Treatment of Crohn's disease with recombinant human interleukin 10 induces the proinflammatory cytokine interferon gamma.

BACKGROUND: Interleukin 10 (IL-10) exerts anti-inflammatory actions by counteracting many biological effects of interferon gamma (IFN-gamma). AIMS: To investigate this in humans, we studied the effects of human recombinant IL-10 administration on IFN-gamma production by patient leucocytes. Furthermore, we assessed the IFN-gamma inducible molecule neopterin and nitrite/nitrate serum levels, which are indicative of endogenous nitric oxide formation. METHODS: As part of two placebo controlled double blind studies, we analysed patients with chronic active Crohn's disease (CACD) who received either subcutaneous recombinant human IL-10 (n=44) or placebo (n=10) daily for 28 days, and patients with mild to moderate Crohn's disease (MCD) treated with either subcutaneous IL-10 (n=52) or placebo (n=16) daily for 28 days. Neopterin and nitrite/nitrate concentrations were measured in serum, and ex vivo IFN-gamma formation by lipopolysaccharide or phytohaemagglutinin (PHA) stimulated whole blood cells were investigated before, during, and after IL-10 therapy. RESULTS: In patients with CACD, the highest dose of 20 microg/kg IL-10 caused a significant increase in serum neopterin on days +15 and +29 of therapy compared with pretreatment levels. No changes were observed for nitrite/nitrate levels under either condition. In MCD, treatment with 20 microg/kg IL-10 resulted in a significant increase in PHA induced IFN-gamma production. CONCLUSIONS: High doses of IL-10 upregulate the production of IFN-gamma and neopterin. This phenomenon may be responsible for the lack of efficacy of high doses of IL-10 in the treatment of CACD and MCD.

Chemiluminescence in inflammatory bowel disease patients: a parameter of inflammatory activity.

BACKGROUND: Reactive oxygen species (ROS) are produced in excess in the inflamed mucosa and peripheral blood of patients with inflammatory bowel disease. These species have emerged as a common pathway of tissue injury in a wide variety of inflammatory and other disease processes. The present study was conducted to assess ROS production and to correlate this with parameters of inflammatory activity. METHODS: In 25 patients with Crohn's disease (CD), 20 patients with ulcerative colitis (UC) and 65 age- and sex-matched healthy volunteers ROS production was measured using the whole blood luminol enhanced chemiluminescence assay (LECA). Disease activity was assessed using the Crohn's disease activity index and the Ulcerative Colitis Symptoms Score (UCSS) for CD and UC, respectively. Furthermore, the effect of various scavengers, enzymes and enzyme inhibitors on LECA was studied to assess the contribution of different ROS. RESULTS: LECA was significantly higher in CD and UC patients compared with healthy controls (7.1+/-4.7 and 9.8+/-6 vs. 5.2+/-2.8 x 10(3) counts per minute (cpm), p<0.05 and <0.001). In CD, relative LECA (patient/control) was correlated with the Crohn's disease activity index and C-reactive protein (CRP) (r=0.54, p=0.001 and r=0.51, p=0.01). In UC, CRP but not LECA was correlated with the Ulcerative Colitis Symptoms Score (C-reactive protein: r=0.42, p=0.01). Addition of azide, superoxide dismutase, deferoxamine and dimethylthiourea resulted in a decrease of LECA values. CONCLUSION: Whole blood LECA is increased in patients with CD and UC. This parameter is correlated with disease activity in CD. The observed chemiluminescence is probably due to generation of superoxide and the hydroxyl radical.

Iron and inflammatory bowel disease.

Both anaemia of iron deficiency and anaemia of chronic disease are frequently encountered in inflammatory bowel disease. Anaemia of iron deficiency is mostly due to inadequate intake or loss of iron. Anaemia of chronic disease probably results from decreased erythropoiesis, secondary to increased levels of proinflammatory cytokines, reactive oxygen metabolites and nitric oxide. Assessment of the iron status in a condition associated with inflammation, such as inflammatory bowel disease, is difficult. The combination of serum transferrin receptor with ferritin concentrations, however, allows a reliable assessment of the iron deficit. The best treatment for anaemia of chronic disease is the cure of the underlying disease. Erythropoietin reportedly may increase haemoglobin levels in some of these patients. The anaemia of iron deficiency is usually treated with oral iron supplements. Iron supplementation may lead to an increased inflammatory activity through the generation of reactive oxygen species. To date, data from studies in animal models of inflammatory bowel disease support the theoretical disadvantage of iron supplementation in this respect. The results, however, cannot easily be extrapolated to the human situation, because the amount of supplemented iron in these experiments was much higher than the dose used in patients with iron deficiency.

Safety and efficacy of recombinant human interleukin 10 in chronic active Crohn's disease. Crohn's Disease IL-10 Cooperative Study Group.

BACKGROUND & AIMS: Interleukin (IL)-10 is a cytokine with potent anti-inflammatory properties. We investigated the safety and efficacy of different doses of human recombinant (rhu)IL-10 in patients with Crohn's disease (CD). METHODS: A prospective, multicenter, double-blind, placebo-controlled study was conducted in 329 therapy-refractory patients with CD. Clinical improvement was defined by a reduction of the Crohn's Disease Activity Index (CDAI) by 100 points or more and clinical remission by a decrease of the CDAI to <150 points. At selected centers, patients underwent ileocolonoscopies and activation of the nuclear factor-kappa B (NF-kappa B) system was assessed in biopsy specimens. RESULTS: Subcutaneous treatment with rhuIL-10 over 28 days induced a fully reversible, dose-dependent decrease in hemoglobin and thrombocyte counts but no clinically significant side effects. No differences in the induction of remission were observed between rhuIL-10 groups (1 microg, 18% [9.6-29.2]; 4 microg, 20% [11.3-32.2]; 8 microg, 20% [11.1-31.8]; 20 microg, 28% [18-40.7]; and placebo, 18% [9.6-29.6]). Clinical improvement was observed in 46% (33.7-59) in the 8-microg/kg rhuIL-10 group in comparison with 27% (17-39.6) in patients taking placebo. Responders to rhuIL-10 showed inhibition of NF-kappaB p65 activation in contrast to nonresponders. CONCLUSIONS: Up to 8 microg/kg of rhuIL-10 was well tolerated. A tendency toward clinical improvement but not remission was observed in the 8-microg/kg dose group. Further studies should delineate which subgroups of patients with CD benefit from rhuIL-10 therapy.

Homocysteine in inflammatory bowel disease: a risk factor for thromboembolic complications?

OBJECTIVE: Patients with inflammatory bowel disease (IBD) are at increased risk for thromboembolic events. Hyperhomocysteinemia, which is an established risk factor for arterial as well as venous thrombosis, may be more prevalent in IBD because of vitamin deficiencies. METHODS: In this retrospective study, we studied the concentrations of total homocysteine (tHcy), cobalamin, folate, and pyridoxine in 231 consecutive patients with IBD, of whom 16 patients had a history of venous thrombosis, and nine a history of arterial thrombosis. Age- and gender-matched healthy volunteers served as controls (n = 102). RESULTS: Homocysteine concentrations in patients were higher (12.3 micromol/L [range 4.6-51.3] vs 11.1 micromol/L [range 3.9-27.6], p = 0.001) and hyperhomocysteinemia tended to be more prevalent in patients than in the controls (11.1% vs 5%, p = 0.07). Folate, cobalamin, creatinine, and pyridoxine concentrations were correlated with tHcy. Folate deficiency was infrequently encountered in IBD patients (4.3%). The tHcy concentration in patients with a history of venous or arterial thrombosis was not higher than in patients without a history of thrombosis (12.7 micromol/L [range 4.6-40.1] and 15.2 micromol/L (range 10.5-26.8) vs 12.3 micromol/L [range 10.5-26.8], not significant). Hyperhomocysteinemia was found in 18.8% of the patients with venous thrombosis, 11.1% of the patients with arterial thrombosis, and 10.5% of the patients without thrombosis (not significant). CONCLUSIONS: Hyperhomocysteinemia is a common phenomenon in IBD and correlates with serum folate, cobalamin, creatinine, and pyridoxine concentrations. No correlation between tHcy and a history of venous or arterial thromboembolic complications is found. Hyperhomocysteinemia does not seem to be a major contributory factor in the development of venous or arterial thrombosis in IBD patients.

Iron supplementation affects the production of pro-inflammatory cytokines in IL-10 deficient mice.

BACKGROUND: Iron supplements may increase disease activity in inflammatory bowel disease through the production of the hydroxyl radical because of its catalytic activity in the Fenton reaction. The purpose of this study was to assess the effect of dietary and locally administered iron in the IL-10 knock-out (-/-) mouse, a model of chronic inflammatory bowel disease. MATERIALS AND METHODS: IL10-/- and wild-type mice received a standard or a high-iron diet (35 mg kg(-1) ferrosulphate vs. 500 mg kg(-1) ferrosulphate) after weaning. After 4 weeks the mice were sacrificed. Furthermore, a group of adult IL-10 knock-out mice was given three iron-containing enema's (0.2 mL of 1 mM ferrous-ammonium sulphate) or phosphate buffered saline. These mice were sacrificed after 1 week. Production of pro-inflammatory cytokines by colon tissue cultures, haematological parameters and histology was determined to assess inflammatory activity. RESULTS: Oral as well as rectal administration of iron resulted in increased pro-inflammatory cytokine production in IL-10-/- mice. Neutrophil counts in IL10-/- on a high iron diet increased as well. No enhanced colonic inflammation was noted on histology after iron supplementation. CONCLUSION: We conclude that dietary or topical administered iron increases pro-inflammatory cytokine production in the colon of IL10-/- mice. No significant increase of histological intestinal inflammation was observed.

A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage-displayed antibody fragments.

A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab). The purified huMab had an affinity of 5 nM and effectively mediated tumor cell killing in in vitro and in vivo assays. These experiments show that nonimmunized phage antibody display libraries can be used to obtain high-affinity, functional, and clinically applicable huMabs directed against a tumor-associated antigen.

Pancreatitis and agenesis of the dorsal pancreas.

We report a case of agenesis of the dorsal pancreas, complicated by pancreatitis and diabetes mellitus. A 39-year-old woman was referred for evaluation of a chronic pancreatitis. Abdominal spiral CT and ERP and MRCP demonstrated agenesis of the dorsal pancreas. The pathogenesis, clinical features and diagnosis of this very rarely reported disease are discussed.

TCR expression in human fetal intestine and identification of an early T cell receptor beta-chain transcript.

TCR expression by human fetal intestinal intraepithelial lymphocytes (ilELs) and intestinal lamina propria lymphocytes was analyzed to address whether T cell development occurs in human fetal intestine, the diversity of human fetal iIELs, and whether human fetal iIELs may contribute to the adult iIEL repertoire. ilELs and intestinal lamina propria lymphocytes from second trimester human fetal intestine were analyzed for TCR-alphabeta transcripts. Rearranged TCR-alpha transcripts were undetectable at 14 wk in the intraepithelial lymphocytes (IELs), whereas multiple TCR-beta transcripts were found at this stage. The TCR-alpha repertoire remained restricted relative to TCR-beta at later stages, and the IEL repertoire was restricted relative to the lamina propria lymphocytes at all stages. A previously reported T early alpha message was the major transcript from the TCR-alpha locus early in gestation. A previously undescribed TCR-beta transcript initiating upstream of the Dbeta1 locus and spliced to Cbeta1 or Cbeta2 was also identified and may represent a T early beta message. These results provide evidence for ongoing TCR gene rearrangement in human fetal intestine and suggest that transcription from the TCR-beta locus initiates with a T early beta transcript. The TCR-alpha repertoire (and hence the repertoire of potentially functional IELs) was limited through the second trimester.

Exogenous protoporphyrin inhibits Hep G2 cell proliferation, increases the intracellular hydrogen peroxide concentration and causes ultrastructural alterations.

Ultrastructural hepatocellular abnormalities in early stages of erythropoietic protoporphyria lead to hepatobiliary changes that cause overt liver disease in 5-10% of patients, not infrequently progressing to fulminant hepatic failure. This cannot be attributed solely to protoporphyrin crystal deposition in the liver. Hepatic redox systems have therefore been postulated as an equivalent for the photoreaction of protoporphyrin. We studied the dark effects of protoporphyrin and hematoporphyrin on HL60 and Hep G2 cells. Cell proliferation and intracellular H2O2 concentrations were assessed and related to the ultrastructural morphology. The incubation with protoporphyrin and hematoporphyrin resulted in a dose- and time-dependent inhibition of proliferation indices of Hep G2 cells. Flow cytometric analyses of intracellular H2O2 concentrations demonstrated a dose-dependent increase in both cell lines upon incubation with protoporphyrin. Hep G2 cells displayed ultrastructural alteration of the endoplasmatic reticulum and plasma membranes. Also 'cell blebbing' occurred. We conclude that exogenous protoporphyrin increases the intracellular H2O2 concentration and exerts a cytotoxic dark effect. This may contribute to the liver injury observed in erythropoietic protoporphyria.

Intestinal T lymphocytes.

The intestine is largely colonized by bacteria and further exposed to an immense array of ingested and shed immunogenic material. Therefore, the gut associated lymphoid tissue plays a major role in the human immune system. It may even constitute a unique immune system of its own, since it has been demonstrated to differ anatomically, phenotypically, functionally and on a molecular basis from its systemic counterpart and other peripheral lymphoid tissue. This is ultimately reflected by the observation in (transgenic) mice that intraepithelial T cells can develop independently of the thymus. Along the same lines, a rapidly growing body of evidences suggests that human bone marrow precursors can home to the gut epithelium, rearrange their T cell receptor genes and further differentiate in the mucosal micro environment. This, and other features that characterize the 'diffuse' mucosal T cell infiltrate will be discussed.

Copper, zinc-superoxide dismutase and hydrogen peroxide: a hydroxyl radical generating system.

Because superoxide (O2-.) is a mediator of inflammation, Cu,Zn-superoxide dismutase (Cu,Zn-SOD) has been employed as an anti-inflammatory compound. However, Cu,Zn-SOD can increase intra- and extracellular H2O2. This may react with the Cu atom of SOD in a Fenton-type reaction producing the hydroxyl radical (.OH). With a non-physiological concentration of H2O2 (0.8 mmol/l) to stimulate chemiluminescence (CL) at a level < 2 mV, it was observed that the addition of Cu,Zn-SOD (100 micrograms/ml) yielded an increase of 204.7 +/- 78.2 mV (P < 0.05). This increase in CL depended on the concentrations of H2O2 and Cu,Zn-SOD and was only seen with luminol (reacts with O2-. and .OH) but not with lucigenin (reacts with O2-.). No CL was observed when Cu,Zn-SOD was heat inactivated, or when Mn-SOD was used. Dissipators of H2O2, copper chelators and .OH scavengers attenuated this CL. In electron paramagnetic resonance, with the use of the spin-trap dimethylpyrroline-N-oxide (DMPO), it was demonstrated that, in the reaction between H2O2 and Cu,Zn-SOD, .OH was generated. The oxidation of keto-methylthiobutyric acid (KMB) to ethylene, assessed by gas chromatography, demonstrated that H2O2/Cu,Zn-SOD-generated .OH can react with KMB and not only with the SOD molecule itself. We conclude that H2O2 reduces SOD-bound Cu2+ to Cu1+ which, in reaction with H2O2 catalyses its reduction to OH. Whether this 'pro-inflammatory' reaction occurs in vivo remains to be established.

Does color vision deficiency in the endoscopist influence the accuracy of endoscopic diagnosis? An anonymous study with Dutch gastrointestinal endoscopists.

Colors play a major role in the endoscopic diagnosis of many gastrointestinal conditions. Gastrointestinal endoscopists in the Netherlands are predominantly male (> 90%), and from population data it is to be expected that approximately 8% will have a color vision deficiency. The present study was designed to assess the prevalence of color vision deficiencies amongst Dutch gastrointestinal endoscopists and to determine whether color vision deficiency affects an endoscopist's diagnostic skill. One hundred and thirty-nine gastroenterologists and physicians of internal medicine took an F2 color vision test and assessed nine videofragments of endoscopies. Color vision deficiencies were detected in 8% of Dutch gastrointestinal endoscopists. In one out of the nine video excerpts of endoscopies, a statistically significant difference was detected between test subjects with and without a color vision deficiency. However, this video excerpt showed a green pea, which could not be mistaken for a polyp at polypectomy. The study therefore does not show any effect of color vision deficiencies on endoscopic skills, nor does it show any deviant prevalence of color vision deficiencies amongst Dutch gastrointestinal endoscopists.

The effect of porphyrins on cellular redox systems: a study on the dark effect of porphyrins on phagocytes.

Erythropoietic protoporphyria (EPP) and porphyria cutanea tarda (PCT) are characterized by skin morbidity, induced by pro-inflammatory reactive oxygen species generated by the photosensitizing properties of protoporphyrin IX and uroporphyrin I. How these porphyrins exert a toxic effect on the liver in the absence of light is poorly understood. We tested the hypothesis that porphyrins can interference with cellular redox systems, by studying the dark effects of protoporphyrin (PP), haematoporphyrin (HP), deuteroporphyrin (DP) and uroporphyrin (UP) on the cellular redox system of phagocytes, and on enzymatic oxyradical generating systems. Both in phagocytic cells and enzymatic systems, a dose-dependent inhibition of chemiluminescence was observed by all porphyrins added. Catalase and SOD-like activity of porphyrins was excluded by oxygraph and ferricytochrome c reduction. However, ferrocytochrome c oxidation was inhibited by porphyrins indicating ferrireductase-like activity. In a Fenton type reaction between H2O2 and PP, we could demonstrate the generation of .OH, or an electronically excited porphyrin species. No influence on phagocyte chemotaxis, phagocytosis and killing-capacity was observed. We conclude that porphyrins do interfere with (cellular) redox systems and can both inhibit and enhance oxygen free radical generation, dependent on the type of redox reaction. Porphyrins can thus affect cellular metabolism. Since H2O2 and PP both readily dissolve in biological membranes, their interaction in the presence of transition metals may contribute to the toxic dark effects of porphyrins as observed in patients with EPP and PCT.

Phenotypic and molecular characterization of human monoclonal TCR gamma/delta T-cell lines from jejunum and colon of healthy individuals.

We have performed a phenotypic and molecular analysis of monoclonal TCR gamma/delta T-cell lines derived from jejunal and colonic biopsies of healthy individuals. Flow cytometric analysis employing a panel of 24 monoclonal antibodies (MoAbs) demonstrated that intestinal TCR gamma/delta intraepithelial lymphocytes (IEL) constitute a phenotypically heterogeneous population. Nucleotide sequence analysis of expressed TCR delta variable (V) regions revealed the dominant utilization of the V delta 2 and D delta 3 gene segments and frequent rearrangement of J delta 3. IEL V delta regions displayed extensive junctional diversity as a result of N and P insertion and the utilization of D delta 3 in all three reading frames. The results demonstrate that intestinal TCR gamma/delta T cells from healthy individuals constitute a phenotypically heterogeneous population expressing V delta regions that differ from their systemic counterparts.

Accumulation of iron in erythroblasts of patients with erythropoietic protoporphyria.

We have studied the iron metabolism in nine patients with erythropoietic protoporphyria (EPP) and three patients with sideroblastic anaemia (SA). All, except one EPP patient were iron deficient. The SA patients had a secondary haemochromatosis. The bone marrow aspirates of patients with SA and also three patients with EPP had a high incidence of ring sideroblasts. Ultrastructural examination of the bone marrow consistently showed finely dispersed electron-dense deposits localized in mitochondria of erythroblasts in all patients with EPP and SA. Mitochondrial electron energy-loss spectroscopy (EELS) indicated identical iron compounds in erythroblasts of all EPP and SA patients. These findings indicate that the mitochondrial iron utilization is disturbed in EPP and SA. The observation of mitochondrial iron deposition in erythroblasts in EPP and SA suggests that this failure is not of pathognomonic value for diagnosis of SA, but is apparently the result of an inefficient haem synthesis, in EPP due to a defective ferrochelatase. The mitochondrial iron deposition does not depend on the iron status (iron overload or iron deficiency) of the EPP patient.

Protoporphyrin photosensitivity cannot be attenuated by oral N-acetylcysteine.

The photodermatosis in erythropoietic protoporphyria (EPP) is caused by the accumulation of photosensitizing protoporphyrin (PP) in the skin, due to a defect in ferrochelatase, the enzyme that inserts ferrous iron into PP to form heme. Hydroxyl radical (.OH) and singlet oxygen generation with subsequent lipid peroxidation are thought to play a major role in the pathogenesis of the photodermatosis in EPP. Hydrogen peroxide (H2O2) can generate .OH in the Haber-Weiss as well as the Fenton reaction, and is thus a potentially harmful intermediate in the photoreduction of O2. The use of oxyradical scavengers, such as beta-carotene, has been reported to be beneficial in the treatment of EPP photodermatosis. In this study, N-acetylcysteine (NAC) 1800 mg/day was used for 3 reasons: (i) its -SH groups directly scavenge H2O2; (ii) ferrochelatase can be activated by sulfhydryl groups; (iii) NAC was reported to upregulate the glutathione redox system, which is a major endogenous anti-oxidant system. However, in a double-blind crossover placebo controlled study on 6 EPP patients, we could neither demonstrate an effect through photosensitivity tests, nor on light hypersensitivity as reported by the patients. This dosage of NAC could not increase reduced glutathione and did not affect the red blood cell PP content nor the excretion of PP in the feces. Neither were adverse effects observed. We conclude that the oral administration of NAC, in the relatively low dose used here, is not effective in the treatment of photodermatosis in EPP.