F.W.M. de Rooij

Afamelanotide for Erythropoietic Protoporphyria

BACKGROUND Erythropoietic protoporphyria is a severe photodermatosis that is associated with acute phototoxicity. Patients with this condition have excruciating pain and a markedly reduced quality of life. We evaluated the safety and efficacy of an α-melanocyte-stimulating hormone analogue, afamelanotide, to decrease pain and improve quality of life. METHODS We conducted two multicenter, randomized, double-blind, placebo-controlled trials of subcutaneous implants containing 16 mg of afamelanotide. Patients in the European Union (74 patients) and the United States (94 patients) were randomly assigned, in a 1:1 ratio, to receive a subcutaneous implant containing either afamelanotide or placebo every 60 days (a total of five implants in the European Union study and three in the U.S study). The type and duration of sun exposure, number and severity of phototoxic reactions, and adverse events were recorded over the respective 180-day and 270-day study periods. Quality of life was assessed with the use of validated questionnaires. A subgroup of U.S. patients underwent photoprovocation testing. The primary efficacy end point was the number of hours of direct exposure to sunlight without pain. RESULTS In the U.S. study, the duration of pain-free time after 6 months was longer in the afamelanotide group (median, 69.4 hours, vs. 40.8 hours in the placebo group; P=0.04). In the European Union study, the duration of pain-free time after 9 months was also longer in the afamelanotide group than in the placebo group (median, 6.0 hours vs. 0.8 hours; P=0.005), and the number of phototoxic reactions was lower in the the afamelanotide group (77 vs. 146, P=0.04). In both trials, quality of life improved with afamelanotide therapy. Adverse events were mostly mild; serious adverse events were not thought to be related to the study drug. CONCLUSIONS Afamelanotide had an acceptable side-effect and adverse-event profile and was associated with an increased duration of sun exposure without pain and improved quality of life in patients with erythropoietic protoporphyria. (Funded by Clinuvel Pharmaceuticals and others; ClinicalTrials.gov numbers, NCT01605136 and NCT00979745.).

Primary bile acid diarrhoea without an ileal carrier defect: quantification of active bile acid transport across the ileal brush border membrane.

Unexplained bile acid malabsorption associated with diarrhoea that responds to cholestyramine was first described in 1973 but convincing evidence of the proposed mechanism--a defective active ileal bile acid transport--has never been substantiated. Active bile acid transport was quantified in vitro using brush border membrane vesicles prepared from terminal ileal biopsy specimens from 10 patients who fulfilled the criteria of idiopathic bile acid diarrhoea. They were recruited from 181 patients with bile acid malabsorption of various causes. Transport was quantified as in vitro Na+ dependent bile acid transport (INBAT), expressed as pmol taurocholate/mg brush border membrane protein/15 seconds, and in vitro Na+ dependent bile acid local transport capacity (INBALTC), expressed as pmol taurocholate/g ileal biopsy tissue/15 seconds. The lowest INBAT and INBALTC values in the 10 patients with idiopathic bile acid diarrhoea were well above the 10th centile values of a control group of 132 patients. Both INBAT (mean (range) 88 (30-136)) and INBALTC (158 (85-268)) values were significantly higher in the 10 patients than in the control group (INBAT: mean (range) 63 (1-244), INBALTC: mean (range) 98 (1-408)). Quantification of active ileal bile acid transport in these 10 patients with idiopathic bile acid malabsorption suggests that a genetic (carrier) defect is rare in adults.

Na+-dependent bile acid transport in the ileum: the balance between diarrhea and constipation.

Ileal Na+-dependent bile acid transport was quantified in vitro as the uptake of 3H-taurocholate into brush-border membrane vesicles. Vesicles were prepared from ileal biopsies of 158 patients placed in 10 diagnostic categories. Active bile acid transport (expressed as picomoles taurocholate uptake per milligram brush-border membrane protein per 15 s, median and interquartile ranges indicated) did not differ significantly in 6 categories: irritable bowel syndrome (71, 35-97; n = 21), colon polyps (42, 30-89; n = 29), colitis (62, 33-91; n = 31), postvagotomy or postcholecystectomy (69, 37-97; n = 11), diarrhea without increased bile acid loss (58, 48-85; n = 12), and lack of gastrointestinal pathology (74, 45-103; n = 22). A decreased active bile acid transport was found in 3 categories: ileal disease (4, 1-36; n = 11), partial ileal resection (5, 1-35; n = 5), and constipation (41, 22-50; n = 8). Bile acid transport was increased in patients with bile acid-losing diarrhea with endoscopically and histologically normal ilea (111, 94-135; n = 8). These findings indicate that a low fecal bile acid loss, presumed to be present in constipated patients, is associated with a low Na+-dependent ileal bile acid transport and a high bile acid loss is associated with a high active bile acid transport. Ileal bile acid transport might be regulated by the availability of bile acids to the ileal enterocytes.

Primary Bile Acid Malabsorption: A Pathophysiologic and Clinical Entity?

Primary bile acid malabsorption is defined as chronic diarrhoea with bile acid malabsorption of unknown cause and a symptomatic response to cholestyramine. Convincing evidence of the proposed pathophysiology-a defect of the active bile acid absorption in the distal ileum-has never been substantiated. We found no evidence of a bile acid transport defect across the ileal brush border membrane in 10 patients with primary bile acid malabsorption; moreover, transport was significantly higher than in a control group. In the patients with primary bile acid malabsorption the estimated bile acid pool was significantly larger than in a control group and in a group of patients with ileal disease. In addition, the oroanal transit time of radiopaque markers was shorter in the primary bile acid malabsorption group than in both other groups. This suggests that the bile acid pool size as well as intestinal motility could play a role in the pathophysiology of primary bile acid malabsorption.

A Gly15Arg mutation in the interleukin-10 gene reduces secretion of interleukin-10 in Crohn disease.

BACKGROUND Genetic susceptibility, probably involving cytokines and their receptors, plays an important role in inflammatory bowel disease (IBD). In this study we examine the potential role of the interleukin-10 (IL-10) gene as a susceptibility gene in IBD. METHODS We studied 17 sib-pairs with either Crohn disease (CD) or ulcerative colitis. After microsatellite analysis for allele-sharing, the IL-10 gene of sib-pairs who shared alleles was screened for nucleotide alterations in and around exons and the promoter region. The IL-10 promoter polymorphism at position -1082 was also determined. Function was evaluated by measuring IL-10 secretion by peripheral blood mononuclear cells stimulated with lipopolysaccharide or phorbol ester. The activity of recombinant immature wild-type and mutated IL-10 was tested in a proliferation assay with a human monocytic leukaemia cell line (HL60 cells). RESULTS DNA sequencing revealed a G --> A point mutation in exon 1 at base position 43 in one sib-pair, both affected with CD. It was also found in 2 of their healthy siblings, but not in 75 unrelated healthy controls. This mutation results in a glycine to arginine substitution at amino acid position 15 of the leader sequence (Gly15Arg). The in vitro IL-10 secretion by mononuclear cells of the IL-10 Gly15Arg carriers was about 50% of healthy controls, matched for the -1082 polymorphism in the IL-10 promoter region. Incubation of HL60 cells with recombinant mutated IL-10 showed a markedly reduced cell proliferation compared to wild-type IL-10. CONCLUSION A Gly15Arg mutation in the leader sequence of IL-10 was found in a multiple CD-affected family. This altered leader sequence decreases IL-10 secretion, thereby reducing the anti-inflammatory effect.Background: Genetic susceptibility, probably involving cytokines and their receptors, plays an important role in inflammatory bowel disease (IBD). In this study we examine the potential role of the interleukin-10 (IL-10) gene as a susceptibility gene in IBD. Methods: We studied 17 sib-pairs with either Crohn disease (CD) or ulcerative colitis. After microsatellite analysis for allele-sharing, the IL-10 gene of sib-pairs who shared alleles was screened for nucleotide alterations in and around exons and the promoter region. The IL-10 promoter polymorphism at position m 1082 was also determined. Function was evaluated by measuring IL-10 secretion by peripheral blood mononuclear cells stimulated with lipopolysaccharide or phorbol ester. The activity of recombinant immature wild-type and mutated IL-10 was tested in a proliferation assay with a human monocytic leukaemia cell line (HL60 cells). Results: DNA sequencing revealed a G r → r A point mutation in exon 1 at base position 43 in one sib-pair, both affected with CD. It was also found in 2 of their healthy siblings, but not in 75 unrelated healthy controls. This mutation results in a glycine to arginine substitution at amino acid position 15 of the leader sequence (Gly15Arg). The in vitro IL-10 secretion by mononuclear cells of the IL-10 Gly15Arg carriers was about 50% of healthy controls, matched for the m 1082 polymorphism in the IL-10 promoter region. Incubation of HL60 cells with recombinant mutated IL-10 showed a markedly reduced cell proliferation compared to wild-type IL-10. Conclusion: A Gly15Arg mutation in the leader sequence of IL-10 was found in a multiple CD-affected family. This altered leader sequence decreases IL-10 secretion, thereby reducing the anti-inflammatory effect.

Purification of porphobilinogen deaminase from human erythrocytes by fast protein liquid chromatography

A four-step procedure using fast protein liquid chromatography is described for the isolation of porphobilinogen deaminase from human erythrocytes. The specific activity of the porphobilinogen deaminase is increased about 13,000-fold, and the preparation is electrophoretically pure on SDS-polyacrylamide gel electrophoresis.

Lysosomal damage by gliadin and gliadin peptides; An activity not related to coeliac disease

Rat-liver lysosomes have been used to determine the toxicity of gliadin fractions in relation to coeliac disease. In this study we compared the activity in acid phosphatase release from rat-liver lysosomes by casein, gliadin and by their peptic-tryptic digests. The release of acid phosphatase is not specific for gliadin.

[75Se]-Selenohomotaurocholic Acid Degradation by Bacterial Enzymes in vitro and in vivo

The stability of the bile acid analogue [75Se]-selenohomotaurocholic acid (75SeH-CAT) was studied in man. When 75SeHCAT was administered to patients for diagnostic purposes the majority of labeled material present in the feces was found deconjugated. In vitro incubation of 75SeHCAT, by addition of fecal homogenate or with addition of purified enzyme, showed identical deconjugation. The relative differences in polarities of 75SeHCAT, [75Se]-selenohomocholic acid (75SeHCA), [14C]-taurocholic acid (14C-TCA) and [14C]-cholic acid (14C-CA) were estimated by isoelectric focusing and selective chloroform extractions at various pH values. The pI values representing the pH where these molecules become uncharged were for 75SeHCA and 75SeHCAT 3.1, for 14C-TCA 3.0 and for 14C-CA 3.9. These results suggest that from these bile acids only 14C-CA is a candidate for passive absorption in the colon, while 75SeHCA would be far too polar for passive diffusion. Indeed, we could demonstrate the inability of 75SeHCA for passive absorption in healthy persons. In conclusion, 75SeHCAT, specifically selected to monitor active ileal bile acid transport, functions as a good indicator of this process in its conjugated form. In contrast to published data it is susceptible to bacterial degradation, and therefore gives rise to a diminished whole-body retention.

Bile Acid Transport in Distal Ileum

Abstract Bile acid active transport in ileum of animals and man is well documented by in vivo studies. The carrier proteins involved in this transport are not defined yet. In vitro investigations on isolated cells or cell membranes however are limited. The aim of this project is to study the transport parameters of bile acids at the membrane level with in vitro experiments using material from rat and man. Therefore we adapted the Ca ++ precipitation method of Kessler and co-workers (1978) for the preparation of brush border membrane vesicles (BBMVs) from ileal biopsy specimen to study the parameters of active bile acid transport over the ileal brush border membrane in vitro. Active Na + -dependent transport of glyco- and taurocholate will be shown with rat ileal vesicle preparation. In our system we find an “overshoot” in the uptake of bile acids upto 300% in the presence of a Na + -gradient. On a molar bases uptake is 2-18 pmoles per milligram membrane protein. Our results show that the Na + -dependent uptake of conjugated bile acids occurs exclusively in the most distal part of the ileum.