Increased potency of a novel complement factor 5a receptor antagonist in a rat model of inflammatory bowel disease

We have previously shown that complement factor 5a(C5a) plays a role in the pathogenesis of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats by using the selective, orally active C5a antagonist AcF-[OP(D-Cha) WR]. This study tested the efficacy and potency of a new C5a antagonist, hydrocinnamate (HC)-[OP(D-Cha) WR], which has limited intestinal lumenal metabolism, in this model of colitis. Analogs of AcF-[OP(D-Cha) WR] were examined for their susceptibility to alimentary metabolism in the rat using intestinal mucosal washings. One metabolically stable analog, HC-[OP(D-Cha)WR], was then evaluated pharmacokinetically and investigated at a range of doses (0.03 - 10 mg/kg/ day p.o.) in the 8-day rat TNBS- colitis model, against the comparator drug AcF-[OP(D-Cha) WR]. Using various amino acid substitutions, it was determined that the AcF moiety of AcF-[OP(D-Cha) WR] was responsible for the metabolic instability of the compound in intestinal mucosal washings. The analog HC-[OP( D-Cha) WR], equiactive in vitro to AcF-[OP(D-Cha) WR], was resistant to intestinal metabolism, but it displayed similar oral bioavailability to AcF-[OP(D-Cha) WR]. However, in the rat TNBS- colitis model, HC-[OP(D-Cha) WR] was effective at reducing mortality, colon edema, colon macroscopic scores, and increasing food consumption and body weights, at 10- to 30- fold lower oral doses than AcF-[OP( D-Cha) WR]. These studies suggest that resistance to intestinal metabolism by HC-[OP(D-Cha) WR] may result in increased local concentrations of the drug in the colon, thus affording efficacy with markedly lower oral doses than AcF-[OP(D-Cha) WR] against TNBS-colitis. This large increase in potency and high efficacy of this compound makes it a potential candidate for clinical development against intestinal diseases such as inflammatory bowel disease.

Il-1 beta breaks tolerance through inhibition of regulatory T cell function

IL-1 is a key proinflammatory driver of several autoimmune diseases including juvenile inflammatory arthritis, diseases with mutations in the NALP/cryopyrin complex and Crohn’s disease, and is genetically or clinically associated with many others. IL-1 is a pleiotropic proinflammatory cytokine; however the mechanisms by which increased IL-1 signaling promotes autoreactive T cell activity are not clear. Here we show that autoimmune-prone NOD and IL-1 receptor antagonist-deficient C57BL/6 mice both produce high levels of IL-1, which drives autoreactive effector cell expansion. IL-1beta drives proliferation and cytokine production by CD4+CD25+FoxP3– effector/memory T cells, attenuates CD4+CD25+FoxP3+ regulatory T cell function, and allows escape of CD4+CD25– autoreactive effectors from suppression. Thus, inflammation or constitutive overexpression of IL-1beta in a genetically predisposed host can promote autoreactive effector T cell expansion and function, which attenuates the ability of regulatory T cells to maintain tolerance to self.

Orofacial granulomatosis: A diagnostic problem for the unwary and a management dilemma. Case reports

Orofacial granulomatosis is a condition that, may be difficult to diagnose for those unfamiliar with the entity. This paper describes two cases and addresses the presentation, pathogenesis and treatment. The clinical recognition of his condition is important as is the subsequent investigation by an appropriate specialist. Management of patients needs to take into account the results of further investigations, the patient's expectations, and the severity of the condition.

CD40 and CD86 upregulation with divergent CMRF44 expression on blood dendritic cells in inflammatory bowel diseases

OBJECTIVE: Dendritic cells (DC) are the only antigen-presenting cells that can activate naive T lymphocytes and initiate a primary immune response. They are also thought to have a role in immune tolerance. DC traffic from the blood to peripheral tissue where they become activated. They then present antigen and the costimulating signals necessary to initiate an immune response. In this study, we investigated the number, subsets, and activation pattern of circulating and intestinal DC from patients with clinically mild ulcerative colitis (UC) or Crohn's disease. METHODS: Patients were recruited, if they were not taking immunosuppressive therapy, and were assessed for clinical severity of their disease using for UC, the Clinical Activity Index, and for Crohn's disease, the Crohn's Disease Activity Index. Blood CD11c(+) and CD11c(-) DC subsets, expression of costimulatory antigens, CD86 and CD40, and the early differentiation/activation antigen, CMRF44, were enumerated by multicolor flow cytometry of lineage negative (lin(-) = CD3(-), CD19(-), CD14(-), CD16(-)) HLA-DR+ DC. These data were compared with age-matched healthy and the disease control groups of chronic noninflammatory GI diseases (cGI), acute noninflammatory GI diseases (aGI), and chronic non-GI inflammation (non-GI). In addition, cryostat sections of colonoscopic biopsies from healthy control patients and inflamed versus noninflamed gut mucosa of inflammatory bowel disease (IBD) patients were examined for CD86(+) and CD40(+)lin(-) cells. RESULTS: Twenty-one Crohn's disease and 25 UC patients, with mean Crohn's Disease Activity Index of 98 and Clinical Activity Index of 3.1, and 56 healthy controls, five cGI, five aGI, and six non-GI were studied. CD11c(+) and CD11c(-) DC subsets did not differ significantly between Crohn's, UC, and healthy control groups. Expression of CD86 and CD40 on freshly isolated blood DC from Crohn's patients appeared higher (16.6%, 31%) and was significantly higher in UC (26.6%, 46.3%) versus healthy controls (5.5%, 25%) (p = 0.004, p = 0.012) and non-GI controls (10.2%, 22.8%) (p = 0.012, p = 0.008), but not versus cGI or aGI controls. CD86(+) and CD40(+) DC were also present in inflamed colonic and ileal mucosa from UC and Crohn's patients but not in noninflamed IBD mucosa or normal mucosa. Expression of the CMRF44 antigen was low on freshly isolated DC, but it was upregulated after 24-h culture on DC from all groups, although significantly less so on DC from UC versus Crohn's or healthy controls (p = 0.024). The CMRF44(+) antigen was mainly associated with CD11c(+) DC, and in UC was inversely related to the Clinical Activity Index (r = -0.69, p = 0.0002). CONCLUSIONS: There is upregulation of costimulatory molecules on blood DC even in very mild IBD but surprisingly, there is divergent expression of the differentiation/activation CMRF44 antigen. Upregulation of costimulatory molecules and divergent expression of CMRF44 in blood DC was also apparent in cGI and aGI but not in non-GI or healthy controls, whereas intestinal CD86(+) and CD40(+) DC were found only in inflamed mucosa from IBD patients. Persistent or distorted activation of blood DC or divergent regulation of costimulatory and activation antigens may have important implications for gut mucosal immunity and inflammation. (Am J Gastroenterol 2001;96:2946-2956. (C) 2001 by Am. Coll. of Gastroenterology).

Activation of ion secretion via proteinase-activated receptor-2 in human colon

Proteinase-activated receptor (PAR) type 2 (PAR-2) has been shown to mediate ion secretion in cultured epithelial cells and rat jejunum. With the use of a microUssing chamber, we demonstrate the role of PAR-2 for ion transport in native human colonic mucosa obtained from 30 normal individuals and 11 cystic fibrosis (CF) patients. Trypsin induced Cl- secretion when added to the basolateral but not luminal side of normal epithelia. Activation of Cl- secretion by trypsin was inhibited by indomethacin and was further increased by cAMP in normal tissues but was not present in CF colon, indicating the requirement of luminal CF transmembrane conductance regulator. Effects of trypsin were largely reduced by low Cl-,by basolateral bumetanide, and in the presence of barium or clotrimazole, but not by tetrodotoxin. Furthermore, trypsin-induced secretion was inhibited by the Ca2+-ATPase inhibitor cyclopiazonic acid and in low-Ca2+ buffer. The effects of trypsin were almost abolished by trypsin inhibitor. Thrombin, an activator of PAR types 1, 3, and 4, had no effects on equivalent short-circuit currents. The presence of PAR-2 in human colon epithelium was confirmed by RT-PCR and additional experiments with PAR-2-activating peptide. PAR-2-mediated intestinal electrolyte secretion by release of mast cell tryptase and potentiation of PAR-2 expression by tumor necrosis factor-alpha may contribute to the hypersecretion observed in inflammatory processes such as chronic inflammatory bowel disease.

CCR5-Delta 32 mutation is strongly associated with primary sclerosing cholangitis

CCR5 plays a key role in the distribution of CD45RO+ T cells and contributes to generation of a T helper 1 immune response. CCR5-Delta32 is a 32-bp deletion associated with significant reduction in cell surface expression of the receptor. We investigated the role of CCR5-Delta32 on susceptibility to ulcerative colitis (UC), Crohn's disease ( CD) and primary sclerosing cholangitis (PSC). Genotype and allelic association analyses were performed in 162 patients with UC, 131 with CD, 71 with PSC and 419 matched controls. There was a significant difference in CCR5 genotype (OR 2.27, P = 0.003) between patients with sclerosing cholangitis and controls. Similarly, CCR5-Delta32 allele frequency was significantly higher in sclerosing cholangitis (17.6%) compared to controls (9.9%, OR 2.47, P = 0.007) and inflammatory bowel disease patients without sclerosing cholangitis ( 11.3%, OR 1.9, P = 0.027). There were no significant differences in CCR5 genotype or allele frequency between those with either UC or CD and controls. Genotypes with the CCR5-Delta32 variant were increased in patients with severe liver disease defined by portal hypertension and/or transplantation (45%) compared to those with mild liver disease (21%, OR 3.17, P = 0.03). The CCR5-Delta32 mutation may influence disease susceptibility and severity in patients with PSC.

Tumour necrosis factor inhibitors

The cytokine, tumour necrosis factor-alpha (TNF-alpha) plays a key role in the pathogenesis of many chronic inflammatory and rheumatic diseases, in particular, Crohn's disease, rheumatoid arthritis, ankylosing spondylitis and psoriatic arthritis. Controlled trials have shown that the TNF inhibitors (etanercept, infliximab and adalimumab) significantly reduce symptoms and signs, improve function and quality of life, and reduce radiologically evident damage in patients with rheumatoid diseases. For reasons that are not entirely clear, etanercept does not work in Crohn's disease. Injection site and intravenous reactions and increased risk of infection (in particular, reactivation of tuberculosis) are associated with the use of these agents. Increased risk of lymphoproliferative disease, the development of lupus-like syndromes and demyelination, including optic neuritis and reactivation of multiple sclerosis, are under evaluation in long-term follow-up studies. The TNF inhibitors are expensive (about $18000 per year), and in some patients need to be given continuously to maintain benefit, even in the presence of other immunosuppressive therapy.

Inflammation suppressor genes: please switch out all the lights

An effective immune system requires rapid and appropriate activation of inflammatory mechanisms but equally rapid and effective resolution of the inflammatory state. A review of the canonical host response to gram-negative bacteria, the lipopolysaccharide-Toll-like receptor 4 signaling cascade, highlights the induction of repressors that act at each step of the activation process. These inflammation suppressor genes are characterized by their induction in response to pathogen, typically late in the macrophage activation program, and include an expanding class of dominant-negative proteins derived from alternate splicing of common signaling components. Despite the expanse of anti-inflammatory mechanisms available to an activated macrophage, the frailty of this system is apparent in the large numbers of genes implicated in chronic inflammatory diseases. This apparent lack of redundancy between inflammation suppressor genes is discussed with regard to evolutionary benefits in generating a heterogeneous population of immune cells and consequential robustness in defense against new and evolving pathogens.

Characterization of tumour-infiltrating lymphocytes and apoptosis in colitis-associated neoplasia: comparison with sporadic colorectal cancer

The development of colorectal cancer is a major complication for patients with chronic idiopathic colitis. Colitis-associated tumours tend to occur at a younger age and be more aggressive than sporadic colorectal cancers. While we have previously associated the presence of tumour-infiltrating lymphocytes (TILs) and increased apoptosis in sporadic colorectal cancer with high-level microsatellite instability and improved prognosis, little is known of the relationship between these variables in colitis-associated colorectal cancer. The aim of this study was to correlate TILs and tumour cell apoptosis in colitis-associated neoplasms stratified according to microsatellite instability. Twenty tumour and 11 dysplastic samples resected from 21 patients with long-standing colitis were analysed for microsatellite instability at 10 microsatellite markers. TIL distribution (CD3, CD8) and function (granzyme B) were quantified by immunohistochemistry. Neoplastic cell apoptosis was assessed using the M30 CytoDEATH antibody. These findings were compared with 40 microsatellite stable (MSS) sporadic colorectal cancers previously evaluated for TILs and neoplastic apoptosis. Low-level microsatellite instability was found in 1/20 colitis-associated tumours. All other colitis-associated lesions were designated MSS. CD3(+) and CD8(+) TIL counts were significantly higher in colitis-associated lesions compared with NISS sporadic colorectal cancer (p < 0.0001, p = 0.001 respectively). Despite their higher TIL density, colitis-associated tumours were more likely to present late (Dukes' stage C or D) (P = 0.02). Functionally, colitis-associated TILs demonstrated significantly less granzyme B expression compared to sporadic cancers (p = 0.002). The level of tumour cell apoptosis was similar between the two groups (sporadic, 1.53%; colitis cancers, 1.45%). In conclusion, NISS colitis-associated tumours have a higher prevalence of CD3(+)/CD8(+) TILs but no associated increase in tumour cell killing by apoptosis. Unlike cytotoxic T cells in sporadic colorectal cancer, TILs do not appear to enhance the prognosis of colitis-associated colorectal cancer. This may be related to an impairment of granzyme B expression within these lesions. Copyright (c) 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.