This work aimed to study the opioid peptide β-casomorphin-7 (BCM7) degradation or stability during digestion using human gastrointestinal (GI) juices and porcine jejunal brush border membrane (BBM) peptidases. Synthetic BCM7 was subjected to in vitro digestion by GI fluids obtained from human volunteers for 180 min, and to downstream degradation with porcine BBM vesicles. The BCM7 was sampled at 4 time points over 24 h after BBM addition. The digests were profiled by HPLC-electrospray ionization mass spectrometry (ESI/MS) to monitor BCM7 during GI digestion, and intact BCM7 through BBM digestion was quantified by reverse-phase (RP)-HPLC. We found that BCM7 was partly digested with human GI enzymes, as 3 proteolytic fragments in addition to f(60-66) YPFPGPI were detected: f(62-66) FPGPI, f(60-65) YPFPGP and f(61-66) PFPGPI. The RP-HPLC analysis revealed that 42% of the initial peptide was degraded after only 2 h of BBM digestion, and as much as 79% was degraded after 4-h digestion with supplementation of BBM. In conclusion, this study showed that most of BCM7 was degraded during GI and BBM digestion, although a small amount (5%) was still detected after 24-h digestion. It remains to be studied whether the small amount of intact BCM7 detected after in vitro digestion is transported via active transceptors in the human intestinal epithelial cells and enters blood circulation.

Background: Fecal calprotectin (FC) correlates well with mucosal healing and risk of relapse in inflammatory Bowel Disease (IBD). An obstacle for a broader use of FC measurement in routine practice is the need to bring stool samples at the hospital or the send them by mail. The aim of our work was to test the usability of a home-based test for the measurement of FC in IBD patients. Methods: IBD patients in clinical remission or mild disease activity and declaring motivation to perform home-based FC measurements were prospectively recruited in three IBD centres in Oslo, Barcelona and Liège. They received a standardized training. They were instructed to collect and extract stools and to measure FC with a dedicated tool and smartphone application, 5 times at two weeks intervals over a 8 weeks period. The included patients had to fill in a usability questionnaire made of simple questions and Linkert scales at the first and the last FC measurement. Two global scores were calculated integrating the different aspects of usability: the System Usability Scale (SUS: 0–100) and the Global Usability Score (GUS: 0–85). FC was also centrally measured by ELISA. Results: 58 patients were recruited, including 18 ulcerative colitis (UC) and 40 Crohn's disease (CD), 30 females. Median (IQR) age was 35 yrs (27–40), median (IQR) HBI in CD was 0 (0–4), median (IQR) Clinical Mayo in UC was 0 (0–1). Over the 58 included patients, 42 performed at least one FC measurement and 27 performed all the FC requested measurements. The median (IQR) GUS (0–85) at the first and last use were 74 (69–80) and 77 (68–83), respectively; the median (IQR) SUS (0–100) at the first and last use were 85 (78–90) and 81 (70–88), respectively. Adherence to the planned measurements and usability of the tool were higher in females and in less severe disease. The inter-class correlation coefficient between home-based and centrally measured FC was 0.85. Conclusions: Around three quarters of the patients who declared themselves motivated to use home-based test of FC measurement actually did it, but only half of them fully adhered to the planned measurements. Usability scores for the home-based test were high. There was a very good correlation with the centrally measured FC by ELISA.

Background: Therapeutic drug monitoring (TDM) has become standard clinical practice and overwhelming clinical evidence indicates that dose-optimization improve clinical outcome by decreasing the risk for anti-drug-antibodies (ADA) and improves the efficacy of the drug itself. There is also another aspect for advocating TDM, that is improving the health economic aspect of these very expensive drugs. However, this has been hampered by the high cost of and the absence of a near patient testing. Aims of the study: The study had two aspects; first is to correlate a CE -marked rapid test for IFX trough level, the Quantum Blue Infliximab test (QB-IFX) (BÜHLMANN Laboratories, AG, Basel, Switzerland) to an assay very similar to the Loeven assay. Secondly, to correlate the performance of such a test done by; A) a nurse and B) a trained laboratory person Methods: The study comprised 64 pts with IBD receiving IFX treatment; 14 Remicade and 50 Remsina. At the day of infusion, blood for IFX-trough level was collected in addition to 3 ml serum for QB-IFX rapid test. Part A: A nurse (IL) received one hour of “laboratory” training before running the QB-IFX under supervision of AR. The serum was diluted 10uL in 190 uL assay buffer and vortexed for 5 sec. 70uL was applied to the rapid test cassette and read after 15 min using the Q B reader. Part B: The same procedure was followed by an experienced lab technician (GHM). In addition, 5 aliquots from three sera-levels was collected (3, 7 and 10ug/ml) and tested to establish CV for the upper and lower trough level values as well as for one high level value. Results: The was a very good correlation between the QB-IFX rapid test and the laboratory ELISA test, r=0.90, p<0.001, the slope was 1.1. Furthermore, the correlation between the results obtained by the nurse and a skilled lab person was acceptable with r=0.92, p<0.001. The CV for the upper and lower trough level was 4.7 and 7.8% respectively. CV for the high level was 14.7% Conclusions: This is the first study that documents a close correlation between a 15 min. rapid test for IFX trough level with that of an standard lab-test. We have shown that such a test can accurately be performed by a nurse. The CV for the three different serumlevels was way lower than expected for a lateral flow rapid test. This means that TDM now can be moved from a distant laboratory to the near patient facility like the infusion centre to ensure correct dosing in IBD and other patients on IFX treatment. References: [1] Moore, C., Corbett, G., & Moss, A. C. (2016). Systematic Review and Meta-Analysis: Serum Infliximab Levels During Maintenance Therapy and Outcomes in Inflammatory Bowel Disease. J of Crohn's & Colitis, 10(5), 619–625.

Background: Necrotizing enterocolitis (NEC) is a neonatal emergency that affects preterm newborns during the 1st weeks of life. Diagnosis is made mainly by clinical criteria since no specific diagnostic tests are available. Objective: The objective was to evaluate fecal calprotectin (fCal) as a biomarker of gut inflammation to predict NEC in preterm neonates. Methods: Design: Diagnostic test evaluation. Inclusion criteria: 102 preterm neonates <36 weeks gestation and within 7 days of birth admitted in Level III neonatal intensive care unit (NICU) were recruited from January 2010 to May 2011. Exclusion criteria: Congenital anomalies and overt infection. Paired stool samples at day 3 and 7 were analyzed by lateral quantum blue rapid calprotectin assay. Cut-off values of fCal were determined among 30 term healthy infants. A structured questionnaire which included gestational age, symptoms at admission, and modified Bell’s staging was used to record NEC episodes on day 3 and 7 of admission. Septic screen and radiological tools were done as per NICU protocol. Results: 48% were above 34 weeks gestation; 31.3% were of very low birth weight. As per modified Bell’s staging on day 3 and 7, 22 and 11 neonates had 1a or above stage, respectively. 15 had features of NEC; of these, 12 were managed appropriately and discharged and 3 died. In the receiver-operated curve with fCal >279 ?g/g as cutoff, the area under the curve was 0.652 (95% confidence interval: 0.516-0.789). Day 3 fCal levels were high in 65.7% neonates. Using NEC as outcome, sensitivity of the test was 93.3%; specificity was 39%; positive predictive value was 20.8% and negative predictive value was 97.14%. Conclusion: fCal has high sensitivity for diagnosing NEC in preterm neonates. However, further research is needed to establish its clinical usefulness.

Science S81Conclusions: These data show a strong correlation of inflammatory responses between the colitis and radiationinduced models of GI injury, with a common TNF-mediated, neutrophilic component in the colon and jejunum, respectively.Further studies will investigate time course and tissue differences.The efficacy of AVX-470m in both models highlights the central role of TNF in GI tract inflammation, and supports the therapeutic potential of an oral anti-TNF antibody in GI inflammatory disease.

A better understanding of the genotype-phenotype correlation of Atlantic salmon is of key importance for a whole range of production, life history and conservation biology issues attached to this species. High-density linkage maps integrated with physical maps and covering the complete genome are needed to identify economically important genes and to study the genome architecture. Linkage maps of moderate density and a physical bacterial artificial chromosome (BAC) fingerprint map for the Atlantic salmon have already been generated. Here, we describe a strategy to combine the linkage mapping with the physical integration of newly identified single nucleotide polymorphisms (SNPs). We resequenced 284 BAC-ends by PCR in 14 individuals and detected 180 putative SNPs. After successful validation of 152 sequence variations, genotyping and genetic mapping were performed in eight salmon families comprising 376 individuals. Among these, 110 SNPs were positioned on a previously constructed linkage map containing SNPs derived from expressed sequence tag (EST) sequences. Tracing the SNP markers back to the BACs enabled the integration of the genetic and physical maps by assigning 73 BAC contigs to Atlantic salmon linkage groups.

A linkage map was constructed for bovine chromosome 6 (BTA6), using 399 single nucleotide polymorphisms (SNPs) detected primarily from PCR-resequencing. The efficiency of SNP detection was highly dependent on the source of sequence information chosen for primer design (BAC-end sequences, introns or promoters). The SNPs were used to build a linkage map comprising 104 cM on BTA6. The SNP order in the linkage map corresponded very well with radiation hybrid (RH) maps available for BTA6 as well as with expected positions in the human comparative map, but diverged significantly from the current assembly of the bovine genome (Btau_3.1). When performing linkage analysis with the marker order suggested from the Btau_3.1 we observed an expansion of the genetic map from 104 cM to 137 cM, strongly suggesting a reordering of scaffolds in the current version of the bovine genome assembly. The extent of LD on BTA6 was evaluated by calculating the average r(2) for SNP pairs separated by given distances. The decline of LD was rapid with distance, such that r(2) was 0.1 at 100 kb. Our results indicate that linkage mapping will be a valuable source of information for correcting errors in the current bovine assembly. These errors were sufficiently frequent to be of concern for the accuracy of mapping QTL with panels of SNPs whose positions are based on the current assembly.

The production of chimeras, by use of cell transplantation, has proved to be highly valuable in studies of development by providing insights into cell fate, differentiation, and developmental potential. So far, chimeric honeybees have been created by nuclear transfer technologies. We have developed protocols to produce chimeric honeybees by use of cell transplantation. Embryonic cells were transplanted between pre-gastrula stage embryos (32-34 hr after oviposition) and hatched larvae were reared in vitro for 4 days. Chimeric individuals were detected by use of microsatellite analysis and a conservative estimation approach. 4.8% of embryos, posteriorly injected with embryonic cells, developed into chimeric honeybee larvae. By injection of cells pre-stained with fluorescent cell tracer dye, we studied the integration of transplanted cells in the developing embryos. Number of injected cells varied from 0 to 50 and cells remained and multiplied mainly in the area of injection.

A QTL affecting milk production traits was previously mapped to an interval of 7.5 cM on chromosome 6 in Norwegian dairy cattle. This article aimed to refine this position by increasing the map density in the region by a set of single-nucleotide polymorphisms and analyzing the data with a combined linkage and linkage disequilibrium approach. Through a series of single- and multitrait and single- and multipoint analyses, the QTL was positioned to an interval surrounded by the genes ABCG2 and LAP3. As no recombinations were detected in this interval, physical mapping was required for further refining. By using radiation hybrid mapping as well as BAC clones, the bovine and human comparative maps in the region are resolved, and the QTL is mapped within a distance of 420 kb.

Combined linkage and linkage disequilibrium analysis were used to refine the position of a previously detected QTL affecting milk production traits on bovine chromosome 6. Through a series of single- and multitrait and single- and multipoint QTL analyses, the QTL could be positioned to a 7.5-cM interval surrounded by the markers BMS2508 and FBN12. The most significant results were found for fat percentage and protein percentage. This effect seemed to be caused by a QTL allele embedded in one specific marker haplotype that caused a reduction in fat and protein yields and a concomitant increase of milk yield, thus resulting in a marked reduction of fat and protein percentages.

BACKGROUND:Pouchitis is a common and troublesome condition, and a disturbed microbiological flora and mucosal blood flow in the pouch have been suggested as possible causes. Laser Doppler flowmetry (LDF) has been used successfully to measure gastric and colonic mucosal perfusion in humans. The aim of this study was to evaluate the effect of intervention with probiotics on ileal pouch inflammation and perfusion in the pouch, assessed by endoscopy, histology, fecal calprotectin and LDF.METHODS:A fermented milk product (Cultura; 500 ml) containing live lactobacilli (La-5) and bifidobacteria (Bb-12) was given daily for 4 weeks to 10 patients operated with ileal-pouch-anal anastomosis (IPAA) for ulcerative colitis (UC). Mucosal perfusion was measured with LDF and the degree of inflammation was examined at predefined levels of the distal bowel by endoscopy and histology. Stool samples were cultured for lactobacilli and bifidobacteria and calprotectin were measured before and after intervention.RESULTS:The LDF measurements were reproducible in the pelvic pouch at each of the predefined levels, but did not change after intervention. The mucosal perfusion was reduced in the distal compared to the proximal part of the pouch. Calprotectin levels did not change significantly after intervention. The median endoscopic score for inflammation was significantly reduced by 50% after intervention, whereas the histological score did not change significantly.CONCLUSION:The results suggest that probiotics primarily act superficially, with change of gross appearance of the mucosa at endoscopy, but without significant effect on histological picture, mucosal perfusion or faecal calprotectin, during a relatively short period of 4 weeks.

BACKGROUND AND AIMS:Assessing the presence and degree of intestinal inflammation objectively, simply, and reliably is a significant problem in gastroenterology. We assessed faecal excretion of calprotectin, a stable neutrophil specific marker, as an index of intestinal inflammation and its potential use as a screening test to discriminate between patients with Crohn's disease and those with irritable bowel syndrome.METHODS:The validity of faecal calprotectin as a marker of intestinal inflammation was assessed in 22 patients with Crohn's disease (35 studies) by comparing faecal excretions and concentrations using four day faecal excretion of (111)indium white cells. A cross sectional study assessed the sensitivity of faecal calprotectin concentration for the detection of established Crohn's disease (n=116). A prospective study assessed the value of faecal calprotectin in discriminating between patients with Crohn's disease and irritable bowel syndrome in 220 patients referred to a gastroenterology clinic.RESULTS:Four day faecal excretion of (111)indium (median 8.7%; 95% confidence interval (CI) 7-17%; normal <1.0%) correlated significantly (p<0.0001) with daily (median ranged from 39 to 47 mg; normal <3 mg; r=0.76-0.82) and four day faecal calprotectin excretion (median 101 mg; 95% CI 45-168 mg; normal <11 mg; r=0.80) and single stool calprotectin concentrations (median 118 mg/l; 95% CI 36-175 mg/l; normal <10 mg/l; r=0.70) in patients with Crohn's disease. The cross sectional study showed a sensitivity of 96% for calprotectin in discriminating between normal subjects (2 mg/l; 95% CI 2-3 mg/l) and those with Crohn's disease (91 mg/l; 95% CI 59-105 mg/l). With a cut off point of 30 mg/l faecal calprotectin has 100% sensitivity and 97% specificity in discriminating between active Crohn's disease and irritable bowel syndrome.CONCLUSION:The calprotectin method may be a useful adjuvant for discriminating between patients with Crohn's disease and irritable bowel syndrome.

BACKGROUND:The pathogenesis of NSAID-induced gastrointestinal damage is believed to involve a nonprostaglandin dependent effect as well as prostaglandin dependent effects. One suggestion is that the nonprostaglandin mechanism involves uncoupling of mitochondrial oxidative phosphorylation.AIMS:To assess the role of uncoupling of mitochondrial oxidative phosphorylation in the pathogenesis of small intestinal damage in the rat.METHODS:We compared key pathophysiologic events in the small bowel following (i) dinitrophenol, an uncoupling agent (ii) parenteral aspirin, to inhibit cyclooxygenase without causing a 'topical' effect and (iii) the two together, using (iv) indomethacin as a positive control.RESULTS:Dinitrophenol altered intestinal mitochondrial morphology, increased intestinal permeability and caused inflammation without affecting gastric permeability or intestinal prostanoid levels. Parenteral aspirin decreased mucosal prostanoids without affecting intestinal mitochondria in vivo, gastric or intestinal permeability. Aspirin caused no inflammation or ulcers. When dinitrophenol and aspirin were given together the changes in intestinal mitochondrial morphology, permeability, inflammation and prostanoid levels and the macro- and microscopic appearances of intestinal ulcers were similar to indomethacin.CONCLUSIONS:These studies allow dissociation of the contribution and consequences of uncoupling of mitochondrial oxidative phosphorylation and cyclooxygenase inhibition in the pathophysiology of NSAID enteropathy. While uncoupling of enterocyte mitochondrial oxidative phosphorylation leads to increased intestinal permeability and low grade inflammation, concurrent decreases in mucosal prostanoids appear to be important in the development of ulcers.

Digitoxin is frequently used in Norway in the treatment of cardiac failure. Digitalis glycosides may give rise to a number of side effects difficult to separate from disease in the elderly. Six patients aged 77-93 years, treated with digitoxin 0.05 mg/day, were hospitalized due to digitalis intoxication. Mean digitoxin half-life was 25.2 days. This is significantly more than reported in other studies on younger patients. The symptoms of digitoxin intoxication disappeared on discontinuation of medication. The slow elimination of digitoxin may be related to reduced serum albumin concentration. When digitoxin is used in the treatment of heart failure in the very elderly patients, one should be aware of the possibility of digitoxin intoxication, even at a low dose.

BACKGROUND The diagnosis of non-steroidal anti-inflammatory drug (NSAID) induced enteropathy is difficult, requiring enteroscopy or the use of four day faecal excretion of 111 In labelled white cells. AIMS To assess faecal calprotectin (a non-degraded neutrophil cytosolic protein) as a method for diagnosing NSAID enteropathy. METHODS Single stool faecal calprotectin concentrations were compared with the four day faecal excretion of 111 In labelled white cells in 47 patients taking NSAIDs. The prevalence and severity of NSAID enteropathy was assessed using this method in 312 patients (192 with rheumatoid arthritis, 65 with osteoarthritis, 55 with other conditions) taking 18 different NSAIDs. RESULTS The four day faecal excretion of 111 In white cells correlated significantly with faecal calprotectin concentrations. In the group of 312 patients on NSAIDs faecal calprotectin concentrations were significantly higher than in controls, the prevalence of NSAID enteropathy being 44%. The prevalence and severity of NSAID enteropathy was independent of the particular type or dose of NSAID being taken or other patient variables. CONCLUSIONS Assay of faecal calprotectin provides a simple practical method for diagnosing NSAID enteropathy in man. Forty four per cent of patients receiving these drugs had NSAID induced enteropathy when assessed by this technique; 20% of these had comparable levels of inflammation to that previously reported in patients with inflammatory bowel disease.

The digitoxin half-life in elderly patients in the eight and ninth decade was more prolonged (mean +/- SD: 25 +/- 9 days) than in younger people (6.7 +/- 1.7). These elderly patients accumulated digitoxin even on a dose of 0.05 mg/ day. The symptoms of digitoxin intoxication disappeared on discontinuation of medication. When digitoxin is used in the treatment for heart failure in the very elderly patients, one should be aware of the possibility of digitoxin intoxication, even on a low dose.