J. H. P. Wilson

Biochemical basis of 5-aminolaevulinic acid-induced protoporphyrin IX accumulation : a study in patients with (pre)malignant lesions of the oesophagus

Administration of 5-aminolaevulinic acid (ALA) leads to porphyrin accumulation in malignant and premalignant tissues, and ALA is used as a prodrug in photodynamic therapy (PDT). To understand the mechanism of porphyrin accumulation after the administration of ALA and to investigate whether ALA-induced protoporphyrin IX might be a suitable photosensitizer in Barretts oesophagus and adenocarcinoma, we determined the activities of porphobilinogen deaminase (PBG-D) and ferrochelatase (FC) in various malignant and premalignant as well as in normal tissues of the human oesophagus. A PDT power index for ALA-induced porphyrin accumulation, the ratio of PBG-D to FC normalized for the normal squamous epithelium of the oesophagus, was calculated to evaluate intertissue variation in the ability to accumulate porphyrins. In malignant and premalignant tissue a twofold increased PBG-D activity and a marginally increased FC activity was seen compared with normal squamous epithelium. A significantly increased PDT power index in Barretts epithelium and adenocarcinoma was found. Our results suggest that, after the administration of ALA, porphyrins will accumulate in a greater amount in Barretts epithelium and adenocarcinoma of the oesophagus because of an imbalance between PBG-D and FC activities. The PDT power index here defined might be a useful indicative parameter for predicting the susceptibility of these tissues to ALA-PDT.

Two different point G to A mutations in exon 10 of the porphobilinogen deaminase gene are responsible for acute intermittent porphyria.

Two mutations of the porphobilinogen (PBG) deaminase gene resulting in cross-reacting immunological material (CRIM) positive forms of acute intermittent porphyria (AIP) have been identified by in vitro amplification of cDNA and cloning of the amplified products in a bacterial expression vector. Both mutations resulted from G to A transitions in exon 10 of the gene and produced arginine to glutamine substitutions in the abnormal protein. Expression of mutant cDNA in Escherichia coli reveals that one but not the other of these amino acid changes results in a striking decrease of the optimal pH of the mutated enzyme. One or the other of these two mutations accounted for the defect causing AIP in six unrelated patients among the eight patients evaluated with the CRIM positive subtype of this disorder.

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.).

Porphyrin biosynthesis in human Barrett's oesophagus and adenocarcinoma after ingestion of 5-aminolaevulinic acid

5-Aminolaevulinic acid (ALA)-induced porphyrin biosynthesis, which is used for ALA-based photodynamic therapy (ALA-PDT), was studied in tissues of 10 patients with Barretts oesophagus (BE) and adenocarcinoma of the oesophagus (AC) undergoing oesophagectomy at a mean time interval of 6.7 h after the ingestion of ALA (60 mg kg–1). In BE, AC, squamous epithelium (SQ) and gastric cardia, the activities of the haem biosynthetic enzymes porphobilinogen deaminase (PBG-D) and ferrochelatase (FC) and the PDT power index – the ratio between PBG-D and FC in BE and AC in comparison with SQ – were determined before ALA ingestion. Following ALA administration, ALA, porphobilinogen, uroporphyrin I and PPIX were determined in tissues and plasma. The PDT power index did not predict the level of intracellular accumulation of PPIX found at 6.7 h. In BE, there was no selectivity of PPIX accumulation compared to SQ, whereas in half of patients with AC selectivity was found. Higher haem biosynthetic enzyme activities (i.e. PBG-D) and lower PPIX precursor concentrations were found in BE and AC compared to SQ. It is therefore possible that PPIX levels will peak at earlier time intervals in BE and AC compared to SQ.

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.

Mutation analysis of three genes encoding novel LKB1-interacting proteins, BRG1, STRADα, and MO25α, in Peutz–Jeghers syndrome

Mutations in LKB1 lead to Peutz–Jeghers syndrome (PJS). However, only a subset of PJS patients harbours LKB1 mutations. We performed a mutation analysis of three genes encoding novel LKB1-interacting proteins, BRG1, STRADα, and MO25α, in 28 LKB1-negative PJS patients. No disease-causing mutations were detected in the studied genes in PJS patients from different European populations.

Characterization of a new mutation (R292G) and a deletion at the human uroporphyrinogen decarboxylase locus in two patients with hepatoerythropoietic porphyria

SummaryA deficiency in the activity of uroporphyrinogen decarboxylase (UROD), the fifth enzyme of the haem biosynthetic pathway, is found in familial porphyria cutanea tarda (F-PCT) and hepatoerythropoietic porphyria (HEP). A new mutation (R292G) and a deletion have been found in a pedigree with two HEP patients (two sisters). The R292G mutation was not detected in 13 unrelated affected patients with F-PCT, so it appears to be uncommon. The possibility that the arginine 292 may participate at the active site of the enzyme is discussed. A summary of the 7 mutations/deletions found in the UROD gene with their frequency is presented.

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.

Heme Synthesis in Chronic Renal Failure: The Effects of Hemodialysis, Peritoneal Dialysis and Erythropoietin Treatment

UNLABELLED Increased plasma porphyrins have been described in patients with chronic renal failure (CRF). We measured plasma levels of porphyrins and the activity in erythrocytes of porphobilinogen deaminase (PBG-D), one of the key enzymes in heme biosynthesis, in CRF patients not yet on dialysis and in patients on intermittent hemodialysis (IHD) or chronic ambulatory peritoneal dialysis (CAPD), some of whom were being treated with recombinant human erythropoietin (rHuEPO). In addition, the amount of immuno-detectable PBG-D (Ig PBG-D) per 100 units standard PBG-D activity (Ig PBG-D/100 U) and the total amount of Ig PBG-D, using polyclonal antibodies, were determined in erythrocytes of all patients and controls to detect changes in biodegradation of this enzyme. Plasma porphyrins were increased in CRF patients not yet on dialysis and even higher in both patient groups on dialysis compared with controls. Plasma porphyrins were higher in patients on IHD than in patients on CAPD. The activity of PBG-D was increased and Ig PBG-D/100 U was decreased in patients on IHD compared with CRF patients not yet on dialysis and patients on CAPD. Reticulocyte counts were also greater in patients on IHD than in CRF patients not yet on dialysis and patients on CAPD. Ig PBG-D was increased in both groups of patients on dialysis and treated with rHuEPO compared with patients not treated with rHuEPO. IN CONCLUSION (1) in patients on IHD, an increased production of porphyrins is, at least partly, caused by an increased PBG-D activity, and (2) an increased PBG-D activity and a decrease in Ig PBG-D/100 U in patients on IHD could be explained by the presence of a (relatively) young erythroid cell population in which a larger part of PBG-D has not yet been degraded.

Diagnosis and Treatment of Acute Intermittent Porphyria

Acute intermittent porphyria is a disorder of haem synthesis which is associated with attacks of neurologic dysfunction. The disease is due to an inherited defect in the third enzyme of haem synthesis, porphobilinogen deaminase. The importance of an exact diagnosis, on the basis of urinary amino-laevulinic acid, and porphobilinogen excretion, faecal porphyrin content, and erythrocyte porphobilinogen deaminase activity is stressed. In some families the genetic variation in porphobilinogen deaminase activity requires the use of DNA techniques for firm diagnosis. Treatment is symptomatic. Haem arginate or haematin intravenously is useful in the acute attack. Tin-protoporphyrin, which is a competitive inhibitor of haem breakdown, is a promising new addition to the treatment of the acute attack.