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The aim of this study was to investigate bacterial translocation and its possible role in the development of post-resuscitation inflammatory response following Cardio-Pulmonary Resuscitation (CPR) after cardiac arrest.Munich female swine were employed for a model of cardiac arrest via application of electrical current. After 7 min, CPR was initiated, and animals were either successfully return to spontaneous circulation (ROSC) within 40 min or not (no-ROSC). At the end of experimental period and prior to sacrifice, samples from the intestine, mesenteric lymph nodes (MLN), liver and portal vein blood were obtained. Evaluation of inflammation and gut permeability was performed; MLN, liver and portal vein samples were analyzed for 16 s rRNA detection and cytokine mRNA expression.A decreased expression of the tight junction protein Occludin, with higher levels of inflammation, greater epithelial disintegration, ulceration, loss of crypts and villi height were found in the intestines of the ROSC swine in comparison to no-ROSC. The macrophage surface antigen CD-14 staining was relatively more intense in the ROSC than in no-ROSC. Higher levels of TNF-α mRNA expression were present in the liver of the ROSC group. Finally, despite the inflammatory response and the gut mucosal alterations in ROSC group, no bacterial translocation was detected in liver, MLN and portal vein.We show that resuscitation from cardiac arrest induces inflammatory response and intestinal permeability in swine 4h after resuscitation, but not a bacterial translocation. Bacterial translocation is not an early phase phenomenon but probably part of the pathophysiologic sequelae.

The aim of this study was to examine the role of growth factors associated with angiogenesis and oxidative stress in the pathogenesis of spontaneous miscarriage.We performed a comparative mRNA expression analysis of VEGF, PlGF, Flt-1, Angiogenin and Endoglin using Real-Time PCR, in the placenta and decidua collected from 12 patients presenting with spontaneous abortion and from 14 women undergoing induced abortion, during the first and second trimester of pregnancy.The mRNA expression of Flt-1 was significantly upregulated in the placenta of spontaneous abortions (5.17-fold, IQR: 2.72-9.11,In this study, we observed higher levels of sFlt-1 mRNA expression in the placenta of spontaneous abortions, while expression of other growth factors in placenta and decidua remained constant. This suggests that an imbalance of sFlt-1 expression in the placenta might contribute to the pathogenesis of spontaneous abortion, probably

Miscarriage is one of the most common complications of pregnancy. Although chromosomal abnormalities of the embryo is a well-known cause of miscarriage, a lot of cases remain unexplained, with immunologic and vascular growth alterations being considered as probable causes. Chemokines are produced by a variety of cells and exhibit several functions including both pro and anti-angiogenic properties. In this study, we investigated the role of the angiogenic and angiostatic chemokines in placenta and decidua tissues from spontaneous and induced abortions.Total RNA was extracted from the placenta and decidua tissues, which was then purified and converted into cDNA. Real-time PCR was then performed for the expression of the angiogenic CCL2, CCL5, CCL20, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8 and CXCL4, and the angiostatic CXCL9, CXCL10, CXCL11, CXCL12 and CXCL14 and results were then statistically analyzed.Regarding the placenta, CXCL7 (2.29-fold, 2.16-2.38, p 0.05), CXCL4 (1.01-fold, 0.74-4.447, p 0.05), CXCL9 (0.87-fold, 0.43-1.34, p 0.05) and CXCL11 (0.31-fold, 0.22-0.45, p 0.05) were altered in spontaneous abortions. CCL2, CCL5, CXCL2-3, CXCL8, CXCL10, CXCL12 and CXCL14 were not statistically significant altered. Regarding the decidua, CXCL7 (7.13-fold, 6.32-7.54, p 0.01), CXCL8 (11.02-fold, 8.58-13.45, p 0.05), CCL20 (1.21-fold, 0.29-1.89, p 0.05) and CXCL9 (5.49-fold, 3.67-6.39, p 0.05) were overexpressed in spontaneous abortions. CXCL2-4, CCL2, CCL5, CXCL10-12 and CXCL14 did not show any differences. The expression of the chemokines CXCL1, CXCL5-6 was absent in either tissue or group.Our results show that the overexpression of angiostatic and diminished expression of angiogenic chemokines takes place in the placenta and decidua of spontaneous abortions, suggesting that dysregulation of angiogenesis could be a contributive factor to the pathogenesis of miscarriage.

Polymethylmethacrylate (PMMA) represents the current gold standard as an antibiotic delivery carrier in orthopaedic surgery. Despite the accepted use of local antibiotic carriers, there aren't any conclusive data comparing PMMA to Bone Graft Substitutes. The aim of this in vitro study was to compare the elution profile of gentamicin from various preparations of PMMA cements and Herafill beads. All cements had high initial elution during the first hour which then slowly decreased, Herafill beads on the other hand showed its higher elution around the eighth hour. Herafill, in general, presented the highest elution of gentamicin regardless of its input amount.

Idiopathic pulmonary fibrosis (IPF) is caused by progressive lung tissue impairment due to extended chronic fibrosis, and it has no known effective treatment. The use of conditioned media (CM) from an immortalized human adipose mesenchymal stem cell line could be a promising therapeutic strategy, as it can reduce both fibrotic and inflammatory responses. We aimed to investigate the anti-inflammatory and anti-fibrotic effect of CM on human pulmonary subepithelial myofibroblasts (hPSM) and on A549 pulmonary epithelial cells, treated with pro-inflammatory or pro-fibrotic mediators. CM inhibited the proinflammatory cytokine-induced mRNA and protein production of various chemokines in both hPSMs and A549 cells. It also downregulated the mRNA expression of IL-1α, but upregulated IL-1β and IL-6 mRNA production in both cell types. CM downregulated the pro-fibrotic-induced mRNA expression of collagen Type III and the migration rate of hPSMs, but upregulated fibronectin mRNA production and the total protein collagen secretion. CM’s direct effect on the chemotaxis and cell recruitment of immune-associated cells, and its indirect effect on fibrosis through the significant decrease in the migration capacity of hPSMs, makes it a plausible candidate for further development towards a therapeutic treatment for IPF.

The probiotics Lactiplantibacillus plantarum UBLP-40, Lactobacillus rhamnosus UBLR-58 and Bifidobacterium longum UBBL-64 seem to promote wound healing when applied topically. Our aim was to investigate their effect on the mRNA expression of pro-inflammatory, healing and angiogenetic factors during the healing process of a standardized excisional wound model in rats. Rats subjected to six dorsal skin wounds were allocated to Control; L. plantarum; combined formula of L. rhamnosus plus B. longum; L. rhamnosus; and B. longum treatments, applied every two days, along with tissue collection. The pro-inflammatory, wound-healing, and angiogenetic factors of mRNA expression were assessed by qRT-PCR. We found that L. plantarum exerts a strong anti-inflammatory effect in relation to L. rhamnosus–B. longum, given alone or in combination; the combined regime of L. rhamnosus–B. longum, works better, greatly promoting the expression of healing and angiogenic factors than L. plantarum. When separately tested, L. rhamnosus was found to work better than B. longum in promoting the expression of healing factors, while B. longum seems stronger than L. rhamnosus in the expression of angiogenic factors. We, therefore, suggest that an ideal probiotic treatment should definitively contain more than one probiotic strain to speed up all three healing phases.

Background Self-transfusion has been proven as an effective management of blood loss after total knee arthroplasty (TKA). Considering that the high local concentration of antibiotic from bone cement is delivered intravenously through the self-transfusion process, systematic toxicity has never been evaluated. In addition, the effectiveness of self-transfusion with the routine concomitant use of other modern blood-salvage strategies, like tranexamic acid, should also be assessed. Therefore, we performed a randomised study to assess: 1) the safety of self-transfusion in TKA by comparing the gentamicin concentrations resulting from the use or not of autologous blood transfusion; 2) the efficacy of self-transfusion in TKA, with the concomitant administration of tranexamic acid. Hypothesis Self-transfusion in TKA elevates the serum gentamicin concentration and the potential risk of nephrotoxicity. Methods The serum concentration of aminoglycosides was measured in two groups of 20 patients each, after TKA, according to the use of self-transfusion. Hemoglobin, renal function and calculated blood loss were compared at several points in time between groups. Results The only time where there was a statistically significant difference in serum gentamicin, was at 48 h postoperatively between groups [0.3 ug/mL ± 0.21, range: 0.15 to 0.72 vs. 0.14ug/mL ± 0.1, range: 0 to 0.35 (p = 0.02)]. There were no significant differences in total blood loss [1341 mL ± 501, range: 830 to 2230 vs. 1263 mL ± 459 range: 840 to 2480 (p = 0.67)] and need of allogeneic blood transfusion [3 units vs. 2 units] between groups. Conclusion The use of autologous blood transfusion was found to be safe, in terms of nephrotoxicity of aminoglycosides after TKA, but it seemed to be ineffective as a blood salvage strategy, when used concomitantly with the administration of tranexamic acid. Level of evidence II; low-powered randomised study. ClinicalTrials.gov registration number NCT04505748 .

Background Colonic subepithelial myofibroblasts (cSEMFs) are mesenchymal cells with a pivotal role in the pathophysiology of Crohn's disease (CD) fibrosis. Here, we demonstrate for the first time a complete expression mapping of cytokine receptors, implicated in inflammatory bowel diseases, in primary human cSEMFs and how pro-inflammatory cytokines regulate this expression. Furthermore, we show the effect of Th1-, Th2-, Th17- and Treg-related cytokines on a fibrosis-related phenotype of cSEMFs. Methods Colonic subepithelial myofibroblasts were isolated from healthy individuals' colonic biopsies. Interleukin (IL)-1α- and/or tumor necrosis factor (TNF)-α-induced mRNA and protein expression of cytokine receptors was assayed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunofluorescence, respectively. Th-related cytokine effects on mRNA and protein profibrotic factor expression were analyzed by qRT-PCR and/or colorimetric assays and on the wound-healing capacity of cSEMFs by scratch test. Results In cSEMFs, we observed basal cytokine receptor expression, which was modified by IL-1α and TNF-α. Th1-related cytokines upregulated tissue factor (TF), collagen, fibronectin and matrix metalloproteinase (MMP)-1 and downregulated α-smooth muscle actin (α-SMA), MMP-9, and wound healing rate. Th2-related cytokines upregulated collagen, TF, α-SMA, MMP-1, and wound healing rate and downregulated fibronectin and MMP-9. IL-17 and IL-23 upregulated fibronectin, and IL-22 downregulated TF. IL-17 and IL-22 decreased wound healing rate. Similar to TGF-β, IL-23 upregulated MMP-1, tissue inhibitor of metalloproteinases-1, collagen expression, and wound healing rates. Conclusions Our results suggest that cSEMFs have a central role in inflammation and fibrosis, as they express a great variety of Th-related cytokine receptors, making them responsive to pro-inflammatory cytokines, abundant in the inflamed mucosa of CD patients.

Background Oncostatin M (OSM) may play an important role in Inflammatory Bowel Disease (IBD) pathogenesis. Specifically, both OSM and its receptor are upregulated in inflamed colonic regions of IBD patients, and high OSM expression has been associated with failure to respond to anti-TNF therapy. Our aim was to investigate the effect of OSM in fibrotic factors and chemokine expression on primary colonic subepithelial myofibroblasts (SEMFs) from healthy individuals (HI). Methods Primary SEMFs were isolated from endoscopically-obtained colonic biopsies from HI. SEMFs were stimulated with 1, 10, or 100ng/ml OSM for 6 hours, with or without pre-stimulation with 5ng/ml IL-1α plus 50ng/ml TNF-α for 24h. Total RNA was collected and mRNA transcripts for collagen type I, type III, fibronectin, and the chemokines CCL2, CXCL9, CXCL10 and CXCL11 were measured by reverse transcription quantitative PCR. Results Unstimulated SEMFs had a basal expression of collagen type I, III, fibronectin, CCL2, CXCL9, CXCL10 and CXCL11. OSM stimulation augmented chemokine mRNA expression in a dose-dependent manner (Table 1) but had no effect on fibrotic factors expression. Pre-stimulation of myofibroblasts with TNF-α and IL-1α resulted in augmented expression of collagens I and III and fibronectin, in addition to further increases in chemokine expression in response to subsequent stimulation by OSM (Table 2). Conclusion Our results show that stimulation with OSM induces fibrotic and chemokine responses by SEMFs. Our findings further support the hypothesis that SEMFs may play a key role in regulating chronic intestinal inflammation and response to biological therapy.

Background Self-transfusion has been proven as an effective management of blood loss after total knee arthroplasty (TKA). Considering that the high local concentration of antibiotic from bone cement is delivered intravenously through the self-transfusion process, systematic toxicity has never been evaluated. In addition, the effectiveness of self-transfusion with the routine concomitant use of other modern blood-salvage strategies, like tranexamic acid, should also be assessed. Therefore we performed a randomized study to assess: (1) the safety of self-transfusion in TKA by comparing the gentamicin concentrations resulting from the use or not of autologous blood transfusion; (2) the efficacy of self-transfusion in TKA, with the concomitant administration of tranexamic acid. Hypothesis Self-transfusion in TKA elevates the serum gentamicin concentration and the potential risk of nephrotoxicity. Methods The serum concentration of aminoglycosides was measured in two groups of 20 patients each, after TKA, according to the use of self-transfusion. Hemoglobin, renal function and calculated blood loss were compared at several points in time between groups. Results The only time where there was a statistically significant difference in serum gentamicin, was at 48 h postoperatively between groups [0.3 ug/ml ± 0.21, range: 0.15 to 0.72 vs. 0.14ug/ml ± 0.1, range: 0 to 0.35 (p = 0.02)]. There were no significant differences in total blood loss [1341 ml ± 501, range: 830 to 2230 vs. 1263 ml ± 459 range: 840 to 2480 (p = 0.67)] and need of allogeneic blood transfusion [3 units vs. 2 units] between groups. Conclusion The use of autologous blood transfusion was found to be safe, in terms of nephrotoxicity of aminoglycosides after TKA, but it seemed to be ineffective as a blood salvage strategy, when used concomitantly with the administration of tranexamic acid. Level of Evidence II; low powered randomized study. ClinicalTrials.gov registration number NCT04505748 .

Background Various probiotic strains play a positive role in Inflammatory Bowel Disease and might support mucosal healing. Colonic Subepithelial Myofibroblasts (SEMFs) have a pivotal role in mucosal healing and fibrosis, by migrating to the trauma region and secreting collagen. Our study aimed to investigate whether different strains of probiotics can affect the migration capacity and the fibrotic phenotype of SEMFs isolated from healthy individuals. Methods The probiotic mix of Lactobacillus acidophilus, Lactobacillus plantarum, Bifidobacterium lactis and Saccharomyces boulardii was reconstituted in SEMFs culture medium, they were identified by Gram staining and their viability was assessed by Trypan Blue staining. Primary SEMFs were isolated from colonic biopsies from healthy individuals, and stimulated with 102, 104 or 108 cfu/ml of the mix for 6h or 48h; in 6h total RNA was collected and mRNA expression of collagen type I, III, fibronectin, tissue factor (TF) and α-SMA was measured by quantitative PCR, and in 48h their effect on migration rate through the Wound Healing Assay and collagen secretion via Sircol was assessed. Results Trypan Blue confirmed that the majority of the probiotic mix was alive, and all three bacterial strains and the yeast were stained Gram positive. Stimulation of SEMFs with the probiotic mix resulted in different effects depending on its concentration; 102 cfu/ml downregulated protein collagen secretion (3.4μg/ml, IQR: 2.7–5.0, p Conclusion This study provides evidence that the probiotic strains L. plantarum, L. acidophillus, B. lactis and S. boulardii remain viable in SEMFs’ culture medium and have the ability to interact with and alter the behavior of SEMFs. Moreover, the effect of the probiotic mix on SEMFs fibrotic phenotype is dependent of the concentration of the probiotics used. Further preclinical research is needed in order to identify the effect of each strain, as well as the suitable dose and combinations in various pathological conditions.

Background Organoids are self-renewing, 3D structures, consisting of different cell types, with histology and physiology features very close to the physiology of the studied organ. Specifically, human Intestinal Organoids (HIOs) develop epithelial crypts consisting of all subtypes of intestinal epithelial cells which are surrounded by mesenchymal cells. Our aim was to develop 3D HIOs from human embryonic stem cells (hESCs) and examine the expression of fibrotic and mesenchymal factors during their maturation process. Additionally, we investigated the effect of the pro-inflammatory cytokines, IL-1α and TNF-α on the expression of fibrotic and inflammatory mediators in HIOs. Methods The human ESC line (H1) was cultured and then differentiated towards HIOs using commercially available kit. HIOs were characterized by immunofluorescence in all differentiation stages. In order to examine their maturation process, we compared the mRNA expression of fibrotic and mesenchymal markers from passages 1–10. In order to examine their functionality, HIOs from different passages were stimulated with 5ng/ml IL-1α and 50ng/ml TNF-α for 12 hours, total RNA was collected and the fibrotic and inflammatory mRNA expression was examined. The mRNA transcripts of CD90, collagen type I, III, fibronectin, CXCL8, CXCL10 and CXCL11 were measured by reverse transcription quantitative PCR. Results HIOs were successfully developed as they were stained positive for all tested markers throughout their developmental process. Regarding their maturation process, we observed high expression of CD90, collagen type I, type III and fibronectin that was gradually decreased during passages. As for the fibrotic and inflammatory responses from HIOs, we found that the IL-1α and TNF-α stimulation resulted in statistically significant upregulation of the fibrotic factors, fibronectin, collagen type I and type III in culture passages 2 and 4, but had no effect in culture passages 8 and 10. Similarly, IL-1α and TNF-α stimulation led to the statistically significant induction of the inflammatory chemokines CXCL8, CXCL10 and CXCL11 in culture passages 2 and 4, while no effect was observed in culture passages 8 and 10. Conclusion Our findings indicate that HIOs contain a functional mesenchymal component that is gradually diminished during passages. Inflammatory and fibrotic responses of HIOs seem to depend on the fitness of their mesenchyme. IBD studies using HIOs as in vitro models should be performed on early passages, when HIO’s mesenchymal component is still functional.

The endocannabinoid system has been shown to be a putative therapeutic target for retinal disease. Here, we aimed to investigate the ability of the endocannabinoid 2-arachidonoylglycerol (2-AG) and novel inhibitors of its metabolic enzymes, α/β-hydrolase domain-containing 6 (ABHD6) and monoacylglycerol lipase (MAGL), a) to protect the retina against excitotoxicity and b) the mechanisms involved in the neuroprotection. Sprague-Dawley rats, wild type and Akt2-/- C57BL/6 mice were intravitreally administered with phosphate-buffered saline or (RS)-α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid hydrobromide (AMPA). 2-AG was intravitreally co-administered with AMPA in the absence and presence of AM251 or AM630 (cannabinoid 1 and 2 receptor antagonists, respectively) or Wortmannin [Phosphoinositide 3-Kinase (PI3K)/Akt inhibitor]. Inhibitors of ABHD6 and dual ABHD6/MAGL (AM12100 and AM11920, respectively) were co-administered with AMPA intravitreally in rats. Immunohistochemistry was performed using antibodies raised against retinal neuronal markers (bNOS), microglia (Iba1) and macroglia (GFAP). TUNEL assay and real-time PCR were also employed. The CB2 receptor was expressed in rat retina (approx. 62% of CB1 expression). 2-AG attenuated the AMPA-induced increase in TUNEL+ cells. 2-AG activation of both CB1 and CB2 receptors and the PI3K/Akt downstream signaling pathway, as substantiated by the use of Akt2-/- mice, afforded neuroprotection against AMPA excitotoxicity. AM12100 and AM11920 attenuated the AMPA-induced glia activation and produced a dose-dependent partial neuroprotection, with the dual inhibitor AM11920 being more efficacious. These results show that 2-AG has the pharmacological profile of a putative therapeutic for retinal diseases characterized by neurodegeneration and neuroinflammation, when administered exogenously or by the inhibition of its metabolic enzymes.

Background Septic shock has been found to disrupt circadian rhythms. Moreover, timing of onset has been associated with different circadian profiles in experimental studies. Results In this prospective study, we enrolled 26 patients divided into two groups: Group A (N = 15) included subjects who had septic shock at the time of ICU admission and Group B (N = 11) included patients who developed septic shock during ICU admission. 6-Sulfatoxymelatonin (aMT6s) and cortisol levels were measured in urine samples every 4 h over a 24-h period. Two sets of samples were taken from Group A (entry/septic shock and exit) and three sets from Group B (entry, septic shock and exit). Mean, amplitude that is the difference between peak and mean values, as well as peak time, were estimated for both aMT6s and cortisol. In Group A, amplitude of aMT6s upon entry (septic shock) was reduced in relation to exit (437.2 ± 309.2 vs. 674.1 ± 657.6 ng/4 h, p

Microflora dysbiosis is implicated in the pathophysiology of Crohn's disease. This work analyzes differences in microbial communities and relevant metabolic pathways among the nonstricturing nonpenetrating (B1), stricturing (B2), and penetrating (B3) subphenotypes of Crohn's disease vs healthy controls. We conducted a bioinformatics analysis using the QIIME pipeline and the Calypso, linear discriminant analysis effect size, Phylogenetic Investigation of Communities by Reconstruction of Unobserved States, and STAMP tools on publicly available 16S bacterial rRNA sequencing data from terminal ileum mucosal biopsies of healthy controls and the 3 subphenotypes of Crohn's disease. We analyzed differences in microbial diversity and taxonomy, inferred active metabolic pathways via relevant genes' abundance, and detected bacterial families that could serve as biomarkers. Microbiota α-diversity was decreased within all 3 Crohn's disease subphenotypes vs control samples, with more significant reductions in B2 and B3 compared with B1. β-diversity analysis identified similar microbial patterns in B2 and B3 samples, different from those of B1 and from those of healthy controls. Abundance analysis of microbial families in cohorts, beyond altered abundances compared with healthy controls, highlighted significant differences between the B2 and B3 subphenotypes and the B1 subphenotype. A similar pattern was observed in the inference of microbial metabolomics: the B2 and B3 cohorts had different predicted metabolotypes from the B1 cohort, in addition to differences observed in Crohn's disease vs healthy controls. Our findings indicate distinct microbiome signatures in complicated Crohn's disease subphenotypes and provide the basis for further investigation into the role of gut microflora in the natural course of Crohn's disease.

Peritoneal adhesions, organized as fibrous bands after abdominal surgery, are related with considerable morbidity and repeated hospitalization. Phospholipids, natural constituents of the peritoneal fluid, seem to display excellent antiadhesive properties. The aim of this study was to investigate whether intraperitoneal application of phospholipids is capable of reducing postoperative adhesions and the possible underlying mechanisms.Twenty male Wistar rats were subjected to a midline laparotomy and a standard peritoneal and cecum abrasion trauma. Before laparotomy closure, a bolus of 3 mL of phospholipids (12 mg/mL) or NaCl (placebo) was given intraperitoneally. Seven days later, the quality and the quantity of adhesions, as well as serum proinflammatory and/or profibrotic mediators, were blindly assessed. Human colonic subepithelial myofibroblasts were isolated from normal controls and cultured with transforming growth factor-β1 (TGFβ1, 5 ng/mL) in the presence of phospholipids (30-300 μg/mL). Collagen production in culture supernatants and migratory activity of myofibroblasts were also assessed.Phospholipids reduced intra-abdominal adhesions (P0.001), with respect to their intensity and area, and serum levels of cytokines (interleukin 1β, interleukin 6, platelet-derived growth factor-1, and TGFβ1) compared with placebo-treated rats. Stimulation of myofibroblasts with TGFβ1 significantly increased (P0.001) the basic collagen production. The presence of phospholipids significantly reduced (P0.001) both the TGFβ1 induced and the basic collagen production. Using a wound healing assay, phospholipids were found to reduce the basic and the TGFβ1-induced migration of myofibroblasts in a concentration-dependent manner.Intraperitoneal phospholipids might be involved in the prevention of postoperative adhesions formation via the reduction of proinflammatory and/or profibrotic mediators and by inhibiting fibrogenic properties of mesenchymal cells.

Objective: To measure plasma glutamine (GLN) levels in systemic and portal circulation after combined enteral and parenteral administration in early endotoxemic swine. We hypothesized that this combination will be more efficient than intravenous administration alone in restoring plasma levels during the course of endotoxemia. Materials and Methods: Endotoxemia was induced with Escherichia coli O111:B4 lipopolysaccharide (LPS) (250 μg/kg body weight) in 16 anes­thetized, fasted swine and maintained by constant infusion (2 μg/kg/h) over 180 min. Another 16 swine served as controls. After infusion with LPS or placebo, GLN was administered intravenously, enterally or in combination (0.5 g/kg i.v. plus 0.5 g/kg enterally) over 30 min. At 0, 15, 30, 45, 60, 120 and 180 min, blood was drawn from the systemic and portal circulation for colorimetric assessment of GLN. Results: In healthy, placebo-alone swine, GLN levels remained stable throughout the study. Intravenous and combined infusion increased systemic levels (p = 0.001), but after enteral administration alone, a smaller effect was observed (p = 0.026). Portal levels were increased after combined, enteral and intravenous administration (p = 0.001). In endotoxemia, systemic and portal levels decreased significantly. Intravenous and, to a greater extent, combined administration increased systemic levels (p = 0.001), while enteral administration only had a small effect (p = 0.001). In the portal vein, intravenous and combined treatment increased plasma levels (p = 0.001), whereas enteral supplementation alone had again a small, yet significant effect (p = 0.001). Conclusions: The findings indicate that combined GLN supplementation is superior to intravenous treatment alone, in terms of enhanced availability in systemic and portal circulations. Thus, combined treatment at the onset of endotoxemia is a beneficial practice, ensuring adequate GLN to compensate for the resulting intracellular shortage.

Background and Objectives: Surgery provokes inflammatory and immune responses, so efforts have been made to reduce host response by using less invasive techniques. The purpose of this experimental study was to investigate the surgical stress induced by skin incision and the role of liver response in this process. Methods: Seventy male anesthetized Wistar rats were subjected to a midline incision confined strictly to the skin (dermis) of either 1 cm long (n = 20), 10 cm long (n = 20), or no incision (n = 20) or served as controls (n = 10). Skin trauma was left open for a 20-minutes period, and then was meticulously sutured. At 3 and 24 hours later, laparotomy was performed on half the rats of each group, for blood and liver sampling. In serum and liver homogenates, cytokine-induced neutrophil chemoattractant (CINC)1/interleukin (IL)-8 and tumor necrosis factor (TNF)-α levels were measured with enzyme-linked immunosorbent assays and nitric oxide (NO) using a Griess reaction. Results: Skin trauma was found to significantly (P < .01) increase all inflammatory mediators tested (CINC1/IL-8, TNF-α, NO) in serum of operated rats versus controls, the increase being proportionally dependent on the length of skin incision. In liver homogenates, CINC1/IL-8 was significantly (P < .01) increased in operated animals versus controls, similarly to serum levels. In contrast, liver TNF-α levels were inversely related to serum levels, and a significant (P < .01) decrease in TNF-α was observed in liver homogenates of operated animals compared with the controls, indicating that the increased TNF-α in blood reflects liver TNF-α secretion. Conclusion: Our findings suggest that inflammatory and immune reactions induced by skin-only surgical trauma are closely correlated to the length of skin incision.

Background Oncostatin M (OSM), a cytokine of the IL-6 family, has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Specifically, OSM and its receptor, OSMR, are elevated in inflamed colonic regions of IBD patients; in addition, high OSM expression at baseline has been associated with failure to respond to anti-TNF. OSMR expression localised in stromal cells of the intestinal lamina propria. Our aim was to investigate the expression of OSM and its receptors subunits, OSMR, LIFR and gp-130 in primary subepithelial myofibroblasts (SEMFs) and test whether this expression is regulated by the innate cytokines, IL-1α and TNF-α. Methods Primary SEMFs were isolated from endoscopically-obtained colonic biopsies from healthy controls, set to culture and stimulated with 5ng/ml IL-1α and/or 50ng/ml TNF-α for 6 h. Total RNA was extracted and the mRNA transcripts for OSM, OSMR, LIFR and gp-130 were measured by reverse transcription-quantitative (RT-q) PCR. Results Unstimulated SEMFs had a basal expression of both OSM and its receptors subunits. IL-1α or TNF-α stimulations did not alter LIFR expression, but they induced a statistically significant upregulation of OSM, OSMR and gp-130. Specifically, the expression of OSM in SEMFs was significantly upregulated after stimulation with IL-1α alone or in combination with TNF-α (IL-1α: 11.48-fold increase, 8.29–26.05; IL-1α+TNF-α: 13.42-fold, 11.32–17.16; p < 0.0001). Regarding the OSMR and gp-130 subunits [which form the type II receptor of OSM], OSMR mRNA was induced by TNF-α alone or in combination with IL-1α (TNF-α: 1.75-fold, 1.27–2.02, p < 0.01; IL-1α+TNF-α: 2.06-fold, 1.83–2.34, p < 0.0001), whereas gp-130 mRNA expression was increased under all stimulatory conditions (IL-1α: 1.94-fold, 1.46–2.07; TNF-α: 1.81-fold, 1.46–2.25; IL-1α+TNF-α: 1.59-fold, 1.31–2.51; p < 0.01 for all comparisons). Conclusion Our results show that OSM and OSMR are expressed on primary human SEMFs and that they are upregulated under stimulation with innate pro-inflammatory cytokines. These data further support a potential role of this system of inflammatory mediators in the pathogenesis of intestinal inflammation.

Background Crohn’s disease (CD) and ulcerative colitis (UC), the two main entities of inflammatory bowel disease (IBD), are characterised by chronic and relapsing/remitting inflammation of the gastrointestinal tract, and occasionally ultimately result in debilitating intestinal fibrosis. Apart from their key role in fibrosis, there is evidence that subepithelial myofibroblasts (SEMFs) participate in the IBD inflammatory cascade, as they express various pro-inflammatory cytokine receptors. We examined the effect of pro-inflammatory IL-17—the hallmark cytokine of T-lymphocytes differentiated to Th17—on the expression of lymphocyte-chemotactic chemokines in SEMFs. Methods SEMFs were isolated from endoscopically obtained colonic biopsies from healthy controls, set to culture and stimulated with 100 ng/ml IL-17 for 6 h. Total RNA was extracted and mRNA expression of CCL5, CXCL1 and CXCL11 was assessed with reverse transcription quantitative (RT-q) PCR. Changes in cytokine mRNA are provided as medians (IQR). Results Untreated SEMFs had a basal expression of chemokines of the CCL and CXCL family groups. So far, our study has shown that the IL-17 stimulation leads to a statistically significant upregulation of CCL and CXCL chemokines in SEMFs (p < 0.001). In detail, CCL5 was upregulated 5.7-fold (4.9–7.5), CXCL1 72.9-fold (63.1–90.7) and CXCL11 25.4-fold (17.3–37.3). Conclusion IL-17 induced the expression of chemotactic factors in SEMFs. Our results further support a potential role of SEMFs in the shaping of intestinal mucosal immunity by serving as immunological intermediates that respond to cytokines of adaptive immunity and amplify the recruitment of immune cells via chemokine production.

Th17 cells play a crucial role in neutrophilic inflammation and tissue injury in non-cystic fibrosis (non-CF) bronchiectasis. Clarithromycin demonstrates anti-inflammatory and immunomodulatory properties but the effect of long-term clarithromycin prophylaxis on the Th17 response in non-CF bronchiectasis is still unexplored. Th17 response was studied in 22 patients with stable non-CF bronchiectasis receiving daily 500-mg clarithromycin for 12 weeks. We analysed IL-17 concentrations in exhaled breath condensate (EBC) and peripheral blood Th17 cells, whereas functional parameters and clinical data were recorded in parallel. Both, post-treatment absolute counts of CD4+IL17+ cells in peripheral blood and IL-17 levels in EBC decreased significantly (post-treatment CD4+IL17+ mean 2.418 ± 0.414 cells/μl versus pre-treatment 3.202 ± 0.507 cells/μl, p = 0.036 and post-treatment IL-17 mean levels 7.16 ± 0.47 pg/ml versus pre-treatment 9.32 ± 0.47 pg/ml, p

The diagnosis of X-linked agammaglobulinemia (XLA) is not always clearcut. Not all XLA conform to the classic phenotype and less than 50% of affected boys have a family history of immunodeficiency. Mutations in the gene for Bruton’s tyrosine kinase (BTK) are responsible for the majority of agammaglobulinemia cases. However, a certain proportion of patients may have mutations involving other genes, although they show with an XLA phenotype. We performed BTK gene mutation analysis in 37 males with presumed XLA and analyzed the pattern of X-chromosome inactivation (XCI) in 31 mothers to evaluate the relevance of these approaches to diagnosis and genetic counseling. Twenty affected males with a sporadic occurrence and 17 familial cases belonging to nine families were enrolled within the framework of the Italian Multicenter Clinical Study on XLA. We used non-isotopic RNase cleavage assay (NIRCA), followed by cDNA sequence determination to screen for BTK mutations and X-chromosome inactivation analysis for carrier detection. Using the cDNA-based approach, the identification of BTK gene abnormalities confirmed the clinical diagnosis of XLA in 31 of 37 affected infants. Missense was the most frequent mutational event and the kinase domain was mostly involved. In addition, nine novel mutations were identified. In sporadic cases, BTK gene abnormalities were identified in 9 out of 10 patients whose mothers had a nonrandom pattern of XCI and in 5 out of 6 patients whose mother had a random pattern of XCI. With the exception of one family, all patients with a familial occurrence and born to mothers with a nonrandom pattern of XCI had mutations of the BTK gene. Our findings indicate that in sporadic cases BTK gene sequencing is the only reliable tool for a definitive diagnosis of XLA and support XCI as the first diagnostic tool in the mothers of affected males in multiple generations. Furthermore, our molecular analysis confirms that 10–20% of BTK-unaltered patients have disorders caused by defects in other genes.

Intestinal subepithelial myofibroblasts (SEMFs) exert a profibrotic role in Crohn's disease (CD). Tumor necrosis factor-like cytokine 1A (TL1A) and its receptors, death-domain receptor 3 (DR3) and decoy receptor 3 (DcR3), are mucosal factors with significant involvement in experimental inflammation and CD. We aimed to determine the regulation of expression of this system of proteins in SEMFs and intestinal epithelial cells. The relative amount of mRNA transcripts for TL1A, DR3, and DcR3 was measured by real-time reverse transcription polymerase chain reaction in cultured primary SEMFs, colonic myofibroblast cell line 18CO, and epithelial cell line HT29. Protein expression was determined by immunofluorescence. The effect of various proinflammatory stimuli in mRNA and protein expression was studied. TL1A mRNA and protein expression in primary SEMFs (and 18CO cells) was significantly upregulated after stimulation with interleukin 1-alpha and/or tumor necrosis factor alpha (TNF-α) (32- to 44-fold increase, P 0.05 vs unstimulated). Following stimulation with interleukin 1-alpha + TNF-α + IFN-γ, HT-29 cells highly expressed DR3 (4.1-fold over unstimulated, P = 0.008) and DcR3 (56-fold, P = 0.009) and secreted soluble factors that led to induction of TL1A mRNA in primary SEMFs (28-fold, P = 0.008). Activated epithelial cells significantly upregulated IL-8 expression in response to stimulation with recombinant TL1A. Supernatants from mucosal cultures of patients with CD were able to stimulate the expression of TL1A in cultured primary SEMFs, in comparison to supernatants from healthy controls (3.8-fold increase, P 0.05) or culture media alone (P 0.05). In conclusion, we found that proinflammatory cytokines are important regulators of the expression of TL1A in SEMFs and of its receptors in intestinal epithelial cells. Our results raise the possibility for involvement of TL1A/DR3/DR3-mediated mechanisms in epithelial-mesenchymal interactions and the development of inflammation-induced intestinal fibrosis in CD.

Glutamine (GLN) is recommended in the critically ill for i.v. administration but enteral use is not quite clarified. We decided to measure GLN plasma levels in healthy and septic swine after GLN given by i.v., enteral or both routes, over a 3-hour period.

Chronic myeloid leukaemia (CML) is a malignant clonal disorder of the haematopoietic stem cell. Treatment of CML patients with interferon alpha (IFN-alpha) has induced haematological and cytogenetic remission. Interferons transcriptionally activate target genes through the JAK-STAT and interferon regulated factors (IRFs) family pathways. Interferon regulated factor-1 (IRF-1) is a transcriptional activator of genes critical for cell growth, differentiation and apoptosis. The skipping of exons 2 or 2 and 3 of IRF-1 in patients with myelodysplastic syndromes and acute myelogenous leukaemia suggests that this factor may have a critical role in leukaemogenesis. The role of IRF-1 in CML is currently unknown. Therefore, mutational analysis of IRF-1 was performed and its expression pattern was also studied in CML patients. We studied IRF-1 in peripheral blood mononuclear cells of 21 patients in chronic phase CML. No point mutations were identified at the cDNA level. Surprisingly, fourfold reduction of full-length IRF-1 mRNA expression was established in 17/21 patients compared with normal individuals. Low expression of full-length IRF-1 was observed in conjunction with high levels of aberrantly spliced mRNAs, reported for the first time. In three patients who were also analysed during blastic transformation, further reduction of full-length IRF-1 mRNA was observed. These findings demonstrate that, in CML patients, IRF-1 can produce high levels of aberrant spliced mRNAs with subsequent reduction in the levels of full-length IRF-1 mRNA. This observation is consistent with the notion that exon skipping may constitute another mechanism of tumour suppressor gene inactivation in this disease.

P032 Pro-inflammatory cytokines induce the expression of TL1A/TNFSF15 in primary intestinal subepithelial myofibroblasts G. Bamias1 *, E. Filidou2, D. Goukos3, V. Valatas4, K. Arvanitidis2, M. Panagopoulou2, G. Kouklakis5, G. Daikos3, S. Ladas1, G. Kolios2. 1Laikon Hospital, Ethnikon & Kapodistriakon University of Athens, Academic Dpt. of Gastroenterology, Athens, Greece, 2Democritus University of Thrace, Laboratory of Pharmacology, Faculty of Medicine, Alexandroupolis, Greece, 3Laikon Hospital, Ethnikon & Kapodistriakon University of Athens, 1st. Dpt of Propaedeutic and Internal Medicine, Laboratory of Infectious Diseases, Athens, Greece, 4University of Crete, Laboratory of Gastroenterology, Faculty of Medicine, Heraklion, Greece, 5Democritus University of Thrace, Endoscopy Unit, Faculty of Medicine, University Hospital of Alexandroupolis, Alexandroupolis, Greece

Mutations in the Bruton's tyrosine kinase (BTK ) gene are responsible for X-linked Agammaglobulinemia (XLA), an immunodeficiency caused by a block in B cell differentiation. Non Isotopic RNAse Cleavage Assay (NIRCA), followed by sequencing was used to screen for BTK mutations in 11 Italian XLA patients. Nine novel mutations were identified: 6 missense (Y39S, L512P, L512Q, R544G, S578Y, E589K), one non-sense (Q260X), one frameshift (1599-1602del GCGC) and one in-frame insertion (2037-2038insTTTTAG), that represents the first case of premature stop codon introduction in the BTK coding frame. These data support the high molecular heterogeneity of BTK gene in XLA disease and provide new insight to the diagnosis and to the role of BTK domain in XLA and in B cell signal transduction and development. Hum Mutat 15:117, 2000.