Improving quality of life in patients with rare autoimmune liver diseases by structured peer-delivered support (Q.RARE.LI): study protocol for a transnational effectiveness-implementation hybrid trial.

BACKGROUND: Psychosocial support is a crucial component of adequate rare disease care, but to date psychosocial support needs of this patient population are insufficiently met. Within Q.RARE.LI, we strive to evaluate the effectiveness of a structured, transdiagnostic, and location-independent psychosocial support intervention in routine care of patients with rare autoimmune liver diseases in five countries and prepare its implementation. METHODS: Within an effectiveness-implementation hybrid trial, we aim to a) investigate the effectiveness of the intervention in routine care in five diverse healthcare systems and b) assess implementation outcomes, examine and prepare the implementation context, and develop country-specific implementation strategies. To assess effectiveness, we will include N = 240 patients with rare autoimmune liver diseases. Within a two-armed randomized controlled trial (allocation ratio 1:1), we will compare structured and peer-delivered psychosocial support in addition to care-as-usual (CAU) with CAU alone. Outcomes will be assessed via electronic database entry prior to intervention, directly after, and at a three-month follow-up. Our primary effectiveness outcome will be mental health-related quality of life at post-assessment. Secondary outcomes include depression and anxiety severity, perceived social support, helplessness, and disease acceptance. Implementation outcomes will be assessed within a mixed-methods process evaluation. In a quantitative cross-sectional survey, we will examine perceived acceptability and feasibility in patients, peer-counselors, and healthcare providers involved in delivery of the intervention. In qualitative focus groups, we will analyze the implementation context and determine barriers and facilitators for implementation with different stakeholders (patients and/or representatives, peer-counselors, healthcare providers, health insurers). Based on these results, we will derive country-specific implementation strategies and develop a concrete implementation plan for each country. DISCUSSION: The intervention is expected to help patients adjust to their disease and improve their mental quality of life. The transdiagnostic and location-independent program has the potential to reach patients for psychosocial support who are usually hard to reach. By preparing the implementation in five countries, the project can help to make low-threshold psychosocial support available to many patients with rare diseases and improve comprehensive healthcare for an often neglected group. TRIAL REGISTRATION: ISRCTN15030282.

Mucosal protection by phosphatidylcholine.

The colonic mucus serves a first barrier towards invasion of commensal bacteria in stools to prevent inflammation. One essential component of intestinal mucus is phosphatidylcholine (PC) which represents more than 90% of the phospholipids in mucus indicative for a selective transport of PC into this compartment. It is arranged in lamellar structures as surfactant-like particles which provide a hydrophobic surface on top of the hydrated mucus gel to prevent the invasion of bacteria from intestinal lumen. In ulcerative colitis (UC), the mucus PC content is reduced by 70%, irrespective of the state of inflammation. Thus, it could represent an intrinsic primary pathogenetic condition predisposing to bacterial invasion and the precipitation of inflammation. Since PC was shown to be mainly secreted by the ileal mucosa from where it is assumed to move distally to the colon, the PC content along the colonic wall towards the rectum gradually thins, with the least PC content in the rectum. This explains the start of the clinical manifestation of UC in the rectum and the expansion from there to the upper parts of the colon. In three clinical trials, when missing mucus PC in UC was supplemented by an oral, delayed release PC preparation, the inflammation improved and even resolved after a 3-month treatment course. The data indicate the essential role of the mucus PC content for protection against inflammation in colon.

Reduced hydrophobicity of the colonic mucosal surface in ulcerative colitis as a hint at a physicochemical barrier defect.

PURPOSE: There is increasing evidence that a defect of the gastrointestinal mucosal barrier is important for the development of inflammatory bowel diseases (IBD). The hydrophobicity of the colonic mucosal surface is a measure of its resistance to luminal antigens, e.g. of bacterial origin. Therefore, the purpose of this study was to determine this parameter in patients suffering from IBD. METHODS: Nineteen patients with ulcerative colitis (UC), ten patients with Crohn's disease (CD) and 20 controls were examined. All underwent colonic surgery at the University Hospital Heidelberg. Clinical disease activity was determined. From every subject, colonic tissue specimens were obtained, and hydrophobicity of the mucosal surface was determined with a goniometer by multiple plateau contact angle measurements. Histological evaluation of disease activity was performed in directly adjacent tissue specimens. RESULTS: Hydrophobicity of the colonic mucosal surface, expressed as plateau contact angles, was significantly reduced in patients with UC (mean ± SEM, 47.8° ± 3.4°) compared to those with CD (72.0° ± 5.2°) and controls (72.5° ± 5.6°; over-all P = 0.0004; UC versus controls, P < 0.001; UC versus CD, P < 0.05; CD versus controls, P > 0.05). Between mucosal hydrophobicity and clinical disease activity, as well as mucosal hydrophobicity and histological disease activity, no significant correlation was found. CONCLUSIONS: The results suggest a defective physicochemical barrier as an essential factor in the pathogenesis of UC, but not CD. The fact that no correlation was found between mucosal hydrophobicity and disease activity may indicate that the loss of mucosal hydrophobicity in UC is not exclusively a secondary effect due to inflammation.

Lipid based therapy for ulcerative colitis-modulation of intestinal mucus membrane phospholipids as a tool to influence inflammation.

Ulcerative colitis (UC) is the result of an inappropriate colonic inflammatory response triggered by environmental and genetic factors. We have recently shown that mucus from UC patients has a decreased phosphatidylcholine (PC) content, while clinical trials revealed that therapeutic addition of PC to the colonic mucus alleviated the inflammatory activity. The mechanisms behind this are still unclear. We hypothesized that PC has at least two possible functions in the intestine: First, it establishes the surface hydrophobicity of the mucus and therefore protects the underlying tissue against intraluminal aggressors; recent experiments on surgical specimens revealed reduced surface tension and hydrophobicity in UC patients. Second, mucus phospholipids might also be integrated into the plasma membranes of enterocytes and thereby influence the signaling state of the mucosa. PC has been shown to inhibit TNF-α induced pro-inflammatory responses including: (1) assembly of plasma membrane actin; (2) activation of MAP kinases ERK and p38; and (3) activation of NF-κB and synthesis of pro-inflammatory gene products. Other phospholipids like phosphatidylethanolamine or sphingomyelin had no effect. PC also inhibited latex bead phagosome actin assembly, killing of M. tuberculosis in macrophages, and sphingosine-1-phosphate induced actin assembly in macrophages. Collectively, these results provide a molecular foundation that shows PC, firstly, as an anti-inflammatory, and secondly, as a surface hydrophobicity increasing compound with promising therapeutic potential in the treatment of inflammatory bowel disease.

Delayed release phosphatidylcholine as new therapeutic drug for ulcerative colitis--a review of three clinical trials.

IMPORTANCE OF THE FIELD: As the pathogenesis of ulcerative colitis (UC) is unknown, a causative therapy is lacking. Therefore, some UC patients suffer from disease activity despite symptomatic anti-inflammatory treatment strategies. We claim that reduction of phosphatidylcholine (PC) in colonic mucus impairs the mucosal barrier and, thus, causes attacks of the commensal bacterial flora to induce colitis. Thus, mucus PC substitution could provide a causal therapy for UC. AREAS COVERED IN THIS REVIEW: A delayed released oral PC preparation (rPC) was found to substitute for the lack of PC in rectal mucus. In non-steroid-treated active UC, 53% of rPC-treated patients reached remission compared with 10% of placebo patients (p < 0.001). In a second trial with chronic-active, steroid-dependent UC patients, steroid withdrawal with a concomitant achievement of remission (CAI ≤ 3) or clinical response (≥ 50% CAI improvement) was reached in 15 rPC-treated patients (50%) but only in 3 (10%) placebo patients (p = 0.002). WHAT THE READER WILL GAIN: The concept that missing PC in colonic mucus is the main pathogenetic factor for development of UC. PC can be substituted by rPC, which cures the disease in the majority of patients. TAKE HOME MESSAGE: rPC is, to our knowledge, the first causative therapeutic option for patients with UC.

Phosphatidylcholine (lecithin) and the mucus layer: Evidence of therapeutic efficacy in ulcerative colitis?

Colonic mucus protects against attacks from bacteria in stool. One component of mucus is phosphatidylcholine (PC) which is thought to be arranged as continuous lamellar layer in the apical mucus and to be responsible for establishing a protective hydrophobic surface. This 'intestinal surfactant' plays a key role in mucosal defense. Thus, a defective PC layer contributes to the development of inflammation. Analysis of rectoscopically acquired mucus aliquots revealed a 70% decrease in PC content in ulcerative colitis (UC) compared to Crohn's disease (CD) and healthy controls - independent of disease activity. Accordingly, we propose that lack of mucus PC is a key pathogenetic factor in UC. In clinical studies a delayed-release oral PC preparation (rPC) was found to substitute the lack of PC in rectal mucus. Indeed, in non-steroid-treated active UC, 53% of rPC patients reached remission [clinical activity index (CAI) ≤3] compared to 10% of placebo patients (p ≤ 0.001). Endoscopic and histologic findings improved concomitantly. A second trial with 60 chronic-active, steroid-dependent UC patients was conducted to test for steroid-sparing effects. Complete steroid withdrawal with a concomitant achievement of remission (CAI ≤3) or clinical response (≥50% CAI improvement) was reached in 15 PC-treated patients (50%) but only in 3 (10%) placebo patients (p = 0.002). In conclusion, intrinsic reduction of PC (lecithin) in colonic mucus may be a key pathogenetic feature of UC. Topical supplement of PC by a delayed-released oral PC preparation is effective in resolving inflammatory activity of UC and may develop to a first-choice therapy for this disease.

Phosphatidylcholine as a constituent in the colonic mucosal barrier--physiological and clinical relevance.

Phosphatidylcholine (PC) is an important constituent of the gastrointestinal tract. PC molecules are not only important in intestinal cell membranes but also receiving increasing attention as protective agents in the gastrointestinal barrier. They are largely responsible for establishing the hydrophobic surface of the colon. Decreased phospholipids in colonic mucus could be linked to the pathogenesis of ulcerative colitis, a chronic inflammatory bowel disease. Clinical studies revealed that therapeutic addition of PC to the colonic mucus of these patients alleviated the inflammatory activity. This positive role is still elusive, however, we hypothesized that luminal PC has two possible functions: first, it is essential for surface hydrophobicity, and second, it is integrated into the plasma membrane of enterocytes and it modulates the signaling state of the mucosa. The membrane structure and lipid composition of cells is a regulatory component of the inflammatory signaling pathways. In this perspective, we will shortly summarize what is known about the localization and protective properties of PC in the colonic mucosa before turning to its evident medical importance. We will discuss how PC contributes to our understanding of the pathogenesis of ulcerative colitis and how reinforcing the luminal phospholipid monolayer can be used as a therapeutic concept in humans.

Delayed release phosphatidylcholine in chronic-active ulcerative colitis: a randomized, double-blinded, dose finding study.

BACKGROUND: In 2 preceding studies, delayed release phosphatidylcholine (rPC) was found to (a) improve disease activity and (b) withdraw steroids in patients with chronic-active ulcerative colitis. GOAL: Objective of the study was to determine the most effective rPC dose with least adverse events. STUDY: A randomized, dose-controlled, double-blinded study. Four groups of 10 patients each with nonsteroid-treated, chronic-active ulcerative pancolitis with a clinical activity index (CAI) and endoscopic activity index (EAI) >or=7. Patients were treated with oral rPC at doses of 0.5, 1, 3, and 4 g daily over 12 weeks. RESULTS: The CAI changes from baseline to the end of the study were 2.5 (0.5 g), 7.0 (1 g), 5.5 (3 g), and 6.0 (4 g dose arm). Significant improvement of the CAI was registered between the lowest rPC dose of 0.5 g (control group) and all higher doses of 1.0, 3.0, and 4.0-g rPC (Por=50% CAI improvement) were 70% in all of the effective dose groups (1 to 4 g, P=0.003). This was paralleled by the EAI improvement and by the rates of mucosal healing. Median time to clinical response was 5 (IQR 2 to 8) weeks. Bloating was registered in 40% of the patients irrespective of the treatment dose. Three of the 10 patients in the 4 g dose group reported nausea. CONCLUSION: We found a saturable dose response of rPC in the treatment of chronic-active ulcerative colitis with effective doses >or=1 g per day; doses of 3 and 4 g seem to be superior in achieving remission.

Separate basolateral and apical phosphatidylcholine secretion routes in intestinally differentiated tumor cells.

AIM: To investigate whether the secretion of phosphatidylcholine (PC) in intestinal mucus occurs by apical secretion or via basolateral excretion and to determine its subsequent passage across the tight junctions to the apical mucus. METHODS: We addressed this question using the polarized intestinally differentiated tumor cell line CaCo-2 grown on filters to confluence in Transwell culture chambers. The released PC and sphingomyelin (Sph) from apical and basolateral media were analyzed by mass spectrometry. RESULTS: The secreted PC species were identical in both compartments indicating the same intracellular origin of PC. However, PC secretion into the basolateral compartment was more effective, and the PC:Sph ratio in the basolateral compartment was significantly higher than that in the apical compartment (8.18 +/- 1.84 vs 4.31 +/- 1.22, P = 0.01). Both pathways were temperature sensitive and were unaltered in the presence of cyclosporine. CONCLUSION: The data demonstrate the PC secretion capacity of CaCo-2 cells and indicate two separated apical and basolateral release mechanisms.

TNF-alpha-induced up-regulation of pro-inflammatory cytokines is reduced by phosphatidylcholine in intestinal epithelial cells.

BACKGROUND: Phosphatidylcholine (PC) is a major lipid of the gastrointestinal mucus layer. We recently showed that mucus from patients suffering from ulcerative colitis has low levels of PC. Clinical studies reveal that the therapeutic addition of PC to the colonic mucus using slow release preparations is beneficial. The positive role of PC in this disease is still unclear; however, we have recently shown that PC has an intrinsic anti-inflammatory property. It could be demonstrated that the exogenous application of PC inhibits membrane-dependent actin assembly and TNF-alpha-induced nuclear NF-kappaB activation. We investigate here in more detail the hypothesis that the exogenous application of PC has anti-inflammatory properties. METHODS: PC species with different fatty acid side chains were applied to differentiated and non-differentiated Caco-2 cells treated with TNF-alpha to induce a pro-inflammatory response. We analysed TNF-alpha-induced NF-kappaB-activation via the transient expression of a NF-kappaB-luciferase reporter system. Pro-inflammatory gene transcription was detected with the help of a quantitative real time (RT)-PCR analysis. We assessed the binding of TNF-alpha to its receptor by FACS and analysed lipid rafts by isolating detergent resistant membranes (DRMs). RESULTS: The exogenous addition of all PC species tested significantly inhibited TNF-alpha-induced pro-inflammatory signalling. The expression levels of IL-8, ICAM-1, IP-10, MCP-1, TNF-alpha and MMP-1 were significantly reduced after PC pre-treatment for at least two hours. The effect was comparable to the inhibition of NF-kB by the NF-kB inhibitor SN 50 and was not due to a reduced binding of TNF-alpha to its receptor or a decreased surface expression of TNF-alpha receptors. PC was also effective when applied to the apical side of polarised Caco-2 cultures if cells were stimulated from the basolateral side. PC treatment changed the compartmentation of the TNF-alpha-receptors 1 and 2 to DRMs. CONCLUSION: PC induces a prolonged inhibition of TNF-alpha-induced pro-inflammatory signalling. This inhibition may be caused by a shift of the TNF-alpha receptors at the surface to lipid rafts. Our results may offer a potential molecular explanation for the positive role of PC seen in clinical studies for the treatment of ulcerative colitis.

Alterations of phospholipid concentration and species composition of the intestinal mucus barrier in ulcerative colitis: a clue to pathogenesis.

BACKGROUND: Phospholipids are essential for the normal function of the intestinal mucus barrier. The objective of this study was to systematically investigate phospholipids in the intestinal mucus of humans suffering from inflammatory bowel diseases, where a barrier defect is strongly supposed to be pathogenetic. METHODS: Optimal mucus recovery was first validated in healthy mice and the method was then transferred to the endoscopic acquisition of ileal and colonic mucus from 21 patients with ulcerative colitis (UC), 10 patients with Crohn's disease (CD), and 29 healthy controls. Nano-electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to determine phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and sphingomyelin (SM) in lipid extracts of mucus specimens. RESULTS: Human and rodent mucus contained very similar phospholipid species. In the ileal and colonic mucus from patients suffering from UC, the concentration of PC was highly significantly lower (607 +/- 147 pmol/100 microg protein and 745 +/- 148 pmol/100 microg protein) compared to that of patients with CD (3223 +/- 1519 pmol/100 microg protein and 2450 +/- 431 pmol/100 microg protein) and to controls (3870 +/- 760 pmol/100 microg protein and 2790 +/- 354 pmol/100 microg protein); overall, P = 0.0002 for ileal specimens and P < 0.0001 for colonic specimens. Independent of disease activity, patients suffering from UC showed an increased saturation grade of PC fatty acid residues and a higher LPC-to-PC ratio. CONCLUSIONS: The intestinal mucus barrier of patients with UC is significantly altered concerning its phospholipid concentration and species composition. These alterations may be very important for the pathogenesis of this disease and underline new therapeutic strategies.

Transcript levels of different cytokines and chemokines correlate with clinical and endoscopic activity in ulcerative colitis.

BACKGROUND: A definition of disease activity in ulcerative colitis (UC) is difficult. The clinical activity index (CAI) is only an indirect assessment tool of bowel inflammation and the endoscopic activity index (EAI) sometimes cannot reflect the severity of disease to the full extent. Therefore, there is a need for an objective means to quantify inflammatory activity in mucosal biopsies. In our study, we wanted to examine the correlation between transcript levels of interleukin 8 (CXCL8), interferon gamma inducible protein 10 (CXCL10), myeloid-related protein 14 (calgranulin B), macrophage inflammatory protein 2 alpha (CXCL2) with CAI and EAI in UC. METHODS: Cytokine and chemokine transcripts were quantified using real-time PCR in 49 mucosal biopsies from 27 different patients with UC. Cytokine transcript levels were correlated with CAI and EAI. RESULTS: There was a statistically significant positive correlation between CXCL8 (r = 0.30; p < 0.05), CXCL10 (r = 0.40; p < 0.02), calgranulin B (r = 0.36; p < 0.03), CXCL2 (r = 0.31; p < 0.05) and CAI. Concerning EAI significant positive correlations for CXCL8 (r = 0.37; p < 0.02), CXCL10 (r = 0.33; p < 0.04), calgranulin B (r = 0.31; p < 0.05) and CXCL2 (r = 0.44; p < 0.05) were found. Low clinical and endoscopic activity was accompanied by low cytokine levels whereas high CAI and EAI were associated with high cytokine levels. CONCLUSION: From our data, we conclude that real-time PCR quantification of CXCL8, CXCL10, calgranulin B and CXCL2 in colonic biopsies is a simple and objective method for grading inflammation of intestinal mucosa in UC. CXCL8, CXCL10, calgranulin B and CXCL2 might be used as biomarkers and thus as an objective tool especially in clinical trials to evaluate anti-inflammatory and immunomodulatory regimens.

A mutation in the canalicular phospholipid transporter gene, ABCB4, is associated with cholestasis, ductopenia, and cirrhosis in adults.

UNLABELLED: Cholestatic liver disease (CLD) is a major cause of progressive liver damage and liver failure. Several forms of biliary cirrhosis are caused by mutations in specific genes. We sought to identify a genetic defect in a family with CLD impossible to assign to a distinct pathogenic entity. Clinical and histopathological characterization of the family members, microarray-based single-nucleotide polymorphism genotyping, and analysis of candidate genes were performed. Among six of 11 siblings severely affected by idiopathic CLD in a family from a population isolate in Transylvania, three died of cirrhosis (aged 5, 7, and 43 years) and three had adult-onset disease with small duct cholangiopathy, including ductopenia. Others were mildly affected and experienced intrahepatic cholestasis of pregnancy, miscarriages, or stillbirth. Pedigree studies revealed distant parental consanguinity. Genome-wide linkage analysis and autozygosity mapping yielded a single maximal lod-score of 3.88 on chromosome 7q21.1-7q22, excluding other genomic loci. Sequencing of ABCB4 at this locus revealed a novel missense mutation c.2362C>T (p.Arg788Trp) which cosegregated with severity of disease. Bile from a mutation homozygote showed a reduced phosphatidylcholine/bile acid ratio, consistent with reduced ABCB4 phosphatidylcholine transport activity. CONCLUSION: We show that a missense mutation in ABCB4 is a cause for ductopenic CLD in adulthood. Allelic status correlated with severity of liver disease ranging from intrahepatic cholestasis of pregnancy through fibrosis to cirrhosis and death in childhood and adulthood. Mutational analysis of ABCB4 should be generally considered in all patients with cholestatic liver disease of unknown etiology regardless of age and onset of disease.

Anti-inflammatory effects of phosphatidylcholine.

We recently showed that mucus from patients with ulcerative colitis, a chronic inflammatory disorder of the colon, is characterized by a low level of phosphatidylcholine (PC) while clinical studies reveal that therapeutic addition of PC using slow release preparations is beneficial. The positive role of PC in this disease is still elusive. Here we tested the hypothesis that exogenous application of PC has anti-inflammatory properties using three model systems. First, human Caco-2 cells were treated with tumor necrosis factor-alpha (TNF-alpha) to induce a pro-inflammatory response via activation of NF-kappaB. Second, latex bead phagosomes were analyzed for their ability to assemble actin in vitro, a process linked to pro-inflammatory signaling and correlating with the growth versus killing of mycobacteria in macrophages. The third system used was the rapid assembly of plasma membrane actin in macrophages in response to sphingosine 1-phosphate. TNF-alpha induced a pro-inflammatory response in Caco-2 cells, including 1) assembly of plasma membrane actin; 2) activation of both MAPKs ERK and p38; 3) transport of NF-kappaB subunits to the nucleus; and 4) subsequent up-regulation of the synthesis of pro-inflammatory gene products. Exogenous addition of most PCs tested significantly inhibited these processes. Other phospholipids like sphingomyelin or phosphatidylethanolamine showed no effects in these assays. PC also inhibited latex bead phagosome actin assembly, the killing of Mycobacterium tuberculosis in macrophages, and the sphingosine 1-phosphate-induced actin assembly in macrophages. TNF-alpha induces the activation of signaling molecules and the reorganization of the actin cytoskeleton in human intestinal cells. Exogenous application of PC blocks pro-inflammatory signaling in Caco-2 cells, in phagosomes in vitro and facilitates intracellular survival of mycobacteria. We provide further evidence that actin assembly by membranes is part of the pro-inflammatory response. Collectively, these results provide a molecular foundation for the clinical studies showing a beneficial effect of PC therapy in ulcerative colitis.

Expression profiles of hydrophobic surfactant proteins in children with diffuse chronic lung disease.

BACKGROUND: Abnormalities of the intracellular metabolism of the hydrophobic surfactant proteins SP-B and SP-C and their precursors may be causally linked to chronic childhood diffuse lung diseases. The profile of these proteins in the alveolar space is unknown in such subjects. METHODS: We analyzed bronchoalveolar lavage fluid by Western blotting for SP-B, SP-C and their proforms in children with pulmonary alveolar proteinosis (PAP, n = 15), children with no SP-B (n = 6), children with chronic respiratory distress of unknown cause (cRD, n = 7), in comparison to children without lung disease (n = 15) or chronic obstructive bronchitis (n = 19). RESULTS: Pro-SP-B of 25-26 kD was commonly abundant in all groups of subjects, suggesting that their presence is not of diagnostic value for processing defects. In contrast, pro-SP-B peptides cleaved off during intracellular processing of SP-B and smaller than 19-21 kD, were exclusively found in PAP and cRD. In 4 of 6 children with no SP-B, mutations of SFTPB or SPTPC genes were found. Pro-SP-C forms were identified at very low frequency. Their presence was clearly, but not exclusively associated with mutations of the SFTPB and SPTPC genes, impeding their usage as candidates for diagnostic screening. CONCLUSION: Immuno-analysis of the hydrophobic surfactant proteins and their precursor forms in bronchoalveolar lavage is minimally invasive and can give valuable clues for the involvement of processing abnormalities in pediatric pulmonary disorders.