlated phenotypically with a loss of CD38 and PD-1 expression and acquisition of CCR7 expression: functionally with an enhanced proliferative capacity and clinically with the decline in serum alanine aminotransferase levels and viral clearance. These results suggest that the expression of CD127 is a marker for the development of functionally and phenotypically defined antigen-specific CD8+ memory T cells in cleared human viral infections.

The P2X7 receptor: a key player in IL-1 processing and release.

Human IL-1 family proteins are key mediators of the host response to infections, injury, and immunologic challenges. The mechanism by which IL-1 activates proinflammatory responses in target cells, and the plasma membrane receptors involved, is fairly well known. This has led to the development of innovative drugs that block IL-1 downstream to its synthesis and secretion. On the contrary, the mechanism of IL-1 and other IL-1 family members (e.g., IL-18) maturation and release is incompletely understood. Accruing evidence points to a plasma membrane receptor for extracellular ATP, the P2X(7) receptor, as a key player in both processes. A deeper understanding of the mechanism by which the P2X(7) receptor triggers IL-1 maturation and exteriorization may suggest novel avenues for the treatment of inflammatory diseases and provide a deeper insight in the fundamental mechanism of protease activation and cellular export of proteins lacking a leader sequence.

Activation of human eosinophils via P2 receptors: novel findings and future perspectives.

A growing body of information indicates that release of intracellular nucleotides represents an important way to modulate several cell pathways in physiological or pathological conditions. Nucleotides released as a consequence of cell damage, cell stress, bacterial infection, or other noxious stimuli signal at a class of plasma membrane receptors--P2 receptors--activating diverse intracellular pathways in many tissues and organs. For example, nucleotides secreted in the airway system control chloride/liquid secretion, goblet cell degranulation, and ciliary beat frequency. Several studies indicate that nucleotides play a role in airway diseases through their action on multiple cell types, including mast cells, dendritic cells, neurons, and eosinophils. Recent work by us and other groups led to the identification and characterization of P2 receptors expressed by human eosinophils. In this review, we will summarize recent developments in this field and put forward a hypothesis about the role of P2 receptors in pathophysiological conditions where eosinophils are major players.

Increased expression of the NK cell receptor KLRG1 by virus-specific CD8 T cells during persistent antigen stimulation.

The killer cell lectin-like receptor G1 (KLRG1) is a natural killer cell receptor expressed by T cells that exhibit impaired proliferative capacity. Here, we determined the KLRG1 expression by virus-specific T cells. We found that repetitive and persistent antigen stimulation leads to an increase in KLRG1 expression of virus-specific CD8+ T cells in mice and that virus-specific CD8+ T cells are mostly KLRG1+ in chronic human viral infections (human immunodeficiency virus, cytomegalovirus, and Epstein-Barr virus) but not in resolved infection (influenza virus). Thus, by using KLRG1 as a T-cell marker, our results suggest that the differentiation status and function of virus-specific CD8+ T cells are directly influenced by persistent antigen stimulation.

T cells with a CD4+CD25+ regulatory phenotype suppress in vitro proliferation of virus-specific CD8+ T cells during chronic hepatitis C virus infection.

Chronic hepatitis C virus (HCV) infection is associated with impaired proliferative, cytokine, and cytotoxic effector functions of HCV-specific CD8(+) T cells that probably contribute significantly to viral persistence. Here, we investigated the potential role of T cells with a CD4(+)CD25(+) regulatory phenotype in suppressing virus-specific CD8(+) T-cell proliferation during chronic HCV infection. In vitro depletion studies and coculture experiments revealed that peptide specific proliferation as well as gamma interferon production of HCV-specific CD8(+) T cells were inhibited by CD4(+)CD25(+) T cells. This inhibition was dose dependent, required direct cell-cell contact, and was independent of interleukin-10 and transforming growth factor beta. Interestingly, the T-cell-mediated suppression in chronically HCV-infected patients was not restricted to HCV-specific CD8(+) T cells but also to influenza virus-specific CD8(+) T cells. Importantly, CD4(+)CD25(+) T cells from persons recovered from HCV infection and from healthy blood donors exhibited significantly less suppressor activity. Thus, the inhibition of virus-specific CD8(+) T-cell proliferation was enhanced in chronically HCV-infected patients. This was associated with a higher frequency of circulating CD4(+)CD25(+) cells observed in this patient group. Taken together, our results suggest that chronic HCV infection leads to the expansion of CD4(+)CD25(+) T cells that are able to suppress CD8(+) T-cell responses to different viral antigens. Our results further suggest that CD4(+)CD25(+) T cells may contribute to viral persistence in chronically HCV-infected patients and may be a target for immunotherapy of chronic hepatitis C.

5-Hydroxytryptamine modulates cytokine and chemokine production in LPS-primed human monocytes via stimulation of different 5-HTR subtypes.

The neurotransmitter 5-hydroxytryptamine (5-HT), commonly known as serotonin, is released at peripheral sites from activated enterochromaffin cells, mast cells and platelets. In this study we analyzed the biological activity and intracellular signaling of 5-HT in human monocytes. By reverse transcription (RT) and PCR, messenger RNA (mRNA) expression of 5-HT receptor 1E (5-HTR(1E)), 5-HTR(2A), 5-HTR(3), 5-HTR(4) and 5-HTR(7) could be revealed. Functional studies showed that 5-HT modulates the release of IL-1beta, IL-6, IL-8/CXCL8, IL-12p40 and tumor necrosis factor-alpha (TNF-alpha), while it has no effect on the production of IL-18 and IFN-gamma in LPS-stimulated human blood monocytes. Moreover, RT and PCR revealed that 5-HT modulated mRNA levels of IL-6 and IL-8/CXCL8, but did not influence mRNA levels of IL-1beta and TNF-alpha. Pharmacological studies with isotype-selective receptor agonists allowed us to show that 5-HTR(3) subtype up-regulates the LPS-induced production of IL-1beta, IL-6 and IL-8/CXCL8, while it was not involved in TNF-alpha and IL-12p40 secretion. Furthermore, activation of the G(s)-coupled 5-HTR(4) and 5-HTR(7) subtypes increased intracellular cyclic AMP (cAMP) and secretion of IL-1beta, IL-6, IL-12p40 and IL-8/CXCL8, while, on the contrary, it inhibited LPS-induced TNF-alpha release. Interestingly, 5-HTR(1) and 5-HTR(2) agonists did not modulate the LPS-induced cytokine production in human monocytes. Our results point to a new role for 5-HT in inflammation by modulating cytokine production in monocytes via activation of 5-HTR(3), 5-HTR(4) and 5-HTR(7) subtypes.

P2X(7) receptor: Death or life?

The P2X(7) plasma membrane receptor is an intriguing molecule that is endowed with the ability to kill cells, as well as to activate many responses and even stimulate proliferation. Here, the authors give an overview on the multiplicity and complexity of P2X(7)-mediated responses, discussing recent information on this receptor. Particular attention has been paid to early and late signs of apoptosis and necrosis linked to activation of the receptor and to the emerging field of P2X(7) function in carcinogenesis.

Characterization of the biological activities of uridine diphosphate in human dendritic cells: Influence on chemotaxis and CXCL8 release.

Uridine nucleotides are endogenous nucleotides which are released into the extracellular space from mechanical stressed endothelial and epithelial cells as well as lipopolysaccharide (lps)-stimulated monocytes. Here, we studied the biological activity of the selective purinoreceptor P2Y6 (P2YR6) agonist Uridine 5'diphosphate (UDP) as well as the P2YR2- and P2YR4-activating uridine 5'triphosphate (UTP) on human dendritic cells (DC). These cells in their immature state have the ability to migrate from blood to peripheral target sites where they sense dangerous signals and capture potential antigens. Moreover, mature DC induce innate immune responses and migrate from peripheral tissues to secondary lymphoid organs in order to activate naive T cells and initiate adaptive immunity. Here, we were able to show that uridine nucleotides stimulated Ca(2+) transients, actin polymerization, and chemotaxis in immature DC. Experiments with pertussis toxin, the stable pyrimidine agonist uridine 5'-O-(2-thiodiphosphate) (UDPgammaS) and receptor antagonists, as well as desensitization studies suggested that these uridine nucleotides activities were mediated by different G(i) protein-coupled receptors. During lps-induced maturation, DC lost their ability to respond towards uridine nucleotides with these activities. Instead, UDP, but not UTP, stimulated the release of the CXC-chemokine 8 (CXCL8) from mature DC in a reactive blue sensitive manner. Moreover, our study indicates that UDP stimulates different signaling pathways in immature and mature DC in order to favor the accumulation of immature DC and to augment the capacity to secrete CXCL8 in mature DC.

The serotoninergic receptors of human dendritic cells: identification and coupling to cytokine release.

The neurotransmitter 5-hydroxytryptamine (5-HT), commonly known as serotonin, is stored at peripheral sites in mast cells and released from this peripheral source upon IgE cross-linking. In this study, we investigated the expression of serotoninergic receptors (5-HTR), the signaling pathway, and biological activity of 5-HT on human dendritic cells (DC), showing that immature and mature DC expressed mRNA for different serotoninergic receptors. Thereby, the mRNA of 5-HTR(1B), 5-HTR(1E), 5-HTR(2A), 5-HTR(2B), one splicing variant of the 5-HTR(3), 5-HTR(4), and 5-HTR(7) receptors were detected. Immature DC preferentially expressed mRNA for the heptahelical 5-HTR(1B), 5-HTR(1E), and 5-HTR(2B) receptors, while mature DC mostly expressed 5-HTR(4) and 5-HTR(7). The mRNA expression level of the ligand-gated cation channel 5-HTR(3) and the heptahelical 5-HTR(2A) did not significantly change during maturation. Isotype-selective receptor agonists allowed us to show that 5-HT stimulated 5-HTR(3)-dependent Ca(2+) influx in immature and mature DC. Moreover, we revealed that 5-HTR(1) and 5-HTR(2) receptor stimulation induced intracellular Ca(2+) mobilization via G(i/o) proteins in immature, but not mature, DC. Activation of 5-HTR(4) and 5-HTR(7) induced cAMP elevation in mature DC. Functional studies indicated that activation of 5-HTR(4) and 5-HTR(7) enhanced the release of the cytokines IL-1beta and IL-8, while reducing the secretion of IL-12 and TNF-alpha in mature DC. In summary, our study shows that 5-HT stimulated, in a maturation-dependent manner, different signaling pathways in DC. These data point to a role for 5-HT in regulating the immune response at peripheral sites.

Lysophosphatidic acid induces chemotaxis, oxygen radical production, CD11b up-regulation, Ca2+ mobilization, and actin reorganization in human eosinophils via pertussis toxin-sensitive G proteins.

Lysophosphatidic acid (LPA) is a bioactive lipid mediator, which is generated by secretory type II phospholipase A(2) and is thought to play a major role in the pathogenesis of atopic diseases. In this study, the biological activity of LPA on human eosinophils was characterized. We showed by reverse transcription and PCR that human eosinophils express the mRNA of the LPA receptors endothelial differentiation gene (EDG)-2 and EDG-7. Experiments revealed that LPA has chemotactic activity toward eosinophils, stimulates the production of reactive oxygen metabolites, and induces up-regulation of the integrin CD11b. Signal pathway measurements indicated Ca(2+)-mobilization from intracellular stores and transient actin polymerization upon stimulation with LPA. Cell responses elicited by LPA were inhibited by pertussis toxin indicating that in eosinophils the LPA receptor(s), presumably EDG-2 and/or EDG-7, are coupled to G(i/o) proteins. Moreover, LPA-induced activation of eosinophils could be completely blocked by the EDG-2/EDG-7 antagonist diacylglycerol pyrophosphate. In addition, at optimal doses the changes induced by LPA were comparable to those obtained by the other well-characterized chemotaxins. These results indicate that LPA is a strong chemotaxin and activator of eosinophils. These findings point to a novel role of LPA in the pathogenesis of diseases with eosinophilic inflammation such as atopic diseases as chemotaxin as well as activator of proinflammatory effector functions.

Inosine stimulates chemotaxis, Ca2+-transients and actin polymerization in immature human dendritic cells via a pertussis toxin-sensitive mechanism independent of adenosine receptors.

Inosine is an endogenous purine nucleoside, which is formed by adenosine deaminidase during adenosine breakdown and is released into the extracellular space from the sympathetic nervous system or injured cells. Here, we studied the biological activity of inosine on human dendritic cells (DC), which are specialized antigen presenting cells characterized by their ability to migrate from the blood to peripheral tissues, and then to secondary lymphoid organs where they initiate adaptive immune responses. In immature DC, inosine concentration-dependently stimulated Ca(2+)-transients, actin polymerization, and chemotaxis. Experiments with adenosine receptor antagonists and pertussis toxin (PTX) as well as desensitization studies suggested that the activity of inosine was mediated by a G protein-coupled receptor pathway independent of adenosine receptors. DC, induced to mature by lipopolysaccharide, lost their ability to respond towards inosine with these activities. Moreover, inosine did neither influence membrane expression of CD54, CD80, CD83, CD86, HLA-DR, and MHC class I molecules nor modulated secretion of interleukin (IL)-12, IL-10, and tumor necrosis factor alpha in immature and lipopolysaccharide-matured DC. In aggregate, our study indicates that inosine may be involved in the trafficking control system of immature DC, and mediates its chemotactic activity by a PTX-sensitive mechanism independent of adenosine receptors.

Distinct effects of sphingosine-1-phosphate, lysophosphatidic acid and histamine in human and mouse dendritic cells.

Dendritic cells (DC) are specialized antigen presenting cells characterized by their ability to migrate into target sites and secondary lymphoid organs in order to process antigens and activate naive T cells. Previously, we have shown that several secretion products from platelets and mast cells such as histamine, sphingosine-1-phosphate (S1P), and lysophosphatidic acid (LPA) have chemotactic activity towards immature human DC. Furthermore, they limit the capacity of mature human DC to initiate and amplify T helper cell type 1 (Th1) immune responses by inhibition of interleukin (IL)-12 and upregulation of IL-10 secretion. In this study we focused on the effect of these agents on murine DC. In murine DC no influence on IL-10 and IL-12 release by these agents was observed. Moreover, histamine and LPA failed to stimulate chemotaxis and actin reorganization in mouse DC. Instead, S1P had chemotactic activity and induced actin polymerization in immature as well as mature mouse DC. Therefore, our in vitro data implicate that in contrast to humans the function and immunological capacity of murine DC are not so tightly controlled by mast cell and platelet-derived secretion products such as histamine, S1P and LPA. These findings suggest that mouse models might underestimate the complex regulative network between mast cells, platelets and DC.