Tommy Sundqvist

Gut Microflora Associated Characteristics in Children with Celiac Disease

OBJECTIVES:The aim of the study was to investigate the metabolic function of intestinal microflora in children with celiac disease (CD) in order to find out if there is a deviant gut flora in CD patients compared to healthy controls.METHODS:The study group comprised children with CD, consecutively diagnosed according to current criteria given by the European Society for Paediatric Gastroenterology, Hepatology, and Nutrition. Thirty-six children were studied at presentation, i.e., on a normal gluten-containing diet, with clinical symptoms and signs indicative of CD, positive celiac serology markers, and a small bowel biopsy showing severe enteropathy. Forty-seven patients were studied when they had been on a gluten-free diet (GFD) for at least 3 months. For comparison, a group of 42 healthy controls (HC) were studied. The functional status of the intestinal microflora was evaluated by gas–liquid chromatography of short chain fatty acids (SCFAs) in fecal samples.RESULTS:There was a significant difference between untreated CD children and HC as well as between treated CD children and HC regarding acetic, i-butyric, i-valeric acid, and total SCFAs. The propionic and n-valeric acids differed significantly between CD children on GFD and HC. Moreover, there was a strong correlation between i-butyric and i-valeric acids in all study groups.CONCLUSIONS:This is the first study of the SCFA pattern in fecal samples from children with CD. The results indicate that there is a difference in the metabolic activity of intestinal microbial flora in children with CD compared to that in HC. The finding of a different pattern of some SCFAs in celiacs both at presentation and during treatment with GFD indicates that it is a genuine phenomenon of CD not affected by either the diet, the inflammation, or the autoimmune status of the patient.

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, HgCl2, 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.

Different induction mechanisms of mRNA for inducible nitric oxide synthase in rat smooth muscle cells in culture and in aortic strips

The expression of mRNA for the inducible form of nitric oxide synthase, (iNOS), was studied in rat aortic smooth muscle cells, (SMCs) in cell culture and in strips of rat aorta by reverse transcriptase coupled to the polymerase chain reaction. iNOS mRNA expression was weak in cultured SMCs when exposed to either interferon‐γ (IFNγ) or lipopolysaccharide (LPS), but the combination LPS + IFNγ enhanced the expression. In aortic strips LPS alone induced a pronounced expression, with no further increase by IFNγ. Cycloheximide potentiated the expression of iNOS mRNA in SMCs in culture stimulated with LPS + IFNγ but attenuated the response in aortic strips.The results indicate different cellular signaling pathways for the induction of iNOS mRNA by LPS and/or IFNγ, in cultured SMCs and in rat aortic strips.

Influence of Fasting on Intestinal Permeability and Disease Activity in Patients with Rheumatoid Arthritis

We have investigated the influence of fasting and lactovegetarian diet on intestinal and non-intestinal permeability in 5 patients with rheumatoid arthritis. We used low-molecular weight polyethyleneglycols (PEG 400) as probe molecules and a deterministic mathematical model to assess the permeability characteristics. Both intestinal and non-intestinal permeability decreased after fasting, but increased again during a subsequent lactovegetarian diet regime. Concomitantly it appeared that disease activity, as shown by a clinical six-joint score, first decreased and then increased again. The results indicate that, unlike lactovegetarian diet, fasting may ameliorate the disease activity and reduce both the intestinal and the non-intestinal permeability in rheumatoid arthritis.

Arginine as an adjuvant to chemotherapy improves clinical outcome in active tuberculosis

Nitric oxide (NO) is involved in the host defence against tuberculosis (TB). Patients with TB exhibit increased catabolism and reduced energy intake. Thus the hypothesis for this study was that restoring a relative deficiency in the amino acid arginine, the substrate for mycobactericidal NO production, would improve the clinical outcome of TB by increasing NO production. In a randomised double-blind study, patients with smear-positive TB (n=120) were given arginine or placebo for 4 weeks in addition to conventional chemotherapy. Primary outcomes were sputum conversion, weight gain, and clinical symptoms after week 8. Secondary outcomes were sedimentation rate and levels of NO metabolites, arginine, citrulline, and tumour necrosis factor‐α. Compared with the human immunodeficiency virus (HIV)−/TB+ placebo group, the HIV−/TB+ patients in the arginine group showed significant improvement, defined as increased weight gain, higher sputum conversion rate and faster reduction of symptoms, such as cough. The arginine level increased after week 2 in the HIV−/TB+ arginine group (100.2 µM (range 90.5–109.9) versus 142.1 µM (range 114.1–170.1)) compared with the HIV−/TB+ placebo group (105.5 µM (range 93.7–117.3) versus 95.7 µM (range 82.4–108.9)). HIV seroprevalence was 52.5%. No clinical improvement or increase in serum arginine was detected in arginine supplemented HIV+/TB+ patients compared with placebo. Arginine is beneficial as an adjuvant treatment in human immunodeficiency virus-negative patients with active tuberculosis, most likely mediated by increased production of nitric oxide.

Intestinal permeability in healthy and allergic children before and after sodium-cromoglycate treatment assessed with different-sized polyethyleneglycols (PEG 400 and PEG 1000)

Gastrointestinal permeability was investigated in twenty‐two children on two occasions, before and after treatment with sodium cromoglycate. The children were between 8 and 10 years old; half of them were classified as allergic according to history and laboratory tests, and half of them as healthy. The 6‐hr urinary recovery of different‐sized polyethyleneglycols (PEG 400 and PEG 1000) in combination with a mathematical model was used to assess the intestinal permeability barrier.

Increased Intestinal Permeability to Differently Sized Polyethylene Glycols in Uremic Rats: Effects of Low- and High-Protein Diets

Intestinal mucosa forms an important barrier towards harmful agents in the intestinal lumen, besides being the site for absorption of nutrients. Little is known about the intestinal permeability properties in chronic uremia. The permeability toward differently sized polyethylene glycols (PEGs; range 326-1,162 Da) was studied in uremic groups compared to the control groups. The urinary recovery was also recovery of PEGs was increased in the uremic groups compared to the control groups. The urinary recovery was also increased in the groups on the high-protein diet compared to the corresponding group on the low-protein diet. This study suggests an increased permeability of PEG molecules in the range of 546-1,162 Da in uremic rats and a decreased intestinal permeability after a low-protein diet in both a uremic and nonuremic state. Thus, in chronic renal failure the intestinal barrier is impaired but returns towards normal with low-protein diets.

Effect of exclusive enteral nutrition on gut microflora function in children with Crohn's disease

Abstract Objective. Exclusive enteral nutrition (EEN) is a first-line treatment in children with active Crohns disease (CD) but is seldom used in adults with active disease. The mode of action of EEN in suppressing mucosal inflammation is not fully understood, but modulation of intestinal microflora activity is one possible explanation. The aim of this study was to investigate the effect of 6-week EEN in children with active CD, with special reference to intestinal microflora function. Materials and methods. Fecal samples from 18 children (11 boys, 7 girls; median age 13.5 years) with active CD (13 children with small bowel/colonic and 5 with perianal disease) were analyzed for short chain fatty acid (SCFA) pattern as marker of gut microflora function. The children were studied before and after EEN treatment. Results from 12 healthy teenagers were used for comparison. Results. Eleven (79%) of the children with small bowel/colonic CD responded clinically positively to EEN treatment showing decreased levels of pro-inflammatory acetic acid as well as increased concentrations of anti-inflammatory butyric acids and also of valeric acids, similar to the levels in healthy age-matched children. In children with active perianal CD, however, EEN had no positive effect on clinical status or inflammatory parameters. Conclusions. The authors present new data supporting the hypothesis that the well-documented anti-inflammatory effect of EEN in children with active small bowel/colonic CD is brought about by modulation of gut microflora activity, resulting in an anti-inflammatory SCFA pattern. By contrast, none of the children with perianal disease showed clinical or biochemical improvement after EEN treatment.

Mechanical manipulation of bone and cartilage cells with 'optical tweezers'.

The single beam optical gradient trap (optical tweezers) uses a single beam of laser light to non‐invasively manipulate microscopic particles. Optical tweezers exerting a force of approximately 7 pN were applied to single bone and cartilage derived cells in culture and changes in intracellular calcium levels were observed using Fluo‐3 labelling. Human derived osteoblasts responded to optical tweezers with an immediate increase in [Ca2+]i that was inhibited by the addition of a calcium channel blocker nifedipine. Force applied to different regions of cells resulted in a variable response. [Ca2+]i elevation in response to load was lower in rat femur derived osteoblasts, and not apparent in primary chondrocytes and the osteocytic cell line (MLO Y4).

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.