A comprehensive understanding of carbon-carbon bond formation by alkyne migratory insertion into manganacycles.

Migratory insertion (MI) is one of the most important processes underpinning the transition metal-catalysed formation of C-C and C-X bonds. In this work, a comprehensive model of MI is presented, based on the direct observation of the states involved in the coupling of alkynes with cyclometallated ligands, augmented with insight from computational chemistry. Time-resolved spectroscopy demonstrates that photolysis of complexes [Mn(C^N)(CO)4] (C^N = cyclometalated ligand) results in ultra-fast dissociation of a CO ligand. Performing the experiment in a toluene solution of an alkyne results in the initial formation of a solvent complex fac-[Mn(C^N)(toluene)(CO)3]. Solvent substitution gives an η2-alkyne complex fac-[Mn(C^N)(η2-R1C2R2)(CO)3] which undergoes MI of the unsaturated ligand into the Mn-C bond. These data allowed for the dependence of second order rate constants for solvent substitution and first order rate constants for C-C bond formation to be determined. A systematic investigation into the influence of the alkyne and C^N ligand on this process is reported. The experimental data enabled the development of a computational model for the MI reaction which demonstrated that a synergic interaction between the metal and the nascent C-C bond controls both the rate and regiochemical outcome of the reaction. The time-resolved spectroscopic method enabled the observation of a multi-step reaction occurring over 8 orders of magnitude in time, including the formation of solvent complexes, ligand substitution and two sequential C-C bond formation steps.

Sulphate-reducing bacteria from ulcerative colitis patients induce apoptosis of gastrointestinal epithelial cells.

The human microbiome consists of a multitude of bacterial genera and species which continuously interact with one another and their host establishing a metabolic equilibrium. The dysbiosis can lead to the development of pathology, such as inflammatory bowel diseases. Sulfide-producing prokaryotes, including sulphate-reducing bacteria (SRB) constituting different genera, including the Desulfovibrio, are commonly detected within the human microbiome recovered from fecal material or colonic biopsy samples. It has been proposed that SRB likely contribute to colonic pathology, for example ulcerative colitis (UC). The interaction of SRB with the human colon and intestinal epithelial cell lines has been investigated using Desulfovibrio indonesiensis as a model mono-culture and in a co-culture with E. coli isolate, and with SRB consortia from human biopsy samples. We find that D. indonesiensis, whether as a mono- or co-culture, was internalized and induced apoptosis in colon epithelial cells. This effect was enhanced in the presence of E. coli. The SRB combination obtained through enrichment of biopsies from UC patients induced apoptosis of a human intestinal epithelial cell line. This response was not observed in SRB enrichments from healthy (non-UC) controls. Importantly, apoptosis was detected in epithelial cells from UC patients and was not seen in epithelial cells of healthy donors. Furthermore, the antibody raised against exopolysaccharides (EPS) of D. indonesiensis cross reacted with the SRB population from UC patients but not with the SRB combination from non-UC controls. This study reinforces a correlation between the presence of sulphate-reducing bacteria and an inflammatory response in UC sufferers.

Summary and recommendations from the Australasian guidelines for the management of pancreatic exocrine insufficiency.

AIM: Because of increasing awareness of variations in the use of pancreatic exocrine replacement therapy, the Australasian Pancreatic Club decided it was timely to re-review the literature and create new Australasian guidelines for the management of pancreatic exocrine insufficiency (PEI). METHODS: A working party of expert clinicians was convened and initially determined that by dividing the types of presentation into three categories for the likelihood of PEI (definite, possible and unlikely) they were able to consider the difficulties of diagnosing PEI and relate these to the value of treatment for each diagnostic category. RESULTS AND CONCLUSIONS: Recent studies confirm that patients with chronic pancreatitis receive similar benefit from pancreatic exocrine replacement therapy (PERT) to that established in children with cystic fibrosis. Severe acute pancreatitis is frequently followed by PEI and PERT should be considered for these patients because of their nutritional requirements. Evidence is also becoming stronger for the benefits of PERT in patients with unresectable pancreatic cancer. However there is as yet no clear guide to help identify those patients in the 'unlikely' PEI group who would benefit from PERT. For example, patients with coeliac disease, diabetes mellitus, irritable bowel syndrome and weight loss in the elderly may occasionally be given a trial of PERT, but determining its effectiveness will be difficult. The starting dose of PERT should be from 25,000-40,000 IU lipase taken with food. This may need to be titrated up and there may be a need for proton pump inhibitors in some patients to improve efficacy.

Management of pancreatic exocrine insufficiency: Australasian Pancreatic Club recommendations.

Pancreatic exocrine insufficiency (PEI) occurs when the amounts of enzymes secreted into the duodenum in response to a meal are insufficient to maintain normal digestive processes. The main clinical consequence of PEI is fat maldigestion and malabsorption, resulting in steatorrhoea. Pancreatic exocrine function is commonly assessed by conducting a 3-day faecal fat test and by measuring levels of faecal elastase-1 and serum trypsinogen. Pancreatic enzyme replacement therapy is the mainstay of treatment for PEI. In adults, the initial recommended dose of pancreatic enzymes is 25,000 units of lipase per meal, titrating up to a maximum of 80,000 units of lipase per meal. In infants and children, the initial recommended dose of pancreatic enzymes is 500 units of lipase per gram of dietary fat; the maximum daily dose should not exceed 10,000 units of lipase per kilogram of bodyweight. Oral pancreatic enzymes should be taken with meals to ensure adequate mixing with the chyme. Adjunct therapy with acid-suppressing agents may be useful in patients who continue to experience symptoms of PEI despite high-dose enzyme therapy. A dietitian experienced in treating PEI should be involved in patient management. Dietary fat restriction is not recommended for patients with PEI. Patients with PEI should be encouraged to consume small, frequent meals and to abstain from alcohol. Medium-chain triglycerides do not provide any clear nutritional advantage over long-chain triglycerides, but can be trialled in patients who fail to gain or to maintain adequate bodyweight in order to increase energy intake.

Screening for coeliac disease using anti-tissue transglutaminase antibody assays, and prevalence of the disease in an Australian community.

OBJECTIVES: To determine (i) the prevalence of positive results of anti-tissue transglutaminase (anti-tTG) antibody assays and coeliac disease (CD) in a rural Australian community; and (ii) whether confirmatory testing of a positive assay result with an alternative anti-tTG assay improved the positive predictive value of the test in population screening for CD. DESIGN: Retrospective analysis in December 2004 of stored serum samples taken in 1994-1995 from 3011 subjects in the Busselton Health Study follow-up. Assays for IgA and IgG anti-tTG antibodies were performed, and positive or equivocal samples were retested with a different commercial anti-tTG assay. Available subjects with one or more positive assay results were interviewed, had serum collected for repeat anti-tTG assays and for HLA-DQ2 and HLA-DQ8 haplotyping and, if appropriate, gastroscopy and duodenal biopsy were performed. In unavailable subjects, HLA-DQ2 and -DQ8 haplotyping was performed on stored sera. Total serum IgA levels were assessed in subjects with initially negative assay results. MAIN OUTCOME MEASURE: Prevalence of anti-tTG positivity and biopsy-proven CD. RESULTS: In 47 of 3011 serum samples (1.56%), at least one anti-tTG assay gave positive results: 31 of the subjects who provided these sera were available for clinical review, and 21 were able to have a gastroscopy. Seventeen subjects (0.56%) were diagnosed with definite CD (14 were confirmed at gastroscopy, and three unavailable subjects had three positive results of anti-tTG assays and an HLA haplotype consistent with CD); in a further 12 unavailable subjects, CD status was considered equivocal, with one or more positive anti-tTG assay results and an HLA haplotype consistent with CD. If these subjects were regarded as having CD, the prevalence of CD would be 0.96%. The positive predictive value when all three anti-tTG assays gave positive results was 94%, but fell to 45.2% with only one positive result. CONCLUSIONS: The prevalence of anti-tTG antibodies in this population is 1.56%; the prevalence of CD is at least 0.56%. The utility of a single, positive result of an anti-tTG assay in screening for CD in the community is poor, and repeat and/or collateral assessment with different assays may decrease the need for gastroscopy and distal duodenal biopsy.

Careful patient selection may improve response rates to infliximab in inflammatory bowel disease.

BACKGROUND AND AIM: The use of infliximab in the treatment of Crohn's disease (CD) is acceptable and appears to be effective in ulcerative colitis (UC). Careful patient selection, resulting in infliximab only for truly refractory inflammatory bowel disease (IBD), may improve its efficacy. The present study aimed to determine if careful patient selection improved infliximab efficacy in IBD. METHODS: CD or UC/IBD unclassified patients (Montreal classification) were considered for infliximab treatment only after failure of disease control with conventional therapies and confirmation of active disease. Patients with purely luminal IBD received a single infliximab dose. Patients with fistulizing disease (with or without luminal disease) received infliximab at 0, 2 and 6 weeks. Changes to Harvey Bradshaw (HBI) for inflammatory CD and Colitis Activity Index (CAI) for UC/IBDU were used to determine the response and remission rates. In fistulizing CD, a remission was sustained cessation of drainage and resolution of the fistula. Response was correlated to inflammatory marker levels. RESULTS: Seventy IBD patients were treated. In CD, 85.2% (46/54) had active luminal and 40.7% (22/54) had fistulizing disease. In luminal CD, at 8 weeks a single infliximab dose induced remission in 75% (24/32) of patients compared to 92.9% (13/14) after infliximab at 0, 2 and 6 weeks. Fistulizing disease responded in 77.2% (17/22) and remitted in 50% (11/22) of patients at 8 weeks. In UC/IBDU, 75% (12/16) responded and 43.8% (7/16) of patients were in remission at 8 weeks. CONCLUSION: Careful patient selection may improve infliximab's efficacy and clinical remission appears greater after induction with three infliximab doses in CD. Clinical efficacy is suggested for UC/IBDU.

A double-blind, randomised, controlled trial to study the effects of an enteral feed supplemented with glutamine, arginine, and omega-3 fatty acid in predicted acute severe pancreatitis.

CONTEXT: Current best evidence is in favour of early institution of enteral feeding in acute severe pancreatitis with promising results from trials in immunonutrition on other patient groups. OBJECTIVE: To identify which groups of patients and products are associated with benefit, we investigated immunonutrition in patients with predicted acute severe pancreatitis. DESIGN: A randomised trial of a study feed containing glutamine, arginine, tributyrin and antioxidants versus an isocaloric isonitrogenous control feed was undertaken. PATIENTS: Thirty-one patients with a diagnosis of acute pancreatitis predicted to develop severe disease: 15 study feeds and 16 control feeds. INTERVENTIONS: Enteral feeding via nasojejunal tube for 3 days. If patients required further feeding the study was continued up to 15 days. MAIN OUTCOME MEASURES: Reduction in C-reactive protein (CRP) by 40 mg/L after 3 days of enteral feeding was the primary endpoint. Carboxypeptidase B activation peptide (CAPAP) levels were taken at regular intervals. RESULTS: After 3 days of feeding, in the study group 2/15 (13%) of patients had reduced their CRP by 40 mg/L or more. In the control group 6/16 (38%) of patients had reduced their CRP by this amount. This difference was found to be near the statistical significant limit (P=0.220). CONCLUSIONS: The cause of the unexpectedly higher CRP values in the study group is unclear. The rise in CRP was without a commensurate rise in CAPAP or outcome measures so there was no evidence that this represented pancreatic necrosis. The contrast between the CRP and CAPAP results is of interest and we believe that specific pancreatic indices such as CAPAP should be considered in larger future studies.

Machine learning can improve prediction of severity in acute pancreatitis using admission values of APACHE II score and C-reactive protein.

BACKGROUND: Acute pancreatitis (AP) has a variable course. Accurate early prediction of severity is essential to direct clinical care. Current assessment tools are inaccurate, and unable to adapt to new parameters. None of the current systems uses C-reactive protein (CRP). Modern machine-learning tools can address these issues. METHODS: 370 patients admitted with AP in a 5-year period were retrospectively assessed; after exclusions, 265 patients were studied. First recorded values for physical examination and blood tests, aetiology, severity and complications were recorded. A kernel logistic regression model was used to remove redundant features, and identify the relationships between relevant features and outcome. Bootstrapping was used to make the best use of data and obtain confidence estimates on the parameters of the model. RESULTS: A model containing 8 variables (age, CRP, respiratory rate, pO2 on air, arterial pH, serum creatinine, white cell count and GCS) predicted a severe attack with an area under the receiver-operating characteristic curve (AUC) of 0.82 (SD 0.01). The optimum cut-off value for predicting severity gave sensitivity and specificity of 0.87 and 0.71 respectively. The predictions were significantly better (p = 0.0036) than admission APACHE II scores in the same patients (AUC 0.74) and better than historical admission APACHE II data (AUC 0.68-0.75). CONCLUSIONS: This system for the first time combines admission values of selected components of APACHE II and CRP for prediction of severe AP. The score is simple to use, and is more accurate than admission APACHE II alone. It is adaptable and would allow incorporation of new predictive factors.

A comparative study of goblet cell and pancreatic exocine autoantibodies combined with ASCA and pANCA in Chinese and Caucasian patients with IBD.

BACKGROUND: The incidence of Crohn's disease (CD) and ulcerative colitis (UC) is increasing, but differentiating between them is often extremely difficult. Pancreatic (PAB) and goblet cell autoantibodies (GAB) are specific for CD and UC, respectively, but with low sensitivity. In combination with anti-Saccharomyces cerevisiae (ASCA) and anti-neutrophil cytoplasmic antibodies (pANCA) testing, these antibodies may improve differentiation between the diseases. This study determined the sensitivity, specificity, and positive and negative predictive values (PPV and NPV) of PAB and GAB +/- ASCA and pANCA testing in Chinese and Caucasian IBD populations. RESULTS: Patients were recruited from Caucasian and Chinese populations (CD, n = 100; UC, n = 99; controls, n = 100). PAB was highly specific for CD, and detection was greater in patients less than 35 years old and in Chinese compared with Caucasian patients with CD (CD, 46% versus 22%, P = 0.018; UC, 2% versus 6%; controls, 0% versus 2%). GAB detection was poor in all groups (<2%). PAB showed a PPV of 93% to differentiate all patients with CD from patients with UC, but only 62% for those with isolated colonic disease. The PPV of PAB increased to 100%, specificity was 100%, and sensitivity was 21% for isolated colonic disease when combined with pANCA and ASCA. PAB expression was not associated with stricturing or perforating CD. CONCLUSIONS: This study identified that GAB was not useful in our patients with IBD. PAB expression was highly specific for CD and more sensitive in Chinese than Caucasian patients with CD. The combination of PAB, pANCA, and ASCA testing improved the differentiation between UC and CD, particularly in isolated colonic disease, compared with pANCA and ASCA testing alone.

Using the polymerase chain reaction coupled with denaturing gradient gel electrophoresis to investigate the association between bacterial translocation and systemic inflammatory response syndrome in predicted acute severe pancreatitis.

AIM: To investigate the use of PCR and DGGE to investigate the association between bacterial translocation and systemic inflammatory response syndrome in predicted severe AP. METHODS: Patients with biochemical and clinical evidence of acute pancreatitis and an APACHE II score > or = 8 were enrolled. PCR and DGGE were employed to detect bacterial translocation in blood samples collected on d 1, 3, and 8 after the admission. Standard microbial blood cultures were taken when there was clinical evidence of sepsis or when felt to be clinically indicated by the supervising team. RESULTS: Six patients were included. Of all the patients investigated, only one developed septic complications; the others had uneventful illness. Bacteria were detected using PCR in 4 of the 17 collected blood samples. The patient with sepsis was PCR-positive in two samples (taken on d 1 and 3), despite three negative blood cultures. Using DGGE and specific primers, the bacteria in all blood specimens which tested positive for the presence of bacterial DNA were identified as E coli. CONCLUSION: Our study confirmed that unlike traditional microbiological techniques, PCR can detect the presence of bacteria in the blood of patients with severe AP. Therefore, this latter method in conjunction with DGGE is potentially an extremely useful tool in predicting septic morbidity and evaluating patients with the disease. Further research using increased numbers of patients, in particular those patients with necrosis and sepsis, is required to assess the reliability of PCR and DGGE in the rapid diagnosis of infection in AP.

A comparative study of tissue transglutaminase antibodies and endomysium antibody immunofluorescence in routine clinical laboratory practice.

BACKGROUND: The demand for screening for coeliac disease has grown rapidly over the last few years. Laboratories depending on immunofluorescence assays are faced with an increasing workload using a labour-intensive test, and an alternative to this test has been sought. This study compares tissue transglutaminase (TTG) and endomysium antibodies (EMA) in a routine clinical laboratory situation. METHODS: An immunofluorescence IgA EMA test was compared with a guinea pig TTG antibody ELISA for 816 unselected requests for gut antibody screening. Discrepant results were investigated more fully using a variety of human source TTG antigen kits. RESULTS: Guinea pig TTG ELISA and EMA assays showed agreement for 93.6% of samples. Four samples were misclassified and 48 samples gave false positive TTG results. Study of 46 EMA samples (this group included 39 of the 'discrepant' negative EMA/positive guinea pig TTG group) using three different human purified and/or recombinant TTG sources showed that 42 patients had no TTG antibodies using human sources, three were misclassified and one patient had negative EMA and positive TTG results that could not be readily explained. Further study of 32 EMA positive samples showed almost complete agreement between the human source TTG kits. CONCLUSIONS: We can recommend the replacement of EMA with ELISA for TTG antibodies for the routine screening for coeliac disease, but all positive TTG antibodies should still be followed up with IgA EMA and samples should be screened for IgA deficiency.