Marcin Surmiak

Probiotics in the Mechanism of Protection Against Gut Inflammation and Therapy of Gastrointestinal Disorders

A growing body of experimental and clinical evidence supports the hypothesis that the intestinal microbiota markedly influences function and the structure of the mucosal lining. Intestinal microbiota can potentially cause damage to the mucosa either directly by releasing toxins or indirectly by causing a detrimental immune response. Probiotic bacteria have been defined as live microorganisms, which when consumed in adequate amounts, confer a health benefit for the host. In recent years, the therapeutic and preventive application of probiotics for several gastrointestinal and liver disorders has received increasing attention. Probiotics appear to be beneficial for premature infants who suffer necrotizing enterocolitis. The effectiveness of certain probiotics as treatment for infectious and antibioticassociated diarrhea in adults and for allergic disorders in children has been supported by clinical studies; however, the potential mechanism( s) remains to be studied. Experimental studies and clinical trials for probiotic treatment of inflammatory bowel diseases (IBD) have yielded conflicting results. Daily intake of selected probiotics was effective in the prevention of ulcerative colitis and the attenuation of the active onset of Crohns disease or ulcerative colitis but others failed to show a beneficial effect. The combination of Lactobacillus, Bifidobacterium, Saccharomyces boulardi and the treatment with Escherichia coli Nissle were found beneficial in inducing and maintaining remission of disease activity of gut inflammation and moderately severe ulcerative colitis. Probiotic bacteria were considered in some studies as a safe adjuvant when added to triple eradication therapy against the symptoms induced by the major gastric pathogen, Helicobacter pylori. This review attempted to overview these new exciting advances in the role of these microbes in the pathogenesis, management and treatment of gastrointestinal disorders.

Carbon Monoxide (CO) Released from Tricarbonyldichlororuthenium (II) Dimer (CORM-2) in Gastroprotection against Experimental Ethanol-Induced Gastric Damage

The physiological gaseous molecule, carbon monoxide (CO) becomes a subject of extensive investigation due to its vasoactive activity throughout the body but its role in gastroprotection has been little investigated. We determined the mechanism of CO released from its donor tricarbonyldichlororuthenium (II) dimer (CORM-2) in protection of gastric mucosa against 75% ethanol-induced injury. Rats were pretreated with CORM-2 30 min prior to 75% ethanol with or without 1) non-selective (indomethacin) or selective cyclooxygenase (COX)-1 (SC-560) and COX-2 (celecoxib) inhibitors, 2) nitric oxide (NO) synthase inhibitor L-NNA, 3) ODQ, a soluble guanylyl cyclase (sGC) inhibitor, hemin, a heme oxygenase (HO)-1 inductor or zinc protoporphyrin IX (ZnPPIX), an inhibitor of HO-1 activity. The CO content in gastric mucosa and carboxyhemoglobin (COHb) level in blood was analyzed by gas chromatography. The gastric mucosal mRNA expression for HO-1, COX-1, COX-2, iNOS, IL-4, IL-1β was analyzed by real-time PCR while HO-1, HO-2 and Nrf2 protein expression was determined by Western Blot. Pretreatment with CORM-2 (0.5–10 mg/kg) dose-dependently attenuated ethanol-induced lesions and raised gastric blood flow (GBF) but large dose of 100 mg/kg was ineffective. CORM-2 (5 mg/kg and 50 mg/kg i.g.) significantly increased gastric mucosal CO content and whole blood COHb level. CORM-2-induced protection was reversed by indomethacin, SC-560 and significantly attenuated by celecoxib, ODQ and L-NNA. Hemin significantly reduced ethanol damage and raised GBF while ZnPPIX which exacerbated ethanol-induced injury inhibited CORM-2- and hemin-induced gastroprotection and the accompanying rise in GBF. CORM-2 significantly increased gastric mucosal HO-1 mRNA expression and decreased mRNA expression for iNOS, IL-1β, COX-1 and COX-2 but failed to affect HO-1 and Nrf2 protein expression decreased by ethanol. We conclude that CORM-2 released CO exerts gastroprotection against ethanol-induced gastric lesions involving an increase in gastric microcirculation mediated by sGC/cGMP, prostaglandins derived from COX-1, NO-NOS system and its anti-inflammatory properties.

Plasma levels of leptin and soluble leptin receptor and polymorphisms of leptin gene -18G > A and leptin receptor genes K109R and Q223R, in survivors of childhood acute lymphoblastic leukemia

BackgroundApproximately 20% of children and adolescents in Europe are overweight. Survivors of pediatric acute lymphoblastic leukemia (ALL) are at increased risk of overweight and obesity. The purpose of this study was to assess leptin and leptin soluble receptor levels, as well as polymorphisms of selected genes in survivors of pediatric ALL, and the influence of chemo- and radiotherapy on development of overweight in the context of leptin regulation.MethodsEighty two patients (55% males), of median age 13.2 years (m: 4.8 years; M: 26.2 years) were included in the study. The ALL therapy was conducted according to modified Berlin-Frankfurt-Munster (BFM; n = 69) regimen or New York (n = 13) regimen. In 38% of patients cranial radiotherapy (CRT) was used in median dose of 18.2Gy (m: 14Gy; M: 24Gy). Median age at diagnosis was 4.5 (m: 1 year; M: 16.9 years) and median time from completion of ALL treatment was 3.2 years (m: 0.5 year; M: 4.3 years). Patients with BMI ≥85 percentile were classified as overweight. Correlation of plasma levels of leptin and leptin soluble receptor, and polymorphisms of leptin gene -18G > A, leptin receptor genes K109R and Q223R, and the overweight status were analyzed in relation to gender, intensity of chemotherapy (high intensity vs. standard intensity regimens) and to the use of CRT.ResultsSignificant differences of leptin levels in patients treated with and without CRT, both in the entire study group (22.2+/- 3.13 ng/ml vs. 14.9+/-1.6 ng/ml; p < 0.03) and in female patients (29.9+/-4.86 ng/ml vs. 16.9+/-2.44 ng/ml; p = 0.014), were found. Significant increase of leptin levels was also found in overweight patients compared to the non-overweight patients in the entire study group (29.2+/-2.86 ng/ml vs. 12.6+/-1.51 ng/ml; p < 0.0001), female patients (35.4+/-6.48 ng/ml vs. 18.4+/-2.5 ng/ml; p = 0.005), and male patients (25.7+/-2.37 ng/ml vs. 6.9+/-0.95 ng/ml; p < 0.0001). Negative correlation was observed for plasma levels of soluble leptin receptor and overweight status, with significant differences in overweight and non-overweight patients, both in the entire study group (18.2+/-0.75 ng/ml vs. 20.98+/-0.67 ng/ml; p = 0.017) and in male patients (18.2+/-1.03 ng/ml vs. 21.8+/- 1.11 ng/ml; p = 0.038). Significant (p < 0.05) negative correlation was found between leptin and leptin receptor levels in the entire group (correlation coefficient: 0.393) and in both gender subgroups (correlation coefficient in female patients: -0.427; in male patients: -0.396).ConclusionsThe prevalence of overweight in our cohort was higher than in general European population (31% vs 20%) and increased regardless of the use of CRT. Leptin and leptin receptor levels may be used as useful markers of high risk of becoming overweight in ALL survivors, particularly in females treated with CRT. Polymorphisms of leptin gene -18G > A and leptin receptor genes K109R and Q223R were not associated with overweight status in ALL survivors.

Endogenous prostaglandins and afferent sensory nerves in gastroprotective effect of hydrogen sulfide against stress-induced gastric lesions.

Hydrogen sulfide (H2S) plays an important role in human physiology, exerting vasodilatory, neuromodulatory and anti-inflammatory effects. H2S has been implicated in the mechanism of gastrointestinal integrity but whether this gaseous mediator can affect hemorrhagic lesions induced by stress has been little elucidated. We studied the effect of the H2S precursor L-cysteine, H2S-donor NaHS, the H2S synthesizing enzyme (CSE) activity inhibitor- D,L-propargylglycine (PAG) and the gastric H2S production by CSE/CBS/3-MST activity in water immersion and restraint stress (WRS) ulcerogenesis and the accompanying changes in gastric blood flow (GBF). The role of endogenous prostaglandins (PGs) and sensory afferent nerves releasing calcitonin gene-related peptide (CGRP) in the mechanism of gastroprotection induced by H2S was examined in capsaicin-denervated rats and those pretreated with capsazepine to inhibit activity of vanilloid receptors (VR-1). Rats were pretreated with vehicle, NaHS, the donor of H2S and or L-cysteine, the H2S precursor, with or without the concurrent treatment with 1) nonselective (indomethacin) and selective cyclooxygenase (COX)-1 (SC-560) or COX-2 (rofecoxib) inhibitors. The expression of mRNA and protein for COX-1 and COX-2 were analyzed in gastric mucosa pretreated with NaHS with or without PAG. Both NaHS and L-cysteine dose-dependently attenuated severity of WRS-induced gastric lesions and significantly increased GBF. These effects were significantly reduced by pretreatment with PAG and capsaicin denervation. NaHS increased gastric H2S production via CSE/CBS but not 3-MST activity. Inhibition of COX-1 and COX-2 activity significantly diminished NaHS- and L-cysteine-induced protection and hyperemia. NaHS increased expression of COX-1, COX-2 mRNAs and proteins and raised CGRP mRNA expression. These effects of NaHS on COX-1 and COX-2 protein contents were reversed by PAG and capsaicin denervation. We conclude that H2S exerts gastroprotection against WRS-induced gastric lesions by the mechanism involving enhancement in gastric microcirculation mediated by endogenous PGs, sensory afferent nerves releasing CGRP and the activation of VR-1 receptors.

The Protective Role of Carbon Monoxide (CO) Produced by Heme Oxygenases and Derived from the CO-Releasing Molecule CORM-2 in the Pathogenesis of Stress-Induced Gastric Lesions: Evidence for Non-Involvement of Nitric Oxide (NO)

Carbon monoxide (CO) produced by heme oxygenase (HO)-1 and HO-2 or released from the CO-donor, tricarbonyldichlororuthenium (II) dimer (CORM-2) causes vasodilation, with unknown efficacy against stress-induced gastric lesions. We studied whether pretreatment with CORM-2 (0.1–10 mg/kg oral gavage (i.g.)), RuCl3 (1 mg/kg i.g.), zinc protoporphyrin IX (ZnPP) (10 mg/kg intraperitoneally (i.p.)), hemin (1–10 mg/kg i.g.) and CORM-2 (1 mg/kg i.g.) combined with NG-nitro-l-arginine (l-NNA, 20 mg/kg i.p.), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 mg/kg i.p.), indomethacin (5 mg/kg i.p.), SC-560 (5 mg/kg i.g.), and celecoxib (10 mg/kg i.g.) affects gastric lesions following 3.5 h of water immersion and restraint stress (WRS). Gastric blood flow (GBF), the number of gastric lesions and gastric CO and nitric oxide (NO) contents, blood carboxyhemoglobin (COHb) level and the gastric expression of HO-1, HO-2, hypoxia inducible factor 1α (HIF-1α), tumor necrosis factor α (TNF-α), cyclooxygenase (COX)-2 and inducible NO synthase (iNOS) were determined. CORM-2 (1 mg/kg i.g.) and hemin (10 mg/kg i.g.) significantly decreased WRS lesions while increasing GBF, however, RuCl3 was ineffective. The impact of CORM-2 was reversed by ZnPP, ODQ, indomethacin, SC-560 and celecoxib, but not by l-NNA. CORM-2 decreased NO and increased HO-1 expression and CO and COHb content, downregulated HIF-1α, as well as WRS-elevated COX-2 and iNOS mRNAs. Gastroprotection by CORM-2 and HO depends upon CO’s hyperemic and anti-inflammatory properties, but is independent of NO.

Exogenous Asymmetric Dimethylarginine (ADMA) in Pathogenesis of Ischemia-Reperfusion-Induced Gastric Lesions: Interaction with Protective Nitric Oxide (NO) and Calcitonin Gene-Related Peptide (CGRP)

Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide (NO) synthesis inhibitor and pro-inflammatory factor. We investigated the role of ADMA in rat gastric mucosa compromised through 30 min of gastric ischemia (I) and 3 h of reperfusion (R). These I/R animals were pretreated with ADMA with or without the combination of l-arginine, calcitonin gene-related peptide (CGRP) or a small dose of capsaicin, all of which are known to afford protection against gastric lesions, or with a farnesoid X receptor (FXR) agonist, GW 4064, to increase the metabolism of ADMA. In the second series, ADMA was administered to capsaicin-denervated rats. The area of gastric damage was measured with planimetry, gastric blood flow (GBF) was determined by H2-gas clearance, and plasma ADMA and CGRP levels were determined using ELISA and RIA. ADMA significantly increased I/R-induced gastric injury while significantly decreasing GBF, the luminal NO content, and the plasma level of CGRP. This effect of ADMA was significantly attenuated by pretreatment with CGRP, l-arginine, capsaicin, or a PGE2 analogue. In GW4064 pretreated animals, the I/R injury was significantly reduced and this effect was abolished by co-treatment with ADMA. I/R damage potentiated by ADMA was exacerbated in capsaicin-denervated animals with a further reduction of CGRP. Plasma levels of IL-10 were significantly decreased while malonylodialdehyde (MDA) and plasma TNF-α contents were significantly increased by ADMA. In conclusion, ADMA aggravates I/R-induced gastric lesions due to a decrease of GBF, which is mediated by a fall in NO and CGRP release, and the enhancement of lipid peroxidation and its pro-inflammatory properties.

New satiety hormone nesfatin-1 protects gastric mucosa against stress-induced injury: Mechanistic roles of prostaglandins, nitric oxide, sensory nerves and vanilloid receptors

Nesfatin-1 belongs to a family of anorexigenic peptides, which are responsible for satiety and are identified in the neurons and endocrine cells within the gut. These peptides have been implicated in the control of food intake; however, very little is known concerning its contribution to gastric secretion and gastric mucosal integrity. In this study the effects of nesfatin-1 on gastric secretion and gastric lesions induced in rats by 3.5h of water immersion and restraint stress (WRS) were determined. Exogenous nesfatin-1 (5-40μg/kg i.p.) significantly decreased gastric acid secretion and attenuated gastric lesions induced by WRS, and this was accompanied by a significant rise in plasma NUCB2/nefatin-1 levels, the gastric mucosal blood flow (GBF), luminal NO concentration, generation of PGE2 in the gastric mucosa, an overexpression of mRNA for NUBC2 and cNOS, as well as a suppression of iNOS and proinflammatory cytokine IL-1β and TNF-α mRNAs. Nesfatin-1-induced protection was attenuated by suppression of COX-1 and COX-2 activity, the inhibition of NOS with L-NNA, the deactivation of afferent nerves with neurotoxic doses of capsaicin, and the pretreatment with capsazepine to inhibit vanilloid VR1 receptors. This study shows for the first time that nesfatin-1 exerts a potent protective action in the stomach of rats exposed to WRS and these effects depend upon decrease in gastric secretion, hyperemia mediated by COX-PG and NOS-NO systems, the activation of vagal and sensory nerves and vanilloid receptors.

Circulating mitochondrial DNA in serum of patients with granulomatosis with polyangiitis

Neutrophil is a key cell in pathophysiology of granulomatosis with polyangiitis. Recently, neutrophil extracellular traps were described in this disease. Mitochondrial DNA is also released during traps formation. We measured circulating cell‐free mitochondrial and genomic DNA in serum of patients with granulomatosis with polyangiitis. Subjects with the disease (14 active and 11 in remission stage) and 10 healthy controls were enrolled. Quantitative real‐time polymerase chain reaction (PCR) was used to measure 79 base pairs (bp) and 230 bp mtDNA fragments. Alu repeats were quantified to evaluate abundance of nuclear DNA in serum at the presence of plasmid control. Both fragments of mtDNA (79 bp and 230 bp) and genomic DNA were elevated significantly in granulomatosis with polyangiitis compared to controls. Only the shorter 79bp mtDNA correlated with active stage of granulomatosis with polyangiitis and clinical symptoms. A mechanism of extracellular release of mitochondrial DNA accompanies the active stage of the disease. Circulating mtDNA is extremely high in untreated patients. This suggests that biomarker properties of mtDNA are useful for monitoring of treatment.

Skewing toward Treg and Th2 responses is a characteristic feature of sustained remission in ANCA-positive granulomatosis with polyangiitis

The objective of our study was to evaluate the T‐helper (Th) and regulatory T (Treg) cell profile in ANCA‐positive granulomatosis with polyangiitis (GPA) and its relation to disease activity. In a prospective study, we studied two groups of GPA patients: (i) disease flare (active‐GPA, BVAS>6, n = 19), (ii) sustained remission (≥ 1‐year prior enrollment, inactive‐GPA, BVAS = 0, n = 18). 24 age‐sex matched healthy subjects served as controls. Active‐GPA patients were followed for 6 months and reevaluated during remission (early remission; n = 13). We analyzed subsets of Th‐cells (flow cytometry), production of signature cytokines by in vitro stimulated lymphocytes, and broad spectrum of serum cytokines (Luminex). In all GPA patients we observed expansion of effector Th17 cells, and increased production of IL‐17A by in vitro stimulated T cells, as compared to controls. Disease flare was characterized by marked reduction in Treg cells, whereas in sustained remission we showed expansion of both Treg and Th2 subset. Finally, analyzing the cytokine profile, we identified CCL23 and LIGHT, as potential biomarkers of active disease. We conclude that in GPA, expansion of Treg and Th2 lymphocytes in parallel to increased Th17 response is a characteristic feature of sustained remission. In contrast, Treg cells are markedly decreased in disease flare.

Homozygosity for the rs9939609T allele of the FTO gene may have protective effect on becoming overweight in survivors of childhood acute lymphoblastic leukaemia

Approximately 20% of adolescents and children in Europe are overweight, and 30% of those who are overweight actually fulfill the criteria of obesity (Branca et al. 2007). The prevalence of obesity in survivors of acute lymphoblastic leukaemia (ALL) is 16 to 57% (Gregory and Reilly 2004). Among others, cranial radiotherapy (CRT) and genetic factors have been implicated as risk factors of overweight/obesity (Ross et al. 2004; Chow et al. 2007). The fat mass and obesity associated gene (FTO) has been recently found to contribute to the risk of obesity. Polymorphism of rs9939609 T>A was associated with an increased BMI in children aged ≥7 years. Individuals homozygous for A allele at rs9939609 constituted 16% of the population and population risk was 20.4% for obesity and 12.7% for overweight (Frayling et al. 2007). The high-risk FTO haplotype yielded a proportion of attributable risk of 22% for uncomplicated obesity in adults and children of European ancestry (Dina et al. 2007). Therefore, the influence of rs9939609 T>A polymorphism of FTO gene on overweight in leukaemia survivors was studied. A total of 191 patients were genotyped for the above genetic variant. Significant negative association between the rs9939609T allele and prevalence of overweight status was found in a subset of patients treated with CRT. Patients who receive CRT and are carriers of this variant may be protected from becoming overweight.