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Formerly considered as a passive process, the resolution of acute inflammation is now recognized as an active host response, with a cascade of coordinated cellular and molecular events that promotes termination of the inflammatory response and initiates tissue repair and healing. In a state of immune fitness, the resolution of inflammation is contained in time and space enabling the restoration of tissue homeostasis. There is increasing evidence that poor and/or inappropriate resolution of inflammation participates in the pathogenesis of chronic inflammatory diseases, extending in time the actions of pro-inflammatory mechanisms, and responsible in the long run for excessive tissue damage and pathology. In this review, we will focus on how resolution can be the target for therapy in "Th1/Th17 cell-driven" immune diseases and "Th2 cell-driven" immune diseases, with inflammatory bowel diseases (IBD) and asthma, as relevant examples. We describe the main cells and mediators stimulating the resolution of inflammation and discuss how pharmacological and dietary interventions but also life style factors, physical and psychological conditions, might influence the resolution phase. A better understanding of the impact of endogenous and exogenous factors on the resolution of inflammation might open a whole area in the development of personalized therapies in non-resolving chronic inflammatory diseases.

T regulatory cells (Tregs) play an important role in the regulation of autoimmunity, autoinflammation, allergic diseases, infection, and the tumor environment. Different subsets are characterized that use a number of regulatory mechanisms. Tregs can influence the progression of inflammatory bowel disease and the development of colorectal cancer. Knowledge of Tregs and their regulatory mechanisms can provide new targets for therapeutic intervention., (© 2016 The Authors Journal of Gastroenterology and Hepatology published by Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Background and Aim: The expression of interleukin (IL)-17 is upregulated in inflammatory bowel disease (IBD). Since fibroblasts are known to be responsive to IL-17, they may play a role in the modulation of inflammatory responses in IBD. Here, the effects of IL-17 on ileum and colon fibroblasts from Crohn's disease (CD) and ulcerative colitis (UC) patients are investigated, as compared to controls., Methods: Fibroblasts were isolated from surgical specimens taken from the tissue of 21 CD patients, 5 UC patients, and 14 patients undergoing surgery for colorectal carcinoma (control). The fibroblasts were cultured with and without IL-17. We performed mRNA microarray analysis on cultured fibroblasts, isolated from three CD samples and three control samples. Based on these results, the expression of IL-17 induced genes was validated in a larger selection of samples using qRT-PCR and ELISA., Results: The mRNA microarray showed that IL-17 induced the expression levels of various genes in fibroblasts of CD patients and controls, among which NFKBIZ, CXCL1, and CXCL6 demonstrated the most prominent response. qRT-PCR validated that IL-17 induced the expression of NFKBIZ significantly (p=0.028) in intestinal fibroblasts of CD patients. By performing an ELISA, we also discovered that, following IL-17 stimulation, CXCL1 levels were significantly increased in fibroblasts from CD patients (p=0.048). IL-17 also stimulated secretion of CXCL6 in fibroblasts from UC patients (p=0.053)., Conclusion: The enhanced expression of IL-17 that is observed in patients with Crohn's disease could act on intestinal fibroblasts to induce expression of transcription factor NFKBIZ and proinflammatory chemokine CXCL1. This can have consequences for fibroblast activity and neutrophil chemotaxis., (Copyright © 2014 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.)

Objective: Proline-glycine-proline (PGP) has been shown to have chemotactic effects on neutrophils via CXCR2 in several lung diseases. PGP is derived from collagen by the combined action of matrix metalloproteinase (MMP) 8 and/or MMP9 and prolyl endopeptidase (PE). We investigated the role of PGP in inflammatory bowel disease (IBD)., Design: In intestinal tissue from patients with IBD and mice with dextran sodium sulfate (DSS)-induced colitis, MMP8, MMP9 and PE were evaluated by ELISA, immunoblot and immunohistochemistry. Peripheral blood polymorphonuclear cell (PMN) supernatants were also analysed accordingly and incubated with collagen to assess PGP generation ex vivo. PGP levels were measured by mass spectrometry, and PGP neutralisation was achieved with a PGP antagonist and PGP antibodies., Results: In the intestine of patients with IBD, MMP8 and MMP9 levels were elevated, while PE was expressed at similar levels to control tissue. PGP levels were increased in intestinal tissue of patients with IBD. Similar results were obtained in intestine from DSS-treated mice. PMN supernatants from patients with IBD were far more capable of generating PGP from collagen ex vivo than healthy controls. Furthermore, PGP neutralisation during DSS-induced colitis led to a significant reduction in neutrophil infiltration in the intestine., Conclusions: The proteolytic cascade that generates PGP from collagen, as well as the tripeptide itself, is present in the intestine of patients with IBD and mice with DSS-induced colitis. PGP neutralisation in DSS-treated mice showed the importance of PGP-guided neutrophilic infiltration in the intestine and indicates a vicious circle in neutrophilic inflammation in IBD.

Aim: To investigate if the presence of relevant genetic polymorphisms has effect on the effectual clearance of bacteria by monocytes and granulocytes in patients with Crohn's disease (CD)., Methods: In this study, we assessed the differential responses in phagocytosis by measuring the phagocytic activity and the percentage of active phagocytic monocytes and granulocytes in inflammatory bowel disease patients as well as healthy controls. As both autophagy related like 1 (ATG16L1) and immunity-related guanosine triphosphatase gene are autophagy genes associated with CD and more recently nucleotide-binding ligomerization domain-containing protein 2 (NOD2) has been identified as a potent inducer of autophagy we genotyped the patients for these variants and correlated this to the phagocytic reaction. The genotyping was done with restriction fragment length polymorphisms analysis and the phagocytosis was determined with the pHrodo™ Escherichia coli Bioparticles Phagocytosis kit for flowcytometry., Results: In this study, we demonstrate that analysis of the monocyte and granulocyte populations of patients with CD and ulcerative colitis showed a comparable phagocytic activity (ratio of mean fluorescence intensity) between the patient groups and the healthy controls. CD patients show a significantly higher phagocytic capacity (ratio mean percentage of phagocytic cells) compared to healthy controls (51.91% ± 2.85% vs 37.67% ± 7.06%, P = 0.05). The extend of disease was not of influence. However, variants of ATG16L1 (WT: 2.03 ± 0.19 vs homozygoot variant: 4.38 ± 0.37, P < 0.009) as well as NOD2 (C-ins) (heterozygous variant: 42.08 ± 2.94 vs homozygous variant: 75.58 ± 4.34 (P = 0.05) are associated with the phagocytic activity in patients with CD., Conclusion: Monocytes of CD patients show enhanced phagocytosis associated with the presence of ATG16L1 and NOD2 variants. This could be part of the pathophysiological mechanism resulting in the disease.

Cytokines are immunomodulating proteins involved in cellular communication. The levels of different cytokines reflect the immune capabilities of a person. In literature both whole blood and peripheral blood mononuclear cells (PBMCs) are used, which might lead to different results. The choice between these different sources is not always explained. The goal of our experiments is to determine the cytokine response of whole blood, PBMCs and polymorphonuclear cells (PMNs) after stimulation with lipopolysaccharide (LPS). We used a multiplex analysis to determine a difference in cytokine secretion patterns. In general, PBMCs demonstrated the highest cytokine production and PMNs have an overall low cytokine production. CCL11 and interleukin-23 (IL-23) (and IL-12p40) were exclusively expressed in whole blood. IL-20, VEGF and GM-CSF were expressed only by PBMCs. This difference in expression could be explained by the bioactive components in serum, presence and interaction with granulocytes or platelets in whole blood, the anticoagulant heparin in whole blood and others. The expression of cytokines by cells is dependent on the microenvironment. Different conditions lead to different results. We recommend a thorough examination of the conditions before performing experiments., (© 2013.)

Background: Induced regulatory T (iTreg) lymphocytes show promise for application in the treatment of allergic, autoimmune and inflammatory disorders. iTreg cells demonstrate advantages over natural Treg (nTreg) cells in terms of increased number of starting population and greater potential to proliferate. Different activation methods to generate iTreg cells result in iTreg cells that are heterogeneous in phenotype and mechanisms of suppression. Therefore it is of interest to explore new techniques to generate iTreg cells and to determine their physiological relevance., Methods: Using phorbol myristate acetate (PMA)/ionomycin and anti-CD3 activation of CD4⁺CD25⁻ cells we generated in vitro functional CD4⁺CD25⁻ iTreg (TregPMA) cells. Functionality of the generated TregPMA cells was tested in vivo in a mouse model of inflammatory bowel disease (IBD) - CD45RB transfer colitis model., Results: TregPMA cells expressed regulatory markers and proved to ameliorate the disease phenotype in murine CD45RB transfer colitis model. The body weight loss and disease activity scores for TregPMA treated mice were reduced when compared to diseased control group. Histological assessment of colon sections confirmed amelioration of the disease phenotype. Additionally, cytokine analysis showed decreased levels of proinflammatory colonic and plasma IL-6, colonic IL-1 β and higher levels of colonic IL-17 when compared to diseased control group., Conclusions: This study identifies a new method to generate in vitro iTreg cells (TregPMA cells) which physiological efficacy has been demonstrated in vivo.

Type 2 innate lymphoid cells (ILC2s) are part of a large family of ILCs that are important effectors in innate immunity, lymphoid organogenesis, and tissue remodeling. ILC2s mediate parasite expulsion but also contribute to airway inflammation, emphasizing the functional similarity between these cells and Th2 cells. Consistent with this, we report that the transcription factor GATA3 was highly expressed by human ILC2s. CRTH2(+) ILC2s were enriched in nasal polyps of patients with chronic rhinosinusitis, a typical type 2-mediated disease. Nasal polyp epithelial cells expressed TSLP, which enhanced STAT5 activation, GATA3 expression, and type 2 cytokine production in ILC2s. Ectopic expression of GATA3 in Lin(-)CD127(+)CRTH2(-) cells resulted in induction of CRTH2 and the capacity to produce high amounts of type 2 cytokines in response to TSLP plus IL-33. Hence, we identify GATA3, potently regulated by TSLP, as an essential transcription factor for the function of human ILC2s., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Aim: To explore the anti-inflammatory potential of adeno-associated virus-mediated delivery of Tregitope 167 in an experimental colitis model., Methods: The trinitrobenzene sulfonate (TNBS) model of induced colitis was used in Balb/c mice. Subsequently after intravenous adeno-associated virus-mediated regulatory T-cell epitopes (Tregitope) delivery, acute colitis was initiated by intra-rectal administration of 1.5 mg TNBS in 40% ethanol followed by a second treatment with TNBS (0.75 mg in 20% ethanol) 8 d later. Control groups included mice not treated with TNBS (healthy control group) and mice treated by TNBS only (diseased group). At the time of sacrifice colon weight, the disease activity index and histology damage score were determined. Immunohistochemical staining of the colonic tissues was performed to asses the cellular infiltrate and the presence of transcription factor forkhead Box-P3 (Foxp3). Thymus, mesenteric lymph nodes, liver and spleen tissue were collected and the corresponding lymphocyte populations were further assessed by flow cytometry analysis for the expression of CD4+ T cell and regulatory T cell associated markers., Results: The Tregitope 167 treated mice gained an average of 4% over their initial body weight at the time of sacrifice. In contrast, the mice treated with TNBS alone (no Tregitope) developed colitis, and lost 4% of their initial body weight at the time of sacrifice (P < 0.01). The body weight increase that had been observed in the mice pre-treated with Tregitope 167 was substantiated by a lower disease activity index and a decreased colon weight as compared to the diseased control group (P < 0.01 and P < 0.001, respectively). Immunohistochemical staining of the colonic tissues for CD4+ showed that inflammatory cell infiltrates were present in TNBS treated mice with or without administration with tregitope 167 and that these cellular infiltrates consisted mainly of CD4+ cells. For both TNBS treated groups CD4+ T cell infiltrates were observed in the sub-epithelial layer and the lamina propria. CD4+ T cell infiltrates were also present in the muscularis mucosa layer of the diseased control mice, but were absent in the Tregitope 167 treated group. Numerous Foxp3 positive cells were detected in the lamina propria and sub-epithelium of the colon sections from mice treated with Tregitope 167. Furthermore, the Foxp3 and glycoprotein A repetitions predominant markers were significantly increased in the CD4+ T lymphocyte population in the thymus of the mice pre-treated with adeno-associated virus serotype 5 (cytomegalovirus promoter-Tregitope 167), as cytomegalovirus promoter compared to lymphocyte populations in the thymus of diseased and the healthy control mice (P < 0.05 and P < 0.001, respectively)., Conclusion: This study identifies adeno-associated virus-mediated delivery of regulatory T-cell epitope 167 as a novel anti-inflammatory approach with the capacity to decrease intestinal inflammation and induce long-term remission in inflammatory bowel disease.

Background & Aims: Variants in the genes ATG16L1 and IRGM affect autophagy and are associated with the development of Crohn's disease. It is not clear how autophagy is linked to loss of immune tolerance in the intestine. We investigated the involvement of the immunologic synapse-the site of contact between dendritic cells (DCs) and T cells, which contains molecules involved in antigen recognition and regulates immune response., Methods: DC autophagy was reduced using small interfering RNAs or pharmacologic inhibitors. DC phenotype and function were analyzed by confocal microscopy, time-lapse microscopy, and flow cytometry. We also examined DCs isolated from patients with Crohn's disease who carried the ATG16L1 risk allele., Results: Immunologic synapse formation induced formation of autophagosomes in DCs; the autophagosomes were oriented toward the immunologic synapse and contained synaptic components. Knockdown of ATG16L1 and IRGM with small interfering RNAs in DCs resulted in hyperstable interactions between DCs and T cells, increased activation of T cells, and activation of a T-helper 17 cell response. LKB1 was recruited to the immunologic synapse, and induction of autophagy in DC required inhibition of mammalian target of rapamycine signaling by the LKB1-AMP activated protein kinase (AMPK) pathway. DCs from patients with Crohn's disease who had an ATG16L1 risk allele had a similar hyperstability of the immunologic synapse., Conclusions: Autophagy is induced upon formation of the immunologic synapse and negatively regulates T-cell activation. This mechanism might increase adaptive immunity in patients with Crohn's disease who carry ATG16L1 risk alleles., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Chemokines (chemotactic cytokines) can have direct antimicrobial activity, which is apparently related to the presence of a distinct positively charged patch on the surface. However, chemokines can retain antimicrobial activity upon linearization despite the loss of their positive patch, thus questioning the importance of this patch for activity. Thrombocidin-1 (TC-1) is a microbicidal protein isolated from human blood platelets. TC-1 only differs from the chemokine NAP-2/CXCL7 by a two-amino acid C-terminal deletion, but this truncation is crucial for antimicrobial activity. We assessed the structure-activity relationship for antimicrobial activity of TC-1. Reduction of the charge of the TC-1-positive patch by replacing lysine 17 with alanine reduced the activity against bacteria and almost abolished activity against the yeast Candida albicans. Conversely, augmentation of the positive patch by increasing charge density or size resulted in a 2-3-fold increased activity against Staphylococcus aureus, Escherichia coli, and Bacillus subtilis but did not substantially affect activity against C. albicans. Reduction of TC-1 resulted in loss of the folded conformation, but this disruption of the positive patch did not affect antimicrobial activity. Using overlapping 15-mer synthetic peptides, we demonstrate peptides corresponding to the N-terminal part of TC-1 to have similar antimicrobial activity as intact TC-1. Although we demonstrate that the positive patch is essential for activity of folded TC-1, unfolded TC-1 retained antimicrobial activity despite the absence of a positive patch. This activity is probably exerted by a linear peptide stretch in the N-terminal part of the molecule. We conclude that intact TC-1 and unfolded TC-1 exert antimicrobial activity via distinct structural elements.

Tissue factor (TF) is traditionally known as the initiator of blood coagulation, but TF also plays an important role in inflammatory processes. Considering the pivotal role of coagulation in inflammatory bowel disease, we assessed whether genetic ablation of TF limits experimental colitis. To this end, wild-type and TF-deficient (TFlow) mice were treated with 1.5% dextran sulfate sodium (DSS) for 7 d, and effects on disease severity, cytokine production and leukocyte recruitment were examined. Clinical and histological parameters showed that the severity of colitis was reduced in both heterozygous and homozygous TFlow mice compared with controls. Most notably, edema, granulocyte numbers at the site of inflammation and cytokine levels were reduced in TFlow mice. Although anticoagulant treatment with dalteparin of wild-type mice reduced local fibrin production and cytokine levels to a similar extent as in TFlow mice, it did not affect clinical and histological parameters of experimental colitis. Mechanistic studies revealed that TF expression did not influence the intrinsic capacity of granulocytes to migrate. Instead, TF enhanced granulocyte migration into the colon by inducing high levels of the granulocyte chemoattractant keratinocyte-derived chemokine (KC). Taken together, our data indicate that TF plays a detrimental role in experimental colitis by signal transduction-dependent KC production in colon epithelial cells, thereby provoking granulocyte influx with subsequent inflammation and organ damage.

Monocyte chemoattractant protein 1 (MCP-1) plays an important role in leukocyte recruitment to sites of infection and inflammation. In addition, MCP-1 may attenuate inflammation by virtue of its capacity to inhibit the production of proinflammatory cytokines. We here investigated the role of MCP-1 in lung inflammation induced by lipopolysaccharide (LPS) or lipoteichoic acid (LTA), constituents of the gram-negative and gram-positive bacterial cell wall, respectively. Healthy humans demonstrated elevated MCP-1 concentrations in their bronchoalveolar lavage fluid (BALF) 6h after inhalation of LPS. Similarly, intranasal administration of LPS or LTA to mice resulted in a rise in BALF MCP-1 levels. Murine alveolar macrophage-like cells released significant amounts of MCP-1 upon stimulation with LPS or LTA in vitro. Compared to Wt mice, MCP-1(-/-) mice demonstrated lower TNF-α levels and a diminished neutrophil influx into their bronchoalveolar space after either LPS or LTA instillation. After intrapulmonary delivery of LPS MCP-1(-/-) mice had decreased interleukin-6 and KC concentrations and less severe lung inflammation upon histopathological examination. Remarkably, MCP-1 deficiency was associated with an early enhancement of interleukin-10 release in BALF after both LPS and LTA instillation. These data suggest that MCP-1 is a proinflammatory mediator during pulmonary inflammation induced by either LPS or LTA., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Phosphatidylinositol-3-kinase gamma (PI3Kgamma) is the major PI3K that is activated in response to chemoattractants. It is responsible for the migration of leukocytes from the bloodstream to sites of injury or infection. Constant migration of new leukocytes to the intestinal mucosa may be an important factor in maintenance of inflammation and tissue damage in inflammatory bowel disease (IBD). Reducing this influx, for example by inhibition of PI3Kgamma, might therefore be a potential goal for therapy. Here we investigated the role of PI3Kgamma in the migration of leukocytes to sites of intestinal inflammation. We induced colitis in mice with a point mutation that inactivates PI3Kgamma enzymatic activity ('kinase-dead') by oral administration of dextran sodium sulphate (DSS). Mice were treated with 1.5% DSS for 1 week and effects on cytokine production, leukocyte recruitment and disease severity were examined. Both clinical and histological parameters showed that the severity of colitis was significantly reduced in PI3Kgamma-kinase-dead mice compared to controls. Although mutant mice had a less severe colitis than controls they produced significantly more pro-inflammatory Th1 cytokines such as Il-12, Tnfalpha and Ifngamma and more Il-10. PI3Kgamma mutant mice showed increased numbers of resident macrophages and T cells in the colonic lamina propria in an unstressed condition but failed to recruit new leukocytes to the mucosa upon treatment with DSS despite the increased cytokine levels. These results suggest that PI3Kgamma plays a critical role in lamina propria leukocyte trafficking and that loss of PI3Kgamma-activity ameliorates DSS-induced colitis in mice., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Background: Antibiotic resistance among microbes urgently necessitates the development of novel antimicrobial agents. Since ancient times, honey has been used successfully for treatment of infected wounds, because of its antibacterial activity. However, large variations in the in vitro antibacterial activity of various honeys have been reported and hamper its acceptance in modern medicine., Methods: We assessed the in vitro bactericidal activity of Revamil (Bfactory), a medical-grade honey produced under controlled conditions, and assessed its efficacy for reduction of forearm skin colonization in healthy volunteers in a within-subject-controlled trial., Results: With Bacillus subtilis as a test strain, we demonstrated that the variation in bactericidal activity of 11 batches of medical-grade honey was <2-fold. Antibiotic-susceptible and -resistant isolates of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, and Klebsiella oxytoca were killed within 24 h by 10%-40% (vol/vol) honey. After 2 days of application of honey, the extent of forearm skin colonization in healthy volunteers was reduced 100-fold (P < .001), and the numbers of positive skin cultures were reduced by 76% (P < .001)., Conclusions: Revamil is a promising topical antimicrobial agent for prevention or treatment of infections, including those caused by multidrug-resistant bacteria.

Dendritic cells (DCs) are key cells in innate and adaptive immune responses that determine the pathophysiology of Crohn's disease. Intestinal DCs migrate from the mucosa into mesenteric lymph nodes (MLNs). A number of different markers are described to define the DC populations. In this study we have identified the phenotype and localization of intestinal and MLN DCs in patients with Crohn's disease and non-IBD patients based on these markers. We used immunohistochemistry to demonstrate that all markers (S-100, CD83, DC-SIGN, BDCA1-4, and CD1a) showed a different staining pattern varying from localization in T-cell areas of lymph follicles around blood vessels or single cells in the lamina propria and in the MLN in the medullary cords and in the subcapsular sinuses around blood vessels and in the T-cell areas. In conclusion, all different DC markers give variable staining patterns so there is no marker for the DC.

Background: Chronic hepatitis C virus (HCV) infection is characterized by an insufficient immune response, possibly owing to impaired function of antigen-presenting cells such as myeloid dendritic cells (DCs). Therapeutic vaccination with in vitro generated DCs may enhance the immune response. Subsets of DCs can originate from monocytes, but the presence of HCV in monocytes that develop into DCs in vitro may impair DC function. Therefore, we studied the presence of HCV RNA in monocytes and monocyte-derived DCs from chronic HCV patients., Methods: Monocytes were cultured with granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) for 6 days, and then with GM-CSF, IL-4, tumour necrosis factor-alpha (TNF-alpha), prostaglandin E2, IL-1beta and IL-6 for 2 days to generate mature DCs. HCV RNA was assessed by polymerase chain reaction. Surface molecules were assessed by flow cytometry. Cytokine production was assessed by cytokine bead array., Results: HCV RNA was present in monocytes in 11 of 13 patients, but undetectable in mature DCs in 13 of 13 patients. The morphology of patient DCs was comparable with DCs from healthy controls, but the percentage of cells expressing surface molecules CD83 (P=0.001), CD86 (P=0.023) and human leucocyte antigen-DR (P=0.028) was lower in HCV patients. Compared with control DCs, patient DCs produced enhanced levels of IL-10 (P=0.0079) and IL-8 (P=0.0079), and lower levels of TNF-alpha (P=0.032), IL-6 (P=NS) and IL-1beta (P=0.0079). Patient and control DCs did not produce IL-12., Conclusions: Monocyte-derived DCs from chronic HCV patients are not infected but show an immature phenotype and aberrant cytokine profile.

Various animal models showed that peroxisome proliferator-activated receptor (PPAR)gamma agonists, when given as a gavage shortly preceding colitis induction, protect against inflammatory bowel disease (IBD). We have examined the effects of 16 days rosiglitazone treatment via the diet prior to dextran sodium sulphate (DSS)-induced colitis in mice. After 7 days DSS in the drinking water, rosiglitazone-fed mice had lost significantly more weight than control mice. Rosiglitazone-treated mice had more diarrhea, weight of colon and spleen were increased, and length of colon was decreased. Histology showed that rosiglitazone-treated mice had more severe colitis, mainly caused by more ulceration, crypt loss, and edema. Immunofluorescence showed a loss of tight junction structure Zonula Occludens protein 1 (ZO-1) in colons of rosiglitazone-treated mice as compared to control mice. Also, serum amyloid P component (SAP) concentrations in plasma were increased. However, concentrations of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma in colon homogenates, and TNF-alpha in spleen homogenates were significantly decreased, whereas interleukin (IL)-10 in spleen homogenates was increased. Other cytokines (IL-2, IL-4, IL-6, IL-12p70 and monocyte chemotactic protein (MCP)-1) and myeloperoxidase (MPO) concentrations showed no differences. In conclusion, 16 days pretreatment with rosiglitazone impaired DSS-induced colitis in mice.

Background: The human anti-tumour necrosis factor (TNF) antibody infliximab binds to the membrane TNF and subsequently induces apoptosis of activated lamina propria T lymphocytes in patients with Crohn's disease in vitro., Aim: To test whether the ability of rapid anti-TNF-induced apoptosis in the gut predicts the efficacy of anti-TNF treatment in inflammatory bowel disease., Methods: (99m)Technetium-annexin V single-photon emission computer tomography (SPECT) was performed in 2 models of murine experimental colitis and in 14 patients with active Crohn's disease as assessed by the Crohńs Disease Activity Index (CDAI) to study the effect of anti-TNF treatment on apoptosis in the intestine during active colitis. Disease activity was evaluated 2 weeks after infliximab infusion using the CDAI (definition response: drop of >100 points)., Results: Colonic uptake of (99m)Tc-annexin V significantly increased in 2,4,6-trinitrobenzene sulphonate-induced colitis as well as in transfer colitis on administration of anti-TNF antibodies compared with a control antibody as determined with dedicated animal pinhole SPECT. In addition, uptake of (99m)Tc-annexin V significantly increased in patients with active Crohn's disease responding to infliximab treatment. Colonic (99m)Tc-annexin V uptake ratio (mean (SEM)) increased from 0.24 (0.03) to 0.41(0.07) (p<0.01), 24 h after infliximab infusion (5 mg/kg). A mean increase of 98.7% in colonic uptake of (99m)Tc-annexin V could be detected in 10 of the 14 responding patients (CDAI >100 points at week 2) compared with 15.2% in non-responding patients (p = 0.03). Analysis of the mucosal biopsy specimens identified lamina propria T cells as target cells undergoing apoptosis., Conclusions: These in vivo observations support the notion that colonic uptake of (99m)Tc-annexin V correlates with clinical benefit of anti-TNF treatment and might be predictive of therapeutic success.

Evidence is increasing that a defect in apoptosis is involved in the pathogenesis of inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC). CD seems to be the cause of an intrinsic defect in the apoptotic pathway of (autoreactive) T cells, resulting in excessive T cell responses. In UC, an increased rate of apoptosis of epithelial cells is observed. In this review we will describe apoptotic mechanisms and their association to IBD. In addition, we will review how specific therapeutic approaches interact at different levels with the apoptotic pathway.

Unlabelled: Evaluating the efficacy of therapy in experimental inflammatory bowel disease (IBD) requires information about inflammatory activity in bowel segments or leukocyte recruitment and about kinetics in the follow-up of treatment. This study evaluated a noninvasive scintigraphic technique able to assess neutrophil trafficking in a mouse model of dextran sulfate sodium (DSS)-induced colitis., Methods: Groups of 4 BALB/c mice were assessed at baseline and after 1, 3, 5, and 8 d of treatment with DSS. Donor neutrophils were harvested by rinsing of the peritoneal cavity with phosphate-buffered saline 5 h after intraperitoneal injection of proteose peptone contained in phosphate-buffered saline and labeled with freshly prepared (99m)Tc-hexamethylpropylene amine oxime (HMPAO). Pinhole SPECT of the abdomen was performed 1 h after reinjection of 50 MBq of labeled neutrophils. In addition, the severity of inflammation was determined by histologic examination. The possibilities of the technique were illustrated by scintigraphic assessment of neutrophil trafficking with and without blocking of neutrophil migration by a CD97 monoclonal antibody in mice with DSS-induced colitis., Results: Colonic uptake of (99m)Tc-HMPAO neutrophils was determined with dedicated animal pinhole SPECT in mice with DSS-induced colitis and correlated well with histologic findings (R = 0.81) and wet colon weight (R = 0.87) and moderately with clinical weight loss (R = 0.62). The neutrophil uptake ratio was reduced significantly (P < 0.01) by blocking of neutrophil migration capacity with the CD97 antibody., Conclusion: Animal pinhole SPECT can be used to study inflammatory activity and neutrophil recruitment in vivo in experimental colitis.

Unlabelled: Blockade of lymphocyte recruitment to the intestinal mucosa is considered a useful therapy for inflammatory bowel disease (IBD) and anti-alpha4 antibodies have clinical benefit in patients with active Crohn's disease. The aim of this study was to evaluate a scintigraphic technique to assess lymphocyte homing to the colon in 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced experimental colitis (TNBS colitis) in vivo., Methods: TNBS-sensitized and nonsensitized murine total lymphocytes or CD4+ lymphocytes were radiolabeled with 111In-oxinate. Cells were injected into control mice (n = 5) or mice with TNBS colitis (n = 5). Specific abdominal radioactive uptake was determined by SPECT using a dedicated pinhole system 48 h after cell transfer. Radioactive colon uptake was correlated with histology and colon weight as parameters of inflammation., Results: The radioactive colon uptake was most evident in mice with TNBS colitis that received sensitized lymphocytes (uptake ratio [mean +/- SEM], 0.51 +/- 0.03 vs. 0.22 +/- 0.04; P = 0.004). The sensitized 111In-labeled lymphocytes exacerbated colitis compared with nonsensitized lymphocytes. The colon uptake correlated well with both colon weight and histologic score (R2 = 0.836 and 0.933, respectively). The use of purified 111In labeled CD4+ lymphocytes resulted in a similar scintigraphic pattern. Administration of an anti-alpha4 antibody decreased radioactivity colon uptake of the (111)In-labeled cells compared with the control antibody in mice with TNBS colitis (uptake ratio, 0.72 +/- 0.14 to 0.33 +/- 0.03; P = 0.012)., Conclusion: Animal pinhole SPECT can be applied for temporal and spatial analysis of the lymphocyte homing process in experimental colitis. This technique makes possible the in vivo evaluation of therapeutic efficacy of new drugs that interfere with lymphocyte migration. Moreover, colon uptake of radioactivity can be used as a parameter of disease activity in experimental colitis.

Background: Local expression of anti-inflammatory or immunoregulatory genes may offer an alternative treatment of gastrointestinal inflammation., Discussion: We review the basic requirements for gene therapy, the possible routes of delivery, and the different strategies for specific targeting focusing on gastrointestinal inflammation.

Background and Aims: The increasing knowledge of the molecular basis of leukocyte trafficking results in the development of novel anti-inflammatory strategies for inflammatory bowel disease (IBD). For optimal evaluation of therapy efficacy, information about inflammatory activity in bowel segments or lymphocyte recirculation and kinetics in the follow-up of experimental treatment for IBD is needed. The aim of this study was to evaluate a non-invasive scintigraphic technique, able to assess lymphocyte trafficking in a trinitrobenzene sulfonic acid (TNBS) induced mouse colitis model of IBD., Methods: TNBS sensitized and non-sensitized murine total splenocytes were labeled in vitro with 111In-oxine and injected into either control or TNBS colitis BALB/c mice. Biodistribution and specific radioactive uptake, representing transferred cells, were determined by serial dedicated animal planar scintigraphy and pinhole SPECT of the abdomen 4, 24 and 48h post injection of labeled cells. In addition, the severity of inflammation was determined by histological scoring., Results: Migration of 111In labeled splenocytes to the colon increased in time and was maximal at 48h after administration. The highest specific radioactive uptake ratio in the colon after 48h was observed in mice with TNBS colitis that received TNBS sensitized lymphocytes. Histological scoring confirmed the presence of colitis in the TNBS treated groups., Conclusion: Homing of TNBS-sensitized lymphocytes can be assessed in vivo by means of dedicated animal pinhole SPECT. Generally, this technique enables serial measurement of specific cell trafficking with potential of in vivo evaluation of novel anti-inflammatory strategies in inflammatory bowel disease.

Dentritic cells (DC) as antigen-presenting cells are most likely responsible for regulation of abnormal T cell activation in Crohn's disease (CD), a chronic inflammatory bowel disease. We have analyzed the expression of activation and maturation markers on DC in the colon mucosa from patients with CD compared with normal colon, using immunohistochemical techniques. We found two distinct populations of DC present in CD patients: a DC-specific ICAM-3 grabbing non-integrin (DC-SIGN)(+) population that was present scattered throughout the mucosa, and a CD83(+) population that was present in aggregated lymphoid nodules and as single cells in the lamina propria. In normal colon the number of DC-SIGN(+) DC was lower and CD83(+) DC were detected only in very few solitary lymphoid nodules. Co-expression of activation markers and cytokine synthesis was analyzed with three-color confocal laser scanning microscopy analysis. CD80 expression was enhanced on the majority of DC-SIGN(+) DC in CD patients, whereas only a proportion of the CD83(+) DC co-expressed CD80 in CD as well as in normal tissue. Surprisingly, IL-12 and IL-18 were only detected in DC-SIGN(+) DC and not in CD83(+) DC. A similar pattern of cytokine production was observed in normal colon albeit to a much lesser extent. The characteristics of these in-situ-differentiated DC markedly differ from the in-vitro-generated DC that simultaneously express DC-SIGN, CD83 and cytokines.

Background & Aims: Regulatory CD4(+) cells secreting the anti-inflammatory cytokine interleukin (IL)-10 play a key role in maintaining the immune balance in the intestinal mucosa. In this study we engineered primary CD4(+) cells to express IL-10 and investigated the efficacy of this approach in offering protection against experimental colitis., Methods: Spleen-derived CD4(+) cells were transduced by using a retroviral vector to simultaneously express IL-10 and green fluorescent protein (GFP). The therapeutic benefit of CD4(+) cells transduced with IL-10 GFP was studied in experimental colitis, induced by transfer of CD45RB(high) CD4(+) cells to severe combined immunodeficient mice, and in acute trinitrobenzene sulfonic acid (TNBS)-induced colitis., Results: Transferred engineered GFP fluorescent cells were detected for at least 15 weeks in peripheral blood, spleens, colon, and lymph nodes draining the intestine of recipient SCID mice. IL-10-GFP CD4(+) cells prevented CD45RB(high)-induced transfer colitis effectively, whereas no effect was observed after transfer of nontransduced CD4(+) cells. IL-10-GFP CD45RB(high) CD4(+) cells lost the capacity to induce colitis. By contrast, no therapeutic benefit was observed in TNBS-induced colitis., Conclusions: Primary murine CD4(+) cells that were engineered to express IL-10 by retroviral transduction act as regulatory cells in CD45RB(high)-induced transfer colitis. This approach may induce long-term maintenance of mucosal immune homeostasis in Crohn's disease.

Background & Aims: Systemic treatment of Crohn's disease patients using recombinant interleukin (rIL)-10 has not resulted in significant therapeutic benefit presumably because of limited bioavailability and unexpected proinflammatory effects of high-dose rIL-10. Ex vivo gene transfer of the interleukin (IL)-10 gene to gut-homing CD4(+) cells may lead to improved long-term management., Methods: Peripheral blood mononuclear cells (PBMCs) were transduced with a retroviral vector containing the IL-10 and green fluorescent protein (GFP) gene or a control vector containing GFP only. Transduced CD4(+) cells were sorted and maintained in culture for phenotypic and functional analysis., Results: Stimulated IL-10-GFP CD4(+) cells produced significantly higher levels of IL-10 than control cells for at least 4 months. The IL-10 transgene was biologically active and decreased proliferation of IL-10-GFP CD4(+) cells as well as expression of major histocompatibility class (MHC) class II, proliferation of autologous responder cells, and IL-12 production by dendritic cells (DCs). The majority of transduced CD4(+) cells had a gut-homing potential because they expressed the mucosal integrin alpha4beta7, and displayed efficient binding to MAdCAM-1-expressing cells in vitro., Conclusions: Transduction of peripheral blood CD4(+) lymphocytes with IL-10 results in a regulatory phenotype. The use of regulatory gut-homing human CD4(+) cells may provide a novel approach to local delivery of immunomodulatory signals to the intestine in Crohn's disease.

Apart from its role in the synthesis of protein and nitric oxide (NO), and in ammonia detoxification, the amino acid arginine exerts an immunosupportive function. We have studied the role of arginine in immune defense mechanisms in the developing postnatal immune system. In suckling mice, arginine is produced in the small intestine. In F/A-2(+/+) transgenic mice, which overexpress arginase in their enterocytes, circulating and tissue arginine concentrations are reduced to 30-35% of controls. In these mice, the development and composition of the T cell compartment did not reveal abnormalities. However, in peripheral lymphoid organs and the small intestine, B cell cellularity and the number and size of Peyer's patches were drastically reduced, and serum IgM levels were significantly decreased. These phenotypes could be traced to an impaired transition from the pro- to pre-B cell stage in the bone marrow. Cytokine receptor levels in the bone marrow were normal. The development of the few peripheral B cells and their proliferative response after in vitro stimulation was normal. The disturbance in B cell maturation was dependent on decreased arginine levels, as this phenotype disappeared upon arginine supplementation and was not seen in NO synthase- or ornithine transcarbamoylase-deficient mice. We conclude that arginine deficiency impairs early B cell maturation.

The molecular mechanisms underlying inflammatory bowel diseases (IBD) are incompletely characterized. MRP-1, normally expressed in the large and small bowel epithelium, serves as a multidrug resistance protein. In this report we explored the role of MRP1 in IBD. Mrp1-deficient mice (mrp1-/-) were subjected to two different models of IBD. The mrp1-/- mice and wild-type (WT) mice showed equal induction of TNBS colitis, a hapten-induced T-cell mediated disease. However, in DSS colitis more severe disease was observed in mrp1-/- mice. In a survival study, mortality of mrp1-/- mice was higher. In nonlethal DSS colitis, the mean histological colitis score was significantly higher in mrp1-/- mice and showed particularly severe epithelial damage. Although endogenous LTB4 levels were significantly increased in mrp1-/- mice, treatment with a LTB4 antagonist did not reduce disease. We conclude that MRP-1 has an important role in the intestinal epithelial resistance to exogenous injury, but MRP-1 does not affect T-lymphocyte mediated mucosal damage.

Although signal transduction phenomena are well understood in vitro, their relevance for physiology and pathophysiology remains obscure. Recent years, however, have seen a plethora of attempts to link cell biochemistry and disease. At the Luxembourg conference it was attempted to link basal science to disease. In my presentation I focused on our research on stress activated kinases and other signalling elements in this respect in a variety of pro-inflammatory conditions. Knowledge of these pathways has now led to new therapeutic approaches, especially in Crohn's disease, which will be the subject of this commentary.

IL-12(p70), a heterodimer composed of two subunits (p35 and p40), is a key cytokine for Th1 mediated inflammatory responses. We dissected the role of IL-12 in the development of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis by studying mice deficient in IL-12p40, IL-12p35, or IL-12Rbeta1. TNBS-treated IL-12Rbeta1(-/-) and IL-12p35(-/-) mice developed only a mild disease associated with low level IL-18 expression in IL-12p35(-/-) mice. In contrast, IL-12p40(-/-) mice developed more severe colitis than wild-type mice associated with high level colonic IL-18 expression. Administration of IL-12p40 neutralizing mononuclear antibody dramatically increased pathology in IL-12p35(-/-) mice similar to disease scored in IL-12p40(-/-) mice. Numbers of IFN-gamma-producing cells infiltrating the lamina propria were comparably augmented in the different groups of IL-12-mutant and wild-type mice. These results demonstrate that IL-12p40, in contrast to IL-12p70, inhibits TNBS-induced colitis and IL-18 expression independent of IFN-gamma.

To determine the role of interferon (IFN)-gamma in pneumonia, IFN-gamma receptor-deficient (IFN-gamma R(-/-)) and 129/Sv (wild-type [wt]) mice were inoculated intranasally with Streptococcus pneumoniae. Although mortality did not differ between the groups 48 h after inoculation, IFN-gamma R(-/-) mice had significantly fewer pneumococci in their lungs than the wt mice. Similarly, IFN-gamma(-/-) mice had fewer colony-forming units in lungs than wt mice. The relatively increased resistance of IFN-gamma R(-/-) mice was not related to favorable effects on defense mechanisms known to contribute to antibacterial immunity-that is, the neutrophilic influx was reduced and the cytokine and nitric oxide levels were similar or lower in IFN-gamma R(-/-) mice. In contrast, mice treated with anti-IFN-gamma did not demonstrate a consistently altered bacterial outgrowth, compared with mice treated with a control antibody. These data suggest that endogenous IFN-gamma, despite its protective role in defense against intracellular pathogens, does not serve a protective role during pneumococcal pneumonia.