Shotgun cholanomics of ileal fluid.

In this study we have developed a rapid method for the shotgun analysis of bile acids in intestinal fluid. The method is semi-quantitative, and requires little sample preparation. Bile salts might contribute to the pathogenesis of Crohn's disease. In a pilot study we demonstrate the method by analysing the bile acid content of ileal fluid from seven Crohn's disease patients and three healthy controls. The dominant bile acids observed were di and/or trihydroxycholanoates, di- and/or trihydroxycholanoylglycines, di- and/or tri-hydroxycholanoyltaurines, monosulphated dihydroxycholanoates and monosulphated dihydroxycholanoylglycine. The method can be similarly applied to samples derived from other parts of the intestine.

Lipidomic profiling in Crohn's disease: abnormalities in phosphatidylinositols, with preservation of ceramide, phosphatidylcholine and phosphatidylserine composition.

Crohn's disease is a chronic inflammatory condition largely affecting the terminal ileum and large bowel. A contributing cause is the failure of an adequate acute inflammatory response as a result of impaired secretion of pro-inflammatory cytokines by macrophages. This defective secretion arises from aberrant vesicle trafficking, misdirecting the cytokines to lysosomal degradation. Aberrant intestinal permeability is also well-established in Crohn's disease. Both the disordered vesicle trafficking and increased bowel permeability could result from abnormal lipid composition. We thus measured the sphingo- and phospholipid composition of macrophages, using mass spectrometry and stable isotope labelling approaches. Stimulation of macrophages with heat-killed Escherichia coli resulted in three main changes; a significant reduction in the amount of individual ceramide species, an altered composition of phosphatidylcholine, and an increased rate of phosphatidylcholine synthesis in macrophages. These changes were observed in macrophages from both healthy control individuals and patients with Crohn's disease. The only difference detected between control and Crohn's disease macrophages was a reduced proportion of newly-synthesised phosphatidylinositol 16:0/18:1 over a defined time period. Shotgun lipidomics analysis of macroscopically non-inflamed ileal biopsies showed a significant decrease in this same lipid species with overall preservation of sphingolipid, phospholipid and cholesterol composition.

Defective tumor necrosis factor release from Crohn's disease macrophages in response to Toll-like receptor activation: relationship to phenotype and genome-wide association susceptibility loci.

BACKGROUND: Recent work provides evidence of a failure of acute inflammation in Crohn's disease (CD), and suggests that the primary defect operates at the level of the macrophage and cytokine release. Here we extend the characterization of the innate immune defect in CD by investigating the macrophage response to Toll-like receptor (TLR) agonists and assess potential links between genome-wide association study (GWAS) susceptibility loci, disease phenotype, and therapeutic regimens on tumor necrosis factor α (TNF) release. METHODS: Peripheral blood-derived macrophages were cultured from control subjects and patients with CD, stimulated with TLR ligands, and the release of TNF measured. Genomic DNA was purified from blood and genotyped for 34 single nucleotide polymorphisms (SNPs) identified as being associated with CD by GWAS. RESULTS: All stimuli resulted in a reduction (32%-48%) in TNF release from macrophages derived from CD patients (n = 28-101) compared to those from healthy control (HC) subjects. All phenotypes demonstrated impaired TNF release, with the greatest defect in patients with colonic disease. There was no detectable relationship between the level of TNF released and the presence of GWAS susceptibility loci in CD patients. Reduced TNF levels were not influenced by age, gender, or use of aminosalicylate (5-ASA) medication. CONCLUSIONS: This study supports the hypothesis of defective proinflammatory cytokine secretion and an innate immunodeficiency in CD. Abnormal TNF secretion is evident downstream of multiple TLRs, affects all disease phenotypes, and is unrelated to 34 polymorphisms associated with CD by GWAS.

G6PC3 mutations are associated with a major defect of glycosylation: a novel mechanism for neutrophil dysfunction.

Glucose-6-phosphatase, an enzyme localized in the endoplasmic reticulum (ER), catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate. In humans, there are three differentially expressed glucose-6-phosphatase catabolic genes (G6PC1-3). Recently, it has been shown that mutations in the G6PC3 gene result in a syndrome associating congenital neutropenia and various organ malformations. The enzymatic function of G6PC3 is dependent on G6P transport into the ER, mediated by G6P translocase (G6PT). Mutations in the gene encoding G6PT result in glycogen storage disease type-1b (GSD-1b). Interestingly, GSD-1b patients exhibit a similar neutrophil dysfunction to that observed in G6PC3-deficient patients. To better understand the causes of neutrophil dysfunction in both diseases, we have studied the neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase of patients with G6PC3 and G6PT syndromes. Unexpectedly, sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments indicated hypo-glycosylation of gp91(phox), the electron-transporting component of the NADPH oxidase, in all of these patients. Rigorous mass spectrometric glycomic profiling showed that most of the complex-type antennae which characterize the neutrophil N-glycome of healthy individuals were severely truncated in the patients' neutrophils. A comparable truncation of the core 2 antenna of the O-glycans was also observed. This aberrant neutrophil glycosylation is predicted to have profound effects on the neutrophil function and merit designation of both syndromes as a new class of congenital disorders of glycosylation.

Survival following Treatment of Aortoesophageal Fistula with Dual Esophageal and Aortic Intervention.

Aortoesophageal fistulas are a rare but commonly fatal complication of esophageal cancer. Reports of successfully managed cases are few, with high mortality and morbidity usually resulting from failure to control the initial massive haemodynamic insult. We report the case of a 47-year-old Caucasian man with recently diagnosed advanced esophageal cancer who suffered an episode of massive haematemesis. Emergency gastroscopy revealed an arterial bleeding point in the proximal esophagus. A self-expanding metal esophageal stent was placed to achieve initial partial haemostasis. CT angiography confirmed an aortoesophageal fistula. An endoluminal stent device was thus inserted within the thoracic aorta stabilising the bleeding point. The patient subsequently made an uneventful recovery and was discharged on long-term antibiotics for palliative care. He survived for 2 months at home before dying of disseminated malignancy. The successful use of esophageal stenting as a means of achieving haemostasis, allowing time for endovascular intervention, is as yet a relatively unexplored area of management of this rare condition.

The neutrophil respiratory burst and bacterial digestion in Crohn's disease.

BACKGROUND: Neutrophils are a key part of the innate immune defence against microbes, using the respiratory burst (RB) to optimise killing and digestion. Previous studies of the neutrophil RB in Crohn's disease (CD) have yielded conflicting results. METHODS: Superoxide production in response to phorbol-myristyl acetate (PMA) was measured in neutrophils from 100 patients with CD compared to 50 healthy controls (HCs) and 50 patients with ulcerative colitis (UC). A further 22 CD and 10 HCs were studied using f-Met-Leu-Phe (fMLP), and digestion of E. coli by neutrophils was also evaluated. RESULTS: The mean ± SEM PMA-stimulated RB (nmol O(2)/10(6) cells/min) was 10.86 ± 0.26 in HCs, 9.76 ± 0.23 in CD (P=0.02) and 10.04 ± 0.28 in UC (P=0.09 vs HC and 0.47 vs CD). No significant effect of age, gender or medication was observed. The RB in three patients with presumed CD was found to be in the range expected in patients with inherited neutrophil disorders. Stimulation with fMLP was calcium dependent and attenuated in patients on 5-ASA. Digestion of E. coli by neutrophils was not different in HC vs CD (21.6 vs 20.53%, P=0.60). CONCLUSION: The significant reduction in neutrophil RB in CD does not appear to result in defective bacterial digestion and is therefore unlikely play a major role in pathogenesis. Three patients in this cohort of patients with presumed idiopathic CD were found to have a profound defect of the neutrophil RB. A high index of suspicion for such patients is prudent, as their prognosis can be improved by altering or augmenting the conventional treatment regimens employed for CD.

Inflammatory bowel diseases in patients with adaptive and complement immunodeficiency disorders.

Crohn's disease and ulcerative colitis are idiopathic chronic inflammatory diseases that primarily affect the gastrointestinal tract. The underlying causes remain poorly understood, but there is a growing body of evidence advocating a likely primary pathogenic role for immunodeficiency in the development of Crohn's lesions. Concordantly, a number of congenital immunodeficiencies disrupting the cellular innate immune system strongly predispose to noninfectious, Crohn's-like inflammatory bowel disease. There are case reports and series suggesting that the same may be true for some of the congenital adaptive and complement immunodeficiencies. This review considers and critiques these potential associations.

Crohn's disease as an immunodeficiency.

The pathogenesis of Crohn's disease (CD) has widely been regarded as the consequence of a dysregulated T-cell-mediated response to intestinal microbes, and the majority of the worldwide research effort has focused on characterizing and treating the chronic inflammatory phase of the disease. However, recent molecular biological and clinical investigations indicate that CD is actually a primary immunodeficiency. At first counter-intuitive, the apparent paradox of a pathogenic innate immune defect can be linked mechanistically to the granulomatous chronic inflammation characteristic of the disease. Genome-wide association studies have corroborated the involvement of innate immune dysfunction in the pathogenesis of CD, but less than 20% of the heritable risk is accounted for. By contrast, in vitro and in vivo stimulation of the immune system has highlighted novel areas of interest that may lead to the development of targeted therapeutic and diagnostic tools.

Delayed resolution of acute inflammation in ulcerative colitis is associated with elevated cytokine release downstream of TLR4.

BACKGROUND: Ulcerative colitis (UC) is widely viewed as a leukocyte-mediated disorder. Although strong evidence implicates an exuberant response to microbial components in its pathogenesis, no intrinsic immune defect has been identified and the underlying pathogenic mechanisms remain obscure. METHODOLOGY/PRINCIPAL FINDINGS: The acute immune response to bacterial injection was determined in UC patients with quiescent disease and directly compared to healthy control subjects. Monocyte-derived macrophages were used to investigate bacterial recognition mechanisms in vitro. An exuberant and protracted acute inflammatory response to bacteria was evident in patients with UC, which coincides with increased systemic levels of CXCL10. Macrophages stimulated with bacteria and Toll-like receptor (TLR) ligands revealed a specific defect in the TLR4 response in UC. The defect resulted in the over-expression of a number of pro-inflammatory molecules under transcriptional control of the adaptor TIR-domain containing adaptor inducing interferon-beta (TRIF). CONCLUSION: These findings highlight a dysregulated innate immune response with over-expression of molecules associated with leukocyte recruitment and activation that may eventuate in the hallmark chronic immune-mediated inflammation of UC.

Crohn's disease: an immune deficiency state.

Crohn's disease is a chronic inflammatory disorder primarily affecting the gastrointestinal tract. Its clinical manifestations arise from a substantial infiltration of the intestinal mucosa by activated leukocytes and the downstream consequences of chronic inflammation. The underlying cause driving this immunological reaction remains poorly understood. A number of hypotheses have been proposed, most of which postulate a primary over-activation of the immune response, based on the pathological appearances of active Crohn's lesions. Interestingly, none of these theories have been mechanistically proven. It is possible that the immunological events responsible for disease initiation are quite different from those contributing to its persistence and propagation. A substantial body of data has emerged in recent years to suggest that the primary defect in Crohn's disease is actually one of relative immunodeficiency. This review considers the evidence for such a phenomenon in contrast to alternative prevailing hypotheses and attempts to address some of the potential paradoxes that it generates.

Henoch-Schönlein purpura complicating adalimumab therapy for Crohn's disease.

Anti-tumour necrosis factor-α (TNF) therapy has revolutionised the management of chronic inflammatory conditions. With ever increasing numbers of patients being treated with these agents, uncommon adverse reactions will inevitably occur more frequently. Cutaneous manifestations are associated with many of these chronic conditions and can complicate anti-TNF therapy in about 20% of cases. Vasculitic complications are rarely associated with anti-TNF therapy. Henoch-Schönlein purpura (HSP), a small vessel vasculitis, has been described following infliximab and etanercept therapy but never with adalimumab, a fully humanized TNF antibody. The risk of such immune-mediated reactions is theoretically less with adalimumab compared to infliximab but can still occur. Here we report the first case in the literature of HSP that can be attributed to the use of adalimumab in a 19-year-old male with recalcitrant Crohn's disease.

Diminished macrophage apoptosis and reactive oxygen species generation after phorbol ester stimulation in Crohn's disease.

BACKGROUND: Crohn's Disease (CD) is a chronic relapsing disorder characterized by granulomatous inflammation of the gastrointestinal tract. Although its pathogenesis is complex, we have recently shown that CD patients have a systemic defect in macrophage function, which results in the defective clearance of bacteria from inflammatory sites. METHODOLOGY/PRINCIPAL FINDINGS: Here we have identified a number of additional macrophage defects in CD following diacylglycerol (DAG) homolog phorbol-12-myristate-13-acetate (PMA) activation. We provide evidence for decreased DNA fragmentation, reduced mitochondrial membrane depolarization, impaired reactive oxygen species production, diminished cytochrome c release and increased IL-6 production compared to healthy subjects after PMA exposure. The observed macrophage defects in CD were stimulus-specific, as normal responses were observed following p53 activation and endoplasmic reticulum stress. CONCLUSION: These findings add to a growing body of evidence highlighting disordered macrophage function in CD and, given their pivotal role in orchestrating inflammatory responses, defective apoptosis could potentially contribute to the pathogenesis of CD.

Impaired macrophage function following bacterial stimulation in chronic granulomatous disease.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is critical for phagocyte anti-microbial activity and plays a major role in innate immunity. Defects in genes coding for components of the NADPH oxidase enzyme system are responsible for chronic granulomatous disease (CGD), a rare primary neutrophil immunodeficiency associated with recurrent, life-threatening bacterial and fungal infections. Microbial killing and digestion within the neutrophil phagosomal compartment are defective in these patients. NADPH oxidase activity is also crucial for optimal macrophage and dendritic cell function and has recently been implicated in both cross-presentation and T-cell priming. We present evidence of impaired macrophage function in CGD, with attenuated pro-inflammatory cytokine and increased interleukin-10 secretion following bacterial stimulation. These results highlight additional abnormalities in macrophage function associated with CGD and the importance of NADPH oxidase activity in immunity.

Disordered macrophage cytokine secretion underlies impaired acute inflammation and bacterial clearance in Crohn's disease.

The cause of Crohn's disease (CD) remains poorly understood. Counterintuitively, these patients possess an impaired acute inflammatory response, which could result in delayed clearance of bacteria penetrating the lining of the bowel and predispose to granuloma formation and chronicity. We tested this hypothesis in human subjects by monitoring responses to killed Escherichia coli injected subcutaneously into the forearm. Accumulation of (111)In-labeled neutrophils at these sites and clearance of (32)P-labeled bacteria from them were markedly impaired in CD. Locally increased blood flow and bacterial clearance were dependent on the numbers of bacteria injected. Secretion of proinflammatory cytokines by CD macrophages was grossly impaired in response to E. coli or specific Toll-like receptor agonists. Despite normal levels and stability of cytokine messenger RNA, intracellular levels of tumor necrosis factor (TNF) were abnormally low in CD macrophages. Coupled with reduced secretion, these findings indicate accelerated intracellular breakdown. Differential transcription profiles identified disease-specific genes, notably including those encoding proteins involved in vesicle trafficking. Intracellular destruction of TNF was decreased by inhibitors of lysosomal function. Together, our findings suggest that in CD macrophages, an abnormal proportion of cytokines are routed to lysosomes and degraded rather than being released through the normal secretory pathway.

Inflammatory bowel disease in CGD reproduces the clinicopathological features of Crohn's disease.

OBJECTIVES: Patients with chronic granulomatous disease (CGD), a rare congenital disorder characterized by defective neutrophil function, frequently develop an inflammatory bowel disease similar to Crohn's disease. The clinical presentations and concordance between the features of the bowel disease in these two conditions have never been formally evaluated. METHODS: Retrospective case note analysis of all adult patients with CGD treated at a tertiary care hospital. RESULTS: A total of 25 eligible patients were identified. Of these, 14 (56%) had experienced gastrointestinal symptoms in the preceding 3 years; and 11 (44%) had documented gastrointestinal inflammation not secondary to infection, manifesting throughout the alimentary canal including the upper gastrointestinal tract (45%), small intestine (27%), colon (73%), and rectum (73%). All had discontinuous inflammation and perianal involvement, and approximately half (55%) demonstrated epithelioid granulomata on histology. All patients fulfilled the Lennard-Jones criteria for the diagnosis of Crohn's disease. Therapeutic responses were observed in five patients to 5-aminosalicylates, and in individual patients to thalidomide, interferon-gamma, azathioprine, infliximab, and intestinal resection. CONCLUSIONS: There are striking clinical and pathological resemblances between the bowel diseases observed in CGD and Crohn's disease, supporting the possibility of mechanistic similarities in their pathogenesis. Patients with CGD appear particularly prone to developing perianal disease.

Defective acute inflammation in Crohn's disease: a clinical investigation.

BACKGROUND: The cause of Crohn's disease has not been mechanistically proven. We tested the hypothesis that the disease is a form of immunodeficiency caused by impaired innate immunity. METHODS: We investigated inflammatory responses in patients and controls by quantifying neutrophil recruitment and cytokine production after acute trauma, interleukin 8 secretion by cultured monocyte-derived macrophages after exposure to inflammatory mediators, and local inflammatory and vascular changes in response to subcutaneous injection of heat-killed Escherichia coli. FINDINGS: In patients with Crohn's disease, trauma to rectum, ileum, or skin led to abnormally low neutrophil accumulation (differences from healthy individuals of 79%, n=8, p=0.0003; 57%, n=3, p=0.05; 50%, n=13, p<0.0001, respectively) and lower production of proinflammatory interleukin 8 (63%, n=7, p=0.003; 63%, n=3, p=0.05; 45%, n=8, p<0.0001) and interleukin 1beta (50%, n=8, p=0.0005). Interleukin 8 secretion by cultured macrophages was reduced after exposure to acute wound fluid (38%, n=50, p<0.0001), C5a (48%, n=41, p=0.0005), or tumour necrosis factor alpha (52%, n=27, p<0.0001). Local inflammatory reaction to inoculation with E coli was attenuated, as quantified by changes in bloodflow (ileal disease 50%, n=6, p=0.01; colonic disease 77%, n=6, p=0.0003). This response was mediated by nitric oxide in controls, was increased by sildenafil in patients, and was not related to CARD15 genotype. INTERPRETATION: In Crohn's disease, a constitutionally weak immune response predisposes to accumulation of intestinal contents that breach the mucosal barrier of the bowel wall, resulting in granuloma formation and chronic inflammation. Polymorphisms in CARD15 do not underlie this phenotype, but incapacitate the NOD2 pathway that can compensate for impairment of innate inflammation. Current treatment of secondary chronic inflammation might exaggerate the underlying lesion and promote chronic disease.

Giant cell (temporal) arteritis: an overview and update.

Giant cell arteritis is a systemic disease that continues to be a sight-threatening medical emergency requiring prompt recognition and treatment in order to avoid devastating ophthalmic consequences. Although there have been advances in the genetic and immunologic understanding of the underlying pathogenesis of the disease, the exact etiology of the condition, to date, remains unclear. Visual manifestations of giant cell arteritis are the common mode of presentation, making the ophthalmologist critically responsible for early diagnosis and treatment. Although temporal artery biopsy remains the only confirmatory procedure, newer laboratory investigations and blood flow studies with fundus fluorescein angiography have aided in the diagnosis of temporal giant cell arteritis. Maintenance of a high index of clinical suspicion is essential to institute prompt adequate treatment, especially in atypical cases. Corticosteroids remain the mainstay of treatment of giant cell arteritis. Recently, immunosuppressive agents as secondary steroid-sparing drugs have been used, particularly in some steroid-resistant cases. A wider recognition of the disease will minimize the prevalence of irreversible visual loss among patients with giant cell arteritis.

Obstetric and gynecologic complications in women with Marfan syndrome.

OBJECTIVE: To analyze the maternal and fetal outcomes of pregnancy and gynecologic problems in women with Marfan syndrome. STUDY DESIGN: The outcomes of 14 pregnancies in 4 women with Marfan syndrome were prospectively observed between January 1988 and December 2000. The cardiovascular and obstetric complications were analyzed. During pregnancy all the patients were carefully monitored with serial echocardiography and close attention to symptoms. RESULTS: Of the 14 pregnancies, 5 (35.7%) ended in abortion, and 3 of them occurred in the early second trimester due to cervical incompetence. Premature onset of labor occurred in 2 pregnancies at 31 and 34 weeks. Postpartum hemorrhage complicated 3 deliveries, and inversion of the uterus occurred in 1 patient. Significant cardiovascular complications occurred in 2 patients, who required surgical correction of the aortic aneurysm and replacement of the aortic valve. In one patient the operation was performed within hours of vaginal delivery, and the other patient underwent surgery 8 weeks postpartum. No maternal death occurred in the study. One infant in the series was diagnosed as having Marfan syndrome. A premature infant delivered at 31 weeks died on the second day of life. CONCLUSION: Women with Marfan syndrome are at high risk of aortic dissection in pregnancy even in the absence of preconceptional aortic root dilatation. Obstetric complications in patients with this condition have been underreported in the past. Women with aortic root dilatation of < 40 mm usually tolerate pregnancy well, with good maternal and fetal outcomes. Women with Marfan syndrome should be counseled regarding the risk of pregnancy to both mother and fetus. Patients who have cardiac decompensation or aortic dilatation > 40 mm are advised to avoid pregnancy.