Bacterial flagellin elicits widespread innate immune defense mechanisms, apoptotic signaling, and a sepsis-like systemic inflammatory response in mice.

Introduction: Systemic inflammation in sepsis is initiated by interactions between pathogen molecular motifs and specific host receptors, especially toll-like receptors (TLRs). Flagellin is the main flagellar protein of motile microorganisms and is the ligand of TLR5. The distribution of TLR5 and the actions of flagellin at the systemic level have not been established. Therefore, we determined TLR5 expression and the ability of flagellin to trigger prototypical innate immune responses and apoptosis in major organs from mice. Methods: Male Balb/C mice (n = 80) were injected intravenously with 1-5 mu g recombinant Salmonella flagellin. Plasma and organ samples were obtained after 0.5 to 6 h, for molecular investigations. The expression of TLR5, the activation state of nuclear factor kappa B (NF kappa B) and mitogen-activated protein kinases (MAPKs) [extracellular related kinase (ERK) and c-jun-NH2 terminal kinase (JNK)], the production of cytokines [tumor necrosis alpha (TNF alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), macrophage inhibitory protein-2 (MIP-2) and soluble triggering receptor expressed on myeloid cells (TREM-1)], and the apoptotic cleavage of caspase-3 and its substrate Poly(ADP-ribose) polymerase (PARP) were determined in lung, liver, gut and kidney at different time-points. The time-course of plasma cytokines was evaluated up to 6 h after flagellin. Results: TLR5 mRNA and protein were constitutively expressed in all organs. In these organs, flagellin elicited a robust activation of NF kappa B and MAPKs, and induced significant production of the different cytokines evaluated, with slight interorgan variations. Plasma TNF alpha, IL-6 and MIP-2 disclosed a transient peak, whereas IL-1 beta and soluble TREM-1 steadily increased over 6 h. Flagellin also triggered a marked cleavage of caspase-3 and PARP in the intestine, pointing to its ability to promote significant apoptosis in this organ. Conclusions: Bacterial flagellin elicits prototypical innate immune responses in mice, leading to the release of multiple pro-inflammatory cytokines in the lung, small intestine, liver and kidney, and also activates apoptotic signalling in the gut. Therefore, this bacterial protein may represent a critical mediator of systemic inflammation and intestinal barrier failure in sepsis due to flagellated micro-organisms

Systematic evaluation of risk factors for diagnostic delay in inflammatory bowel

Aim: The diagnosis of inflammatory bowel disease (IBD), comprising Crohn's disease (CD) and ulcerative colitis (UC), continues to present difficulties due to unspecific symptoms and limited test accuracies. We aimed to determine the diagnostic delay (time from first symptoms to IBD diagnosis) and to identify associated risk factors in a national cohort in Switzerland.¦Materials and Methods: A total of 1,591 IBD patients (932 CD, 625 UC, 34 indeterminate colitis) from the Swiss IBD cohort study (SIBDCS) were evaluated. The SIBDCS collects data on a large sample of IBD patients from hospitals and private practice across Switzerland through physician and patient questionnaires. The primary outcome measure was the diagnostic delay.¦Results: Diagnostic delay in CD patients was significantly longer compared to UC patients (median 9 vs. 4 months, P < 0.001). Seventy-five percent of CD patients were diagnosed within 24 months compared to 12 months for UC and 6 months for IC patients. Multivariate logistic regression identified age <40 years at diagnosis (OR 2.15, P = 0.010) and ileal disease (OR 1.69, P = 0.025) as independent risk factors for long diagnostic delay in CD (>24 months). A trend for long diagnostic delay (>12 months) was associated with NSAID intake (OR 1.75, P = 0.093) and male gender (OR 0.59, P = 0.079) in UC patients.¦Conclusions: Whereas the median delay for diagnosing CD, UC, and IC seems to be acceptable, there exists a long delay in a considerable proportion of CD patients. More public awareness work needs to be done in order to reduce patient's and doctor's delay in this target population.

Acute inflammatory response in the stomach of BALB/c mice challenged with coccoidal Helicobacter pylori

An experimental murine model was used to verify the viability and pathogenicity of coccoid Helicobacter pylori. For this purpose, 27 BALB/c mice were inoculated intragastrically with 1 ml broth culture (10(8)organisms/ml) of a coccoid H. pylori clinical isolate. The animals were divided into two groups. Nine were infected on a one-time basis (GA1) and 18 were infected on two consecutive days (GA2). Other 27 mice were inoculated with Brucella broth and divided in the same way; they composed the control group. Mice were killed at 2, 3, 7, 14 and 21 days post inoculation (pi). Fragments of stomach and duodenum were collected, fixed with 12% formalin and stained by hematoxilin-eosin and Giemsa for histopathological examination. Until the 14th()day, only reinfected mice had mild-to-moderate inflammatory infiltrate in the stomach. The infiltration was predominantly lymphomonocytic, although plasma cells and eosinophils could be seen. However, at 21st day, severe eosinophilic infiltration was present in the lamina propria and submucosa of gastric corpus. In subgroup GA1, animals presented lymphomonocytic infiltration in the stomach from 14th()day pi. Our results showed that coccoid H. pylori was able to induce an acute inflammatory response in stomach of reinfected mice since the initial periods of infection.

Experimental lagochilascariosis: histopathological study of inflammatory response to larval migration in the Murine model

The goal of this study was to investigate the pattern of inflammatory response induced by Lagochilascaris minor in murine experimental model. For this purpose 115 mice were given 1000-3000 L. minor infective eggs "per os" and 51 uninfected mice were considered as controls. Four hours post-inoculation (PI), 3rd stage larvae were seen passing through the mucosa of terminal ends of small intestine. Six hours PI larvae were observed as an embolus inside the portal vein and also migrating through the liver parenchyma. During the first 24 h larvae-containing eggs of L. minor were observed in the lumen of intestinal tract. Two days PI larvae were seen migrating through lung parenchyma associated with an initial neutrophilic perivasculitis. From the 13th day of this experimental study, L. minor larvae were found mainly in skeletal muscles, in the center of granulomas. Concentric fibrosis with mixed inflammatory infiltrate involved the larvae after the 47th day PI, persistently. This experimental murine study with L. minor indicated that the 3rd stage larvae penetrated via ileum-cecal mucosa reaching the liver and probably other tissues through the hematogenic via. Throughout its pathway the larvae induced a granulomatous reaction, with abundant polimorphonuclear cells.

Participation of cytokines in the necrotic-inflammatory lesions in the heart and skeletal muscles of Calomys callosus infected with Trypanosoma cruzi

Calomys callosus, a sylvatic reservoir of Trypanosoma cruzi, when infected with the Colombian strain (Biodeme Type III, T. cruzi I ) develops necrotic-inflammatory lesions and intense early fibrogenesis in the heart and skeletal muscles, that spontaneously regress. Participation of pro-inflammatory and pro-fibrogenic cytokines, such as tumor necrosis factor-alpha (TNF-alpha), gamma interferon (IFN-gamma) , and tumor growth factor-beta (TGF-beta), in the pathogenesis of the lesions is herein studied. Eighty C. callosus weighing 20 to 30 g were used. Seventy of them were inoculated with the Colombian strain (10(5) blood forms) and 10 were maintained as intact non-infected controls. After infection, C. callosus were sacrificed at different time-points from 15 to 70 days. The heart and skeletal muscle were processed for histopathology and cryopreserved for immunohistochemistry. Early necrotic lesions of parasitized skeletal muscle and myocardium with intense inflammatory lesions were present. Search for the in situ presence of TNF-alpha and IFN-gamma, was performed using rat-IgG anti-mouse antibodies against these cytokines. For the in situ search of TGF-beta, rabbit IgG anti-mouse antibodies were used. Immunolabeling of the cytokines in tissues of infected C. callosus was successful. The cytokines TNF-alpha, IFN-gamma , and TGF-beta were detected in the cytoplasm of macrophages and in the necrotic material from 15 to 45 days post-infection, decreasing their intensity until complete disappearance by the 65th day, which correlated with subsiding histopathological lesions. These findings suggest the participation of these cytokines in the control of parasite multiplication, in the development of an early fibrogenesis and in the regression of fibrotic-inflammatory lesions observed in C. callosus.

Severity of endoscopically detected esophageal inflammatory and fibrostenotic features correlates with degree of esophageal eosinophilia in adults with eosinophilic esophagitis

Background: Eosinophilic esophagitis (EoE) has emerged as a leading cause of dysphagia in adults. Characteristic esophageal features on endoscopy include structural/fibrostenotic (rings, narrow caliber, strictures) and inflammatory manifestations (longitudinal furrows, exudates, edema). Aim: The purpose of this study was to correlate the clinical, endoscopic and histopathologic features in adult EoE patients. Methods: A total of 106 encounters of 81 patients with EoE were analyzed. Data included an EoE-directed symptom-severity patient questionnaire evaluating symptoms of dysphagia (frequency, intensity, duration), meal duration, chest pain, and overall symptom severity. Video recordings of endoscopies were reviewed in a blinded manner using a classification and grading scheme for the esophageal features of EoE. Histopathology was reviewed for peak eosinophil count/high power field by pathologists blinded to the patients' clinical status. Associations between endoscopic features, histology and symptoms were evaluated using the Spearman rank correlation analysis. Results: The endoscopic severity of both structural and inflammatory esophageal features of EoE, including rings, exudates, longitudinal furrows, and edema, correlated significantly with peak eosinophil counts (see Table 1). Presence of "crepe paper mucosa" did not demonstrate significant association with peak eosinophil counts. Both structural (rings, narrow-caliber esophagus and strictures) and inflammatory (furrows, exudates and edema) composite endoscopic scores demonstrated a strong correlation with peak eosinophil counts. The strongest association with the degree of esophageal eosinophilia was found with a combination of both structural and inflammatory findings (p < 0.0001). The esophageal diameter (in mm) was negatively correlated with overall symptom severity (Spearman's rho = -0.4883, P = 0.0339). None of the individual or combined patient reported symptoms correlated significantly with either endoscopic or histopathologic findings. Conclusion: The severity of both structural and inflammatory endoscopic features associated with EoE is significantly associated with the degree of esophageal eosinophilia. Patient reported symptom severity was not associated with the degree of esophageal eosinophilia. Esophageal stricture diameter was inversely correlated with EoE symptom severity. The prognostic and therapeutic implications of these observations need to be determined.

PPARs as drug targets to modulate inflammatory responses?

The three peroxisome proliferator-activated receptors (PPARs) isotypes (PPAR alpha, beta/delta and gamma) belong to the nuclear hormone receptor family. During the last decade, they have been identified as anti-inflammatory transcription factors. Part of this regulation antiinflammatory is mediated through negative interference between PPARs and other nuclear factors such as NFkB, AP-1 and C/EBP, which regulate innate as well as adaptative immunity. In addition, the PPARs control the functions of macrophages, B cells and T cells. In this review, we summarise the pathways through which the PPARs control inflammatory responses. We also discuss the potential utilisation of PPAR specific ligands in the treatment of inflammatory diseases, such as inflammatory bowel diseases, atherosclerosis, Parkinson's and Alzheimer's diseases.

An overview of the effects of annexin 1 on cells involved in the inflammatory process

The concept of anti-inflammation is currently evolving with the definition of several endogenous inhibitory circuits that are important in the control of the host inflammatory response. Here we focus on one of these pathways, the annexin 1 (ANXA1) system. Originally identified as a 37 kDa glucocorticoid-inducible protein, ANXA1 has emerged over the last decade as an important endogenous modulator of inflammation. We review the pharmacological effects of ANXA1 on cell types involved in inflammation, from blood-borne leukocytes to resident cells. This review reveals that there is scope for more research, since most of the studies have so far focused on the effects of the protein and its peptido-mimetics on neutrophil recruitment and activation. However, many other cells central to inflammation, e.g. endothelial cells or mast cells, also express ANXA1: it is foreseen that a better definition of the role(s) of the endogenous protein in these cells will open the way to further pharmacological studies. We propose that a more systematic analysis of ANXA1 physio-pharmacology in cells involved in the host inflammatory reaction could aid in the design of novel anti-inflammatory therapeutics based on this endogenous mediator.

Lipoxin agonists: turn right! to path of resolving neutrophil

An impressive array of cellular and molecular adaptive responses achieves homeostasis. The inflammatory reaction is an adaptive response triggered by an insult to culminate into the overt cardinal signs of inflammation, eventually leading to resolution and returning the organism back to its original centered state. This article focuses on some aspects of the lipoxin A4 signaling pathway during the resolution phase, to better understand molecular mechanisms by which a neutrophil directs an inflammatory reaction to switch off and resume homeostasis. Defining the resolution state of a neutrophil at the molecular level will aid in treatments of diseases that are associated with an exaggerated and uncontrolled inflammation.

Selective PDE4 inhibitors as potent anti-inflammatory drugs for the treatment of airway diseases

Phosphodiesterases (PDEs) are responsible for the breakdown of intracellular cyclic nucleotides, from which PDE4 are the major cyclic AMP metabolizing isoenzymes found in inflammatory and immune cells. This generated greatest interest on PDE4 as a potential target to treat lung inflammatory diseases. For example, cigarette smoke-induced neutrophilia in BAL was dose and time dependently reduced by cilomilast. Beside the undesired side effects associated with the first generation of PDE4 inhibitors, the second generation of selective inhibitors such as cilomilast and roflumilast showed clinical efficacy in asthma and chronic obstrutive pulmonary diseases trials, thus re-enhancing the interest on these classes of compounds. However, the ability of PDE4 inhibitors to prevent or modulate the airway remodelling remains relatively unexplored. We demonstrated that selective PDE4 inhibitor RP 73-401 reduced matrix metalloproteinase (MMP)-9 activity and TGF-beta1 release during LPS-induced lung injury in mice and that CI-1044 inhibited the production of MMP-1 and MMP-2 from human lung fibroblasts stimulated by pro-inflammatory cytokines. Since inflammatory diseases of the bronchial airways are associated with destruction of normal tissue structure, our data suggest a therapeutic benefit for PDE4 inhibitors in tissue remodelling associated with chronic lung diseases.

Macrophage elastase (MMP-12): a pro-inflammatory mediator?

As many metalloproteinases (MMPs), macrophage elastase (MMP-12) is able to degrade extracellular matrix components such as elastin and is involved in tissue remodeling processes. Studies using animal models of acute and chronic pulmonary inflammatory diseases, such as pulmonary fibrosis and chronic obstrutive pulmonary disease (COPD), have given evidences that MMP-12 is an important mediator of the pathogenesis of these diseases. However, as very few data regarding the direct involvement of MMP-12 in inflammatory process in the airways were available, we have instilled a recombinant form of human MMP-12 (rhMMP-12) in mouse airways. Hence, we have demonstrated that this instillation induced a severe inflammatory cell recruitment characterized by an early accumulation of neutrophils correlated with an increase in proinflammatory cytokines and in gelatinases and then by a relatively stable recruitment of macrophages in the lungs over a period of ten days. Another recent study suggests that resident alveolar macrophages and recruited neutrophils are not involved in the delayed macrophage recruitment. However, epithelial cells could be one of the main targets of rhMMP-12 in our model. We have also reported that a corticoid, dexamethasone, phosphodiesterase 4 inhibitor, rolipram and a non-selective MMP inhibitor, marimastat could reverse some of these inflammatory events. These data indicate that our rhMMP-12 model could mimic some of the inflammatory features observed in COPD patients and could be used for the pharmacological evaluation of new anti-inflammatory treatment. In this review, data demonstrating the involvement of MMP-12 in the pathogenesis of pulmonary fibrosis and COPD as well as our data showing a pro-inflammatory role for MMP-12 in mouse airways will be summarized.

Protease-activated receptors and inflammatory hyperalgesia

Recent advances in basic science pointed to a role for proteinases, through the activation of proteinase-activated receptors (PARs) in nociceptive mechanisms. Activation of PAR1, PAR2 and PAR4 either by proteinases or by selective agonists causes inflammation inducing most of the cardinal signs of inflammation: swelling, redness, and pain. Sub-inflammatory doses of PAR2 agonist still induced hyperalgesia and allodynia while PAR2 has been shown to be implicated in the generation of hyperalgesia in different inflammatory models. In contrast, sub-inflammatory doses of PAR1 increases nociceptive threshold, inhibiting inflammatory hyperalgesia, thereby acting as an analgesic agent. PARs are present and functional on sensory neurons, where they participate either directly or indirectly to the transmission and/or inhibition of nociceptive messages. Taken together, the results discussed in this review highlight proteinases as signaling molecules to sensory nerves. We need to consider proteinases and the receptors that are activated by proteinases as important potential targets for the development of analgesic drugs in the treatment of inflammatory pain.

Inflammatory effects of snake venom metalloproteinases

Metalloproteinases are abundant enzymes in crotaline and viperine snake venoms. They are relevant in the pathophysiology of envenomation, being responsible for local and systemic hemorrhage frequently observed in the victims. Snake venom metalloproteinases (SVMP) are zinc-dependent enzymes of varying molecular weights having multidomain organization. Some SVMP comprise only the proteinase domain, whereas others also contain a disintegrin-like domain, cysteine-rich, and lectin domains. They have strong structural similarities with both mammalian matrix metalloproteinases (MMP) and members of ADAMs (a disintegrin and metalloproteinase) group. Besides hemorrhage, snake venom metalloproteinase induce local myonecrosis, skin damage, and inflammatory reaction in experimental models. Local inflammation is an important characteristic of snakebite envenomations inflicted by viperine and crotaline snake species. Thus, in the recent years there is a growing effort to understand the mechanisms responsible for SVMP-induced inflammatory reaction and the structural determinants of this effect. This short review focuses the inflammatory effects evoked by SVMP.

Good bug, bad bug: in the case of enteric inflammatory disease does the epithelium decide?

Many studies demonstrate that intestinal inflammation is either initiated or exaggerated by a component of the normal microbiota, most likely commensal bacteria or products derived from these organisms. We review the nature of human inflammatory bowel disease, the evidence for the involvement of the normal bacterial flora in these disorders and the relevance of maintaining the integrity of the epithelial barrier. Moreover, we, and others, have shown abnormal mitochondria structure in tissue resections from patients with inflammatory bowel disease and tissues from rodents that demonstrated psychological stress-induced increases in epithelial permeability. Thus, we also consider the possibility that a defect in epithelial mitochondrial function would predispose an individual to respond to their commensal bacteria flora - no longer considering them as a beneficial passive inhabitant, but rather perceiving them as a threatening and pro-inflammatory stimulus. In support of this postulate, we discuss our recent findings from an in vitro model showing that the human colon-derived T84 cell line exposed to the metabolic stressor, dinitrophenol, and the non-pathogenic, non-invasive, Escherichia coli (strain HB101) display a loss of barrier function, increased signal transduction and increased production of the chemokine, interleukin 8.