LACC1 polymorphisms in inflammatory bowel disease and juvenile idiopathic arthritis.

The function of the Laccase domain-containing 1 (LACC1) gene is unknown, but genetic variation at this locus has been reported to consistently affect the risk of Crohn's disease (CD) and leprosy. Recently, a LACC1 missense mutation was found in patients suffering from monogenic forms of CD, but also systemic juvenile idiopathic arthritis. We tested the hypothesis that LACC1 single nucleotide polymorphisms (SNPs), in addition to CD, are associated with juvenile idiopathic arthritis (JIA, non-systemic), and another major form of inflammatory bowel disease, ulcerative colitis (UC). We selected 11 LACC1 tagging SNPs, and tested their effect on disease risk in 3855 Swedish individuals from three case-control cohorts of CD, UC and JIA. We detected false discovery rate corrected significant associations with individual markers in all three cohorts, thereby expanding previous results for CD also to UC and JIA. LACC1's link to several inflammatory diseases suggests a key role in the human immune system and justifies further characterization of its function(s).

Association of celiac disease genes with inflammatory bowel disease in Finnish and Swedish patients.

Some genetic loci may affect susceptibility to multiple immune system-related diseases. In the current study, we investigated whether the known susceptibility loci for celiac disease (CelD) also associate with Crohn's disease (CD) and/or ulcerative colitis (UC), the two main forms of inflammatory bowel disease (IBD), in Finnish patients. A total of 45 genetic markers were genotyped in a Finnish data set comprising 699 IBD patients and 2482 controls. Single-marker association with IBD and its subphenotypes was tested. A meta-analysis with a Swedish UC data set was also performed. A total of 12 single-nucleotide polymorphisms associated with CD and/or UC (P<0.05). In the subphenotype analysis, rs6974491-ELMO1 (P=0.0002, odds ratio (OR): 2.20) and rs2298428-UBE2L3 (P=5.44 × 10(-5), OR: 2.59) associated with pediatric UC and CD, respectively. In the meta-analysis, rs4819388-ICOSLG (P=0.00042, OR: 0.79) associated with UC. In the subphenotype meta-analysis, rs1738074-TAGAP (P=7.40 × 10(-5), OR: 0.61), rs6974491-ELMO1 (P=0.00052, OR: 1.73) and rs4819388-ICOSLG (P=0.00019, OR: 0.75) associated with familial UC, pediatric UC and sporadic UC, respectively. Multiple CelD risk loci also confer susceptibility for CD and/or UC in the Finnish and Swedish populations. Certain genetic risk variants may furthermore predispose an individual for developing a particular disease phenotype.

Contribution of the NOD1/CARD4 insertion/deletion polymorphism +32656 to inflammatory bowel disease in Northern Europe.

BACKGROUND: NOD1/CARD4 and NOD2/CARD15 are both intracellular pattern-recognition receptors. The NOD1/CARD4 gene lies within a previously described inflammatory bowel disease (IBD) locus (7p14). An association has been suggested between the NOD1/CARD4+32656 deletion*1 variant of a complex deletion*1/insertion*2 polymorphism and IBD in 1 recent study in Europe. Our aim was to assess the influence of NOD1/CARD4+32656 on disease susceptibility and phenotype in the Scottish and Swedish IBD populations. METHODS: A total of 3,962 individuals (1,791 IBD patients, 522 parents, 1,649 healthy controls) from 2 independent populations (Scotland and Sweden) were genotyped for NOD1/CARD4+32656 A/C by TaqMan and direct sequencing. Case-control, Transmission Disequilibrium Testing (TDT) and detailed genotype-phenotype (Montreal) analyses were performed. The case-control analysis had 80% power to detect an effect size of odds ratio (OR) 1.21 for IBD. RESULTS: In case-control analyses in Scottish and Swedish patients, none of the genotypes studied in IBD, Crohn's disease (CD) or ulcerative colitis (UC), differed significantly from controls (deletion*1 allelic frequency 73.9%, 73.6%, 73.9%, and 73.6%, respectively: all P > 0.8). No epistatic interaction with NOD2/CARD15 was seen for CD susceptibility. TDT analysis in our Scottish early onset cohort was negative. CONCLUSIONS: This variant allele of NOD1/CARD4+32656 is not associated with a strong effect on susceptibility to IBD in children and adults in Northern Europe. A gene-wide haplotype-based approach may be preferable to analysis of individual variants to assess the contribution of the NOD1/CARD4 gene to IBD.

Low molecular weight heparin as adjuvant therapy in active ulcerative colitis.

BACKGROUND: Heparin given intravenously has shown beneficial effects in the treatment of refractory ulcerative colitis in open trials. Low molecular weight heparin (LMWH) offers advantages in the method of administration but have not been evaluated in inflammatory bowel disease conditions. AIM: To assess the tolerability and safety of subcutaneous self-administered LMWH in outpatients with refractory ulcerative colitis and to evaluate any potential adjuvant therapeutic effect. PATIENTS AND METHODS: Twelve patients with mild to moderately active ulcerative colitis were included in the trial. The patients had either responded poorly to treatment with conventional therapy, including oral and/or rectal glucocorticosteroids, or had experienced a rapid relapse during or shortly after GCS therapy. Dalteparin sodium 5000 units s.c. injection was administered twice daily for 12 weeks. Patients were monitored for possible adverse events and changes in clinical symptoms, and endoscopic and histological scores were analysed. Leucocyte scanning was performed at inclusion and at the end of the study. RESULTS: Tolerability and compliance were excellent and no serious adverse events occurred. Eleven patients improved symptomatically and six (50%) attained complete remission after 12 weeks of treatment. Endoscopic, scintigraphic and histological scores were found to be significantly improved. CONCLUSION: Self-administered LMWH given s.c. may be a safe adjuvant therapy for patients with active, glucocorticosteroids-refractory ulcerative colitis. A controlled trial should be undertaken to confirm the positive effects found in this study.

Contribution of the sensory and sympathetic nervous system to scalding-induced edema in the rat paw.

It has recently been hypothesized that both the sensory and sympathetic nervous system contribute to the inflammatory reaction. A scalding model was developed in anaesthetized rats to investigate the contribution of neuropeptides in heat-induced edema localized to the hindpaw. After immersing the paw in water at 60 degrees C for 10, 20, 30 and 60 s, edemic reactions were registered as change of paw volume in a plethysmograph and hindpaw perfusates collected to measure the content of neuropeptides by radioimmunoassay. A scalding period of 30 s induced the most prominent edemic reaction. There was a marked increase of the sensory neuropeptide neurokinin A and the sympathetic related transmitter neuropeptide Y in hindpaw perfusates after scalding. The effect of peripheral nerve ligation on edemic reaction and on the release of neuropeptides was investigated in rats scalded for 30 s at 60 degrees C. There was a significant decrease of edema formation in the scalded nerve ligated paw as compared with the scalded paw on the non-ligated side. Neurokinin A was not detected in nerve ligated rats before or after scalding, whereas mononeuropathic rats showed increased concentrations of neuropeptide Y. The present results indicate that the sensory as well as the sympathetic nervous system, possibly through the release of neuropeptides, may contribute to scald-induced edema.

Increased tissue survival in experimental skin flaps in mast cell-deficient rats.

AIM: The role of mast cells and their principal mediator, histamine, in surgical skin flap survival was investigated using mast cell-deficient (Ws/Ws); their congenic littermates wild-type (+/+), and Wistar rats. METHODS: A standardized dorsal skin flap was raised and sutured back into position, and 6 days later the percentage of flap survival was assessed. Moreover, endogenous histamine concentration in the dorsal skin during the surgical preparation was determined using in vivo microdialysis technique together with high performance liquid chromatography-fluorometry. Accumulation of skin flap myeloperoxidase (MPO) (reflecting leucocyte recruitment) was determined spectrophotometrically. RESULTS: The experimental skin flaps in genetically mast cell-deficient rats exhibited increased tissue survival and showed little accumulation of MPO and rather low and stable level of histamine output in comparison with skin flaps in the wild-type (+/+) littermates or normal Wistar rats. Antihistamine treatment inhibited but did not prevent leucocyte recruitment in the skin flaps post-surgery in +/+ and Wistar rats. CONCLUSION: It is suggested that mast cell derived histamine plays an important role in leucocyte recruitment in skin flaps. However, mast cell-independent factors should be taken into consideration and needs further investigation as even in mast cell-deficient animals there was some accumulation of leucocytes and tissue necrosis in the skin flaps post-surgery.

Heparin protects against skin flap necrosis: relationship to neutrophil recruitment and anti-coagulant activity.

OBJECTIVE AND DESIGN: Heparin has been shown to improve survival of surgical skin flaps. However, it is not known whether the protective effect of heparin is related to its anticoagulative or anti-inflammatory effects. METHODS: Surgical flaps were raised in the dorsal skin of Sprague-Dawley rats. Neutrophil recruitment was determined by measuring the tissue content of myeloperoxidase (MPO) and clotting time was estimated by assessment of activated partial thromboplastin time (APTT) in plasma. RESULTS: Administration of heparin (150 U/kg) significantly increased skin flap survival from 44% in vehicle-treated controls to 91%. This heparin treatment increased APTT by 4.5 fold. However, administration of 150 U/kg of heparin had no effect on skin flap neutrophil recruitment. In contrast, we found that the polysaccharide fucoidan reduced MPO and also improved skin flap survival. CONCLUSIONS: In conclusion, we demonstrate that protective effect of clinically relevant doses of heparin correlates with its ability to prolong clotting time and not to inhibition of neutrophil accumulation in the healing of skin flaps.

Low molecular weight heparin inhibits tumor necrosis factor alpha-induced leukocyte rolling.

OBJECTIVE: Heparins may exert several anti-inflammatory actions. This study investigated the impact of low molecular weight heparin (LMWH) on tumor necrosis factor (TNF)-alpha-provoked leukocyte rolling, adhesion and extravascular accumulation. MATERIALS AND METHODS: Leukocyte-endothelium interactions and tissue accumulation of leukocytes were induced by TNF-alpha in Balb/c mice and analyzed by the use of intravital microscopy in the mouse cremaster muscle microcirculation. RESULTS: We observed that administration of 5000 units/kg of LMWH, but not 50 and 500 units/kg, markedly reduced TNF-alpha-induced leukocyte rolling, adhesion and tissue infiltration. Notably, when LMWH was given after TNF-alpha only leukocyte rolling was reduced and no effect was observed on firm adhesion of leukocytes. CONCLUSIONS: Taken together, these findings indicate that LMWH inhibits TNF-alpha-induced leukocyte accumulation by inhibiting the rolling adhesive interaction which may help explain the anti-inflammatory effects of LMWH.

Role of CD18-dependent neutrophil recruitment in skin and intestinal wound healing.

CD11/CD18 is an important adhesion molecule mediating recruitment of leukocytes, which, in turn, may cause postoperative injury in the skin and gastrointestinal tract. The objective of the present study was to investigate the effects of inhibiting the function of CD18 on surgery-induced dermal and intestinal infiltration of neutrophils and on the healing of surgical skin flaps and colonic anastomosis. A flap in the dorsal skin or an end-to-end colonic anastomosis were created in Sprague-Dawley rats. Skin necrosis and anastomotic breaking strength were analyzed 6 and 3 days after surgery, respectively. Tissue myeloperoxidase (MPO) was used as a marker of neutrophil recruitment. Administration of a monoclonal antibody directed against rat CD18 (WT.3, 2 mg/kg) significantly decreased dermal and anastomotic MPO activity by more than 80%. Passive immunization against CD18 significantly improved flap survival, i.e. the survival was 80% in the anti-CD18 antibody group as compared to 38% in the control group. In contrast, this passive immunization against CD18 had no effect on the reconstitution of the integrity of the colonic anastomosis, i.e. the anastomotic breaking strength was 1.3 +/- 0.1 and 1.3 +/- 0.3 N in the control and anti-CD18 antibody group, respectively. These findings suggest that specific inhibition of CD18 function and reduced neutrophil recruitment may improve the survival of experimental skin flaps and, thus, may represent a potential target for therapeutic intervention. In contrast, we also found that blocking CD18-dependent neutrophil infiltration in the intestine had no effect on breaking strength of colonic anastomosis. Thus, neutrophils may influence the wound-healing process differently in specific organs and this needs to be considered when applying an anti-inflammatory treatment regime in order to improve tissue healing.

Effects of environmental stress on tissue survival and neutrophil recruitment in surgical skin flaps in relation to plasma corticosterone levels in the rat.

OBJECTIVE: Because glucocorticoid treatment can improve the survival of surgical skin flaps, we examined the influence of environmental stress on skin flap survival in the rat. MATERIAL: Female Sprague-Dawley rats. TREATMENT: Dexamethasone (1 mg/kg i.p.). METHODS: A standardized dorsal skin flap was raised and sutured back into position, and six days latter the percentage of flap survival was assessed. Corticosterone in rat plasma was measured using radioimmuno assay, and skin flap myeloperoxidase accumulation (reflecting neutrophil recruitment) was determined spectrophotometrically. RESULTS: Skin flap survival decreased gradually during a 10 day acclimatization period after transportation of the animals from the supplier, and plasma corticosterone levels were increased during the first 5 days of acclimatization compared to day 7 and 10. Dexamethasone treatment of rats accustomed to their new environment for 10 days increased flap survival to a level close to that observed in animals operated at day 1 after arrival. Flap surgery induced pronounced neutrophil recruitment into flap tissue, and this cell accumulation was greatly reduced in both the dexamethasone treated rats and in rats with elevated corticosterone levels. CONCLUSIONS: Skin flap survival in rats exposed to environmental stress may be significantly increased as compared to animals accustomed to their new environment for one week, possibly as a consequence of anti-inflammatory actions exerted by stress-induced elevations in plasma corticosterone. These findings emphasize the importance of strictly controlling environmental stress factors in studies of inflammation and tissue damage after surgical skin trauma.

Effects of local cooling on microvascular hemodynamics and leukocyte adhesion in the striated muscle of hamsters.

OBJECTIVES: Cellular metabolism is dependent on the local temperature in tissues. Induced hypothermia has been shown to be protective in a number of conditions, especially traumatic, ischemic, burn, and neurological injury. However, the protective mechanisms of cold therapy remain controversial and the hemodynamic changes in the microcirculation of striated muscles in response to hypothermia have not been studied in detail previously. METHODS: In this study, we investigated the microvascular response of local cooling and rewarming in the striated muscle of hamsters by use of the dorsal skinfold preparation and in vivo fluorescence microscopy. RESULTS: We found that reduction of the surface temperature to 8 degrees C for 30 minutes caused arteriolar vasoconstriction with a decrease in diameters by 43+/-7% while the venular and capillary diameters remained unchanged. The cooling procedure also markedly reduced the functional capillary density and the blood flow velocity and diameters in all vessel types, i.e., arterioles, venules, and capillaries. Moreover, the percentage of capillaries with no flow increased from 0.4+/-0.5% to 44+/-14% after 10 minutes of cold therapy. However, these hemodynamic changes induced by local hypothermia were completely reversed to the precooling values after termination of cooling and 30 min of rewarming. Strikingly, we found no increase in the number of adherent leukocytes and vascular permeability after the cooling and rewarming period, while, in contrast, additional experiments with warm ischemia (30 minutes) and reperfusion (30 minutes), i.e., reduced microvascular perfusion and reperfusion at normothermia, caused a sustained decrease in local perfusion and a nine-fold increase in venular leukocyte adhesion. CONCLUSIONS: Taken together, our functional data demonstrate that hypothermia markedly reduces microvascular perfusion, which is completely restored upon rewarming. The reduced microvascular perfusion during hypothermia did not provoke an inflammatory response, whereas leukocyte recruitment was prominent after reduced perfusion at normothermia, indicating that transient hypothermia has no adverse effects on microvascular parameters in the striated muscle in vivo.

Effects of calcitonin gene-related peptide on tissue survival, blood flow and neutrophil recruitment in experimental skin flaps.

Local administration of calcitonin gene-related peptide (CGRP) has been shown to improve tissue survival in surgical skin flaps. Moreover, topical CGRP has been demonstrated to exert anti-inflammatory effects in different animal models of skin inflammation. The aim of the present study was to establish whether systemic treatment with low doses of CGRP may improve survival and reduce neutrophil accumulation in surgical skin flaps. Using a well-established dorsal skin-flap model in the rat, we found that intraperitoneal (i.p.) pretreatment with low doses of CGRP dose-dependently increased flap survival. Thus, in untreated animals flap survival at day 7 after surgery was 42%, as compared to 44%, 60%, 69% and 73% survival after a single preoperative i.p. injection of 10(-15), 10(-12), 10(-9) and 10(-6) mol CGRP, respectively (P < 0.05 versus control for the three highest doses). The three effective doses had no detectable effects on either flap blood flow (laser Doppler) or mean arterial blood pressure. On the other hand, 5 x 10(-9) mol CGRP i.p. significantly reduced the marked surgery-induced accumulation of flap myeloperoxidase (a marker for neutrophil recruitment) without affecting the circulating neutrophil count. Taken together, our findings demonstrate that low systemic doses of CGRP can cause a major improvement in skin-flap survival in the rat, possibly via inhibition of surgically induced neutrophil recruitment.