Oral immunoglobulin treatment improved intestinal permeability in children with active Crohn's disease.

AIM: Crohn's disease (CD) is a chronic mucosal inflammation that affects the intestinal barrier function, for example, by altering the intestinal permeability. This pilot clinical study investigated the impact of oral human immunoglobulin (OHIG) treatment on permeability characteristics in children with active luminal Crohn's disease. METHODS: The study was performed at the Department of Paediatrics, Norrköping Hospital, Sweden. Intestinal permeability was studied in three boys aged 13, 15 and 18 years with active CD, before and after a six-week treatment programme with OHIG, using different-sized polyethylene glycols as the test molecules. Three age- and sex-matched children with active CD treated with exclusive enteral nutrition (EEN) were also studied. RESULTS: OHIG and EEN resulted in virtually similar reductions in the signs and symptoms of mucosal inflammation. However, OHIG, unlike EEN, appeared to normalise mucosal transfer leading to a normalisation of the maximum permeation of the small PEG molecules, as well as less restrictions of the larger PEG molecules. CONCLUSION: Our study found that OHIG appeared to normalise the mucosal barrier. This suggests that it could offer a new additional and versatile treatment for paediatric CD patients, with a minimal risk of adverse effects.

Urinary nitric oxide metabolites in children with celiac disease after long-term consumption of oats-containing gluten-free diet.

OBJECTIVE: Oats are accepted in the gluten-free diet (GFD) for children with celiac disease (CD). Some reports have indicated, however, that not all celiac patients tolerate oats. We have previously shown that some children still have high levels of urinary nitric oxide (NO) metabolites as markers of intestinal inflammation after 1 year on GFD with oats. In this study, we measured urinary NO metabolites in CD children who had been consuming oats-containing GFD for an extended, 2-6-year period, also taking into consideration ordinary consumption of nitrite/nitrate-rich foods close to the urine sampling. MATERIALS AND METHODS: Morning urinary nitrite/nitrate concentrations were measured in 188 pediatric CD patients. A questionnaire was used to elucidate factors possibly affecting the urinary levels, for example, dietary factors, asthma, or urinary tract infection. RESULTS: Oats were consumed by 89.4% of the patients for a median time of 3 years. The median nitrite/nitrate level was 980 µM. The majority (70.2%) who consumed oats had low levels of urinary nitrite/nitrate, that is, <1400 µM, while 29.8% demonstrated high levels, that is, >1400 µM. Nitrite/nitrate-rich foods did not significantly influence the urinary concentrations. CONCLUSION: The urinary levels of NO metabolites revealed two subpopulations, one with high and one with low levels. The high levels could be possibly due to poor adherence to the GFD, sensitivity to oats, or some unknown factor(s). Nitrate-rich foods, asthma, or urinary tract infection did not affect the result. The elevated levels of NO metabolites could indicate mucosal inflammation and pinpoint the need of careful follow-up of children on oats-containing GFD.

Urinary nitric oxide excretion in infants with eczema.

Eczema is characterized by inflammation of the skin and is commonly associated with food allergy. It has been suggested that nitric oxide (NO) is an important player in eczema, food allergy and intestinal inflammation. The aim of this study was to assess the levels of urinary NO breakdown products in infants with eczema and the effect of eczema treatment on NO levels. Ninety-four infants with eczema, 58 boys and 36 girls, with a mean age of 7.5 ± 5.2 months (mean ± s.d.) at inclusion were examined twice with an interval of 6 wk. The sum of nitrite and nitrate was measured colorimetrically in urinary samples from both visits and compared with clinical data concerning eczema severity, nutrition, gastrointestinal symptoms, asthma and skin prick positivity. The levels of NO products increased significantly from the first to the second visit: 289; 374 µm (median; IQR) vs. 457; 678 µm (median; IQR) (p < 0.001) in parallel with a significant improvement of the eczema. After eczema treatment consisting of skin care and elimination diet during the 6-wk interval between evaluations, the NO levels approached the values previously found in healthy children. The results support previous studies indicating that the homeostasis of nitrogen radicals is disturbed in childhood eczema.

Nitric oxide production in the exhaled air of patients with pulmonary tuberculosis in relation to HIV co-infection.

BACKGROUND: Nitric oxide (NO) is essential for host defense in rodents, but the role of NO during tuberculosis (TB) in man remains controversial. However, earlier observations that arginine supplementation facilitates anti-TB treatment, supports the hypothesis that NO is important in the host defense against TB. Local production of NO measured in fractional exhaled air (FeNO) in TB patients with and without HIV co-infection has not been reported previously. Thus, our aim was to investigate levels of FeNO in relation to clinical symptoms and urinary NO metabolites (uNO). METHODS: In a cross sectional study, FeNO and uNO were measured and clinical symptoms, chest x-ray, together with serum levels of arginine, tumor necrosis factor alpha (TNF-alpha) and interleukin 12 (IL-12) were evaluated in sputum smear positive TB patients (HIV+/TB, n = 36, HIV-/TB, n = 59), their household contacts (n = 17) and blood donors (n = 46) from Gondar University Hospital, Ethiopia. RESULTS: The proportion of HIV-/TB patients with an increased FeNO level (> 25 ppb) was significantly higher as compared to HIV+/TB patients, but HIV+/TB patients had significantly higher uNO than HIV-/TB patients. HIV+ and HIV-/TB patients both had lower levels of FeNO compared to blood donors and household contacts. The highest levels of both uNO and FeNO were found in household contacts. Less advanced findings on chest x-ray, as well as higher sedimentation rate were observed in HIV+/TB patients as compared to HIV-/TB patients. However, no significant correlation was found between FeNO and uNO, chest x-ray grading, clinical symptoms, TNF-alpha, IL-12, arginine levels or sedimentation rate. CONCLUSION: In both HIV negative and HIV co infected TB patients, low levels of exhaled NO compared to blood donors and household were observed. Future studies are needed to confirm whether low levels of exhaled NO could be a risk factor in acquiring TB and the relative importance of NO in human TB.

Effects of CNI-1493 on human granulocyte functions.

During acute bacterial infections such as sepsis and meningitis, activation of inflammatory mediators such as nitric oxide (NO) plays a crucial role in both pathogenesis and host defense. We have previously reported that CNI-1493, a macrophage deactivator, reduced mortality in infant rats infected with Haemophilus influenzae type b (Hib) with associated decrease in the number of granulocytes in the infected tissue. The aim of the present study was to investigate how CNI-1493 affects granulocytes and macrophages in vitro. Murine macrophages (RAW 264.7) pre-incubated with CNI-1493 prior to activation with lipopolysaccharide (LPS)/interferon gamma (IFNgamma) had decreased NO production measured as NO(2)(-)/NO(3)(-) levels and reduction in inducible NO-synthase (iNOS) expression. Reactive oxygen species (ROS) production was increased in formylmethionyl-leucyl-phenylalanine (FMLP)-stimulated granulocytes following CNI-1493 treatment, whereas F-actin content, motility and chemotaxis were decreased under the same conditions. The effects of CNI-1493 on both NO production in LPS/IFNgamma-activated macrophages and ROS production, F-actin content, motility and chemotaxis in granulocytes, may contribute to the reduced inflammatory response and increased survival in Hib-infected animals treated with CNI-1493.

Evaluation of Multiple Diagnostic Indicators in Comparison to the Intestinal Biopsy as the Golden Standard in Diagnosing Celiac Disease in Children.

Celiac disease (CD) is a chronic small intestinal enteropathy triggered by gluten in genetically predisposed individuals. The susceptibility is strongly associated with certain human leukocyte antigen (HLA)-genes, but efforts are being made in trying to find non-HLA genes that are predictive for the disease. The criteria for diagnosing CD were previously based primarily on histologic evaluation of small intestinal biopsies, but nowadays are often based only on blood tests and symptoms. In this context, we elucidated the accuracy of three diagnostic indicators for CD, alone or in combination. Genetic analyses of HLA-type and nine single nucleotide polymorphisms (SNPs) known to be associated with CD were performed in 177 children previously investigated for the suspicion of CD. CD was confirmed in 109 children, while 68 were considered non-celiacs. The antibodies and urinary nitrite/nitrate concentrations of all of them were measured. The combinations of all the variables used in the study would classify 93% of the study population in the correct diagnostic group. The single best predictors were antibodies (i.e., anti-endomysium immunoglobulin A (IgA) (EMA) and transglutaminase IgA (TGA)), followed by HLA-type and nitric oxide (NO)-metabolites. The nine SNPs used did not contribute to the right diagnoses. Although our control group consisted of children with mostly gastrointestinal symptoms, the presented methodology predicted a correct classification in more than 90% of the cases.

Neutrophil leukocyte motility requires directed water influx.

The ability of neutrophils to sense and move to sites of infection is essential for our defense against pathogens. For motility, lamellipodium extension and stabilization are prerequisites, but how cells form such membrane protrusions is still obscure. Using contrast-enhanced video microscopy and Transwell assays, we show that water-selective aquaporin channels regulate lamellipodium formation and neutrophil motility. Addition of anti-aquaporin-9 antibodies, HgCl(2), or tetraethyl ammonium inhibited the function(s) of the channels and blocked motility-related shape changes. On human neutrophils, aquaporin-9 preferentially localized to the cell edges, where N-formyl peptide receptors also accumulated, as assessed with fluorescence microscopy. To directly visualize water fluxes at cell edges, cells were loaded with high dilution-sensitive, self-quenching concentrations of fluorophore. In these cells, motile regions always displayed increased fluorescence compared with perinuclear regions. Our observations provide the first experimental support for motility models where water fluxes play a pivotal role in cell-volume increases accompanying membrane extensions.

Inducible nitric oxide synthase (NOS II) is constitutive in human neutrophils.

The objective was to study the expression of inducible nitric oxide synthase (NOS II) in and NO production by human blood neutrophils and in in vivo exudated neutrophils. Cellular expression of NOS II was evaluated by flow cytometry in whole blood, in isolated blood neutrophils, and in neutrophils obtained by exudation in vivo into skin chambers. Neutrophil NOS II was also demonstrated by Western blotting. Uptake of 3H-labelled L-arginine was studied in vitro and NOS activity measured in a whole cell assay by the conversion of 3H-arginine to 3H-citrulline. In contrast to unseparated blood cells, NOS II was demonstrable both in isolated blood neutrophils and exudated cells. The failure to detect NOS II by flow cytometry in whole blood cells thus proved to be due to the quenching effect of hemoglobin. Western blotting revealed a 130 kD band corresponding to NOS II in isolated blood neutrophils, but detection was dependent on diisopropylfluorophosphate for proteinase inhibition. L-arginine was taken up by neutrophils, but enzymatic activity could not be demonstrated. We conclude that human neutrophils constitutively express NOS II, but that its demonstration by FITC-labelling is inhibited by hemoglobin-mediated quenching in whole blood samples.

Enhanced neutrophil expression of annexin-1 in coronary artery disease.

The systemic inflammatory activity in patients with stable coronary artery disease (CAD) is associated with a dysregulated cortisol response. Moreover, an aberrant activation status of neutrophils in CAD has been discussed; and the question of glucocorticoid resistance has been raised. The anti-inflammatory actions of glucocorticoids are mediated by annexin-1 (ANXA1). We investigated the expression of glucocorticoid receptors (GR) and ANXA1, as well as the exogenous effects of ANXA1 on neutrophils in CAD patients and related the data to diurnal salivary cortisol. Salivary cortisol levels were measured in the morning and evening during 3 consecutive days in 30 CAD patients and 30 healthy individuals. The neutrophil expression of GR and ANXA1 was determined by flow cytometry. The effect of exogenous ANXA1 was determined in a neutrophil stimulation assay. The patients showed a flattened diurnal cortisol pattern compared with healthy subjects, involving higher levels in the evening. The neutrophil expression of GR-total and GR-alpha was decreased, whereas the GR-beta expression did not differ compared with controls. The neutrophil expression of ANXA1 was significantly increased in patients. Ex vivo, ANXA1 impaired the leukotriene B(4)-induced neutrophil production of reactive oxygen species in patients but not in controls. Our findings indicate a persistent overactivation of the hypothalamic-pituitary-adrenal axis in CAD patients but do not give any evidence for glucocorticoid resistance, as assessed by the neutrophil expression of GR and ANXA1. The altered neutrophil phenotype in CAD may thus represent a long-term response to disease-related activation.

Intracellular oxidative activation in synovial fluid neutrophils from patients with rheumatoid arthritis but not from other arthritis patients.

OBJECTIVE: To compare total and intracellular oxidative activation of blood and synovial fluid (SF) neutrophils from patients with rheumatoid arthritis (RA) and other arthritides with blood donor neutrophils. METHODS: Peripheral blood and SF samples were obtained from 26 gonarthritis patients (13 RA, 13 non-RA) attending the rheumatology unit for therapeutic joint aspiration. Isolated neutrophils were stimulated by a formylated tripeptide (fMLF) or by microbeads coated with collagen-I. Formation of superoxide-anion-derived reactive oxygen species (ROS) was studied by luminol-enhanced chemiluminescence. Paired samples of blood and SF neutrophils from patients with active arthritis were compared with blood neutrophils from patients in remission and from 47 healthy blood donors. RESULTS: SF neutrophils from patients with RA, but not from non-RA patients, showed high baseline intracellular ROS production. Blood neutrophils from arthritis patients in remission existed in a primed state as revealed by more rapid oxidative response after collagen-bead challenge and a more pronounced response after fMLF stimulation compared to healthy blood donors. Blood neutrophils from RA patients with ongoing gonarthritis, however, did not differ from healthy blood donors concerning oxidative activation, whereas blood neutrophils from non-RA patients with gonarthritis showed a significantly lower peak ROS production. CONCLUSIONS: A novel finding with pathogenetic implications in our study is that SF neutrophils from patients with RA, but not other arthritides, are activated and produce ROS intracellularly. This implies that synovial neutrophils in RA are engaged in the processing of endocytosed material.

Helicobacter pylori SabA adhesin evokes a strong inflammatory response in human neutrophils which is down-regulated by the neutrophil-activating protein.

The human pathogen Helicobacter pylori expresses two dominant adhesins; the Lewis b blood group antigen binding adhesin, BabA, and the sialic acid-binding adhesin, SabA. These adhesins recognize specific carbohydrate moieties of the gastric epithelium, i.e. the Lewis b antigen, Le(b), and the sialyl-Lewis x antigen, sLe(x), respectively, which promote infection and inflammatory processes in the gastroduodenal tract. To assess the contribution of each of BabA, SabA and the neutrophil activating protein (HP-NAP) in a local inflammation, we investigated the traits of H. pylori mutants in their capacity to interact with and stimulate human neutrophils. We thence found that the SabA adhesin was not only the key inducer of oxidative metabolism (Unemo et al. J Biol Chem 280:15390-15397, 2005), but also essential in phagocytosis induction, as evaluated by flow cytometry, fluorescence microscopy and luminol-enhanced chemiluminescence. The napA deletion resulted in enhanced generation of reactive oxygen species and impaired adherence to the host cells. In conclusion, the SabA adhesin stimulates human neutrophils through selectin-mimicry. Interestingly, HP-NAP modulates the oxidative burst, which could tune the impact of the H. pylori infection for establishment of balanced and chronic inflammation of the gastric mucosa.

Urinary nitric oxide during one year of gluten-free diet with or without oats in children with coeliac disease.

OBJECTIVE: Although in both adults and children with coeliac disease (CD) it is now recommended that oats be added to their gluten-free diet, there is still some controversy concerning the possible harmful effects of oats in some individuals. In this study concentrations of nitric oxide metabolites were repeatedly measured in the urine of children under investigation for CD, when on a gluten-free diet with or without oats. MATERIAL AND METHODS: The study included 116 children, randomized to a standard gluten-free diet (GFD-std) or a gluten-free diet supplemented with wheat-free oat products (GFD-oats), over a one-year period. Small-bowel biopsy was performed at the beginning and end of the study. Morning urine samples were collected from 87 children and urinary nitrite/nitrate concentrations were monitored at 0, 3, 6, 9 and 12 months. RESULTS: All patients were in clinical remission after the study period. There was a rapid decline in urinary nitrite/nitrate concentrations in both groups as early as after 3 months. No differences were seen between the study groups at any of the checkpoints. However, at the end of the study, the nitrite/nitrate values of 9 children in the GFD-oats group and 8 children in the GFD-std group had not normalized. CONCLUSIONS: Children with CD on a gluten-free diet with oats display a similar reduction in urinary nitrite/nitrate as those on a traditional gluten-free diet. Some children, however, still demonstrate high nitrite/nitrate excretion after one year on either diet, indicating that long-term follow-up studies of children on an oats-containing diet are needed.