Antigen-specific T cell responses to BK polyomavirus antigens identify functional anti-viral immunity and may help to guide immunosuppression following renal transplantation.

Infection with the polyoma virus BK (BKV) is a major cause of morbidity following renal transplantation. Limited understanding of the anti-viral immune response has prevented the design of a strategy that balances treatment with the preservation of graft function. The proven utility of interferon-gamma enzyme-linked immunospot (ELISPOT) assays to measure T cell responses in immunocompetent hosts was the basis for trying to develop a rational approach to the management of BKV following renal transplantation. In a sample of transplant recipients and healthy controls, comparisons were made between T cell responses to the complete panel of BKV antigens, the Epstein-Barr virus (EBV) antigens, BZLF1 and EBNA1, and the mitogen phytohaemagglutinin (PHA). Correlations between responses to individual antigens and immunosuppressive regimens were also analysed. Antigen-specific T cell responses were a specific indicator of recent or ongoing recovery from BKV infection (P < 0·05), with responses to different BKV antigens being highly heterogeneous. Significant BKV immunity was undetectable in transplant patients with persistent viral replication or no history of BKV reactivation. Responses to EBV antigens and mitogen were reduced in patients with BKV reactivation, but these differences were not statistically significant. The T cell response to BKV antigens is a useful and specific guide to recovery from BKV reactivation in renal transplant recipients, provided that the full range of antigenic responses is measured.

Impaired mucosal defense to acute colonic injury in mice lacking cyclooxygenase-1 or cyclooxygenase-2.

To investigate roles in intestinal inflammation for the 2 cyclooxygenase (COX) isoforms, we determined susceptibility to spontaneous and induced acute colitis in mice lacking either the COX-1 or COX-2 isoform. We treated wild-type, COX-1(-/-), COX-2(-/-), and heterozygous mice with dextran sodium sulfate (DSS) to provoke acute colonic inflammation, and we quantified tissue damage, prostaglandin (PG) E(2), and interleukin-1beta. No spontaneous gastrointestinal inflammation was detected in mice homozygous for either mutation, despite almost undetectable basal intestinal PGE(2) production in COX-1(-/-) mice. Both COX-1(-/-) and COX-2(-/-) mice showed increased susceptibility to a low-dose of DSS that caused mild colonic epithelial injury in wild-type mice. COX-2(-/-) mice were more susceptible than COX-1(-/-) mice, and selective pharmacologic blockade of COX-2 potentiated injury in COX-1(-/-) mice. At a high dose, DSS treatment was fatal to 50% of the animals in each mutant group, but all wild-type mice survived. DSS treatment increased PGE(2) intestinal secretion in all groups except COX-2(-/-) mice. These results demonstrate that COX-1 and COX-2 share a crucial role in the defense of the intestinal mucosa (with inducible COX-2 being perhaps more active during inflammation) and that neither isoform is essential in maintaining mucosal homeostasis in the absence of injurious stimuli.

Protective effects of neurokinin-1 receptor during colitis in mice: role of the epidermal growth factor receptor.

1. The role of substance P and its high affinity neurokinin-1 receptor in colitis has not been fully elucidated. We assessed the participation of neurokinin-1 receptor in colitis using the 2,4,6,-trinitrobenzensulphonic acid and dextran sulphate-induced animal models of colitis and genetically-engineered, neurokinin-1 receptor-deficient mice. 2. Clinical signs, macroscopic and histologic damage associated with 2,4,6,-trinitrobenzensulphonic acid (12 days) and dextran sulphate (5 days) colitis were more severe in neurokinin-1 deficient than in wild-type mice, while immunoreactivities for epidermal growth factor and its receptor were similar in the colon of both mice strains before and after colitis. 3. Substance P, dose-dependently induced intestinal fibroblast proliferation and enhanced epidermal growth factor-induced proliferation in intestinal fibroblasts isolated from wild-type, but not from neurokinin-1 receptor deficient mice. 4. Substance P-induced intestinal fibroblast proliferation required the presence of epidermal growth factor receptor with kinase activity. Furthermore, substance P induced epidermal growth factor tyrosine phosphorylation and activation in normal intestinal fibroblasts. 5. Our results indicate that in mice lacking the neurokinin - 1 receptor, substance P plays a protective role in prolonged experimental colitis.

Influence of 5-HT3 receptor antagonists in visceromotor and nociceptive responses to rectal distension before and during experimental colitis in rats.

We investigated the possible involvement of 5-HT3 receptors in the colonic motor alterations and abdominal pain evoked by rectal distension (RD) in rats, under normal and inflammatory conditions. Responses to RD were evaluated by electromyography in rats treated with 5-HT3 antagonists (ondansetron and cilansetron, 0.1 and 1 mg/kg, intraperitoneally), before and 3 days after intrarectal administration of TNB/ethanol. RD evoked a significant (P < 0.05) and gradual inhibition of the occurrence of colonic spike bursts (SB) and a gradual increase in abdominal SB from 11 mm in diameter on wards. Ondansetron and cilansetron (0.1 mg/kg) significantly reduced both the colonic (62 and 66%, respectively) and the abdominal response (28 and 61%, respectively) for an 11 mm diameter of RD. After TNB/ethanol, both colonic and abdominal responses to RD were significantly (P < 0.05) enhanced and appeared for a lower diameter (9 mm) (colon: 4.8 +/- 0.9 vs 8.4 +/- 1.1, abdomen: 7.7 +/- 1.5 vs 0.5 +/- 0.4). Cilansetron (0.1, 1 mg/kg) significantly (P < 0.05) attenuated the TNB-induced colonic motor inhibition, while ondansetron and cilansetron (0.1, 1 mg/kg) reduced the TNB-induced increase in abdominal response. We conclude that 5-HT and 5-HT3 receptors mediate RD-induced viscerosensitive alterations in rats, both in normal conditions and during TNB-induced rectocolitis. However, the relative efficacy of the 5-HT3 receptor antagonists depends on the experimental conditions (intact or inflamed bowel) and does not appear to increase with the dose.

Prostaglandins and inflammation: the cyclooxygenase controversy.

Prostaglandins (PGs) are arachidonic acid metabolites produced by the action of the enzyme cyclooxygenase (COX). Although PGs are important mediators of inflammation in various diseases, they also are key factors in the physiological regulation of gastrointestinal and renal homeostasis. The finding that two distinct COX isoforms are responsible for PG synthesis has provided basis to the opposite actions of PGs in inflammation and homeostasis regulation. COX-1-derived PGs are thought to mediate cytoprotective actions on the gastrointestinal mucosa, whereas COX-2-derived PGs are assumed to display pro-inflammatory properties. This dichotomy has led to the development of selective inhibitors of COX-2 activity which are safer for the gastrointestinal mucosa than the classic inhibitors of both COX isoforms. However, some COX-2 anti-inflammatory properties have been recently demonstrated in several experimental models of inflammation. These studies have raised some concern about the potential adverse effects of COX-2 selective inhibitors. In addition, there is evidence that COX-1 displays pro-inflammatory properties, depending on the organ and on the stage of the inflammatory response. This review will focus on the roles of COX-1 and COX-2 in inflammation, based on studies involving pharmacologic COX inhibitors as well as COX knock-out mice, with a particular emphasis on the gastrointestinal tract.

Involvement of neurokinin 1 and 2 receptors in viscerosensitive response to rectal distension in rats.

BACKGROUND/AIMS: Tachykinins participate in somatic pain and intestinal motility control. The role of tachykinin receptors in both colonic motor disturbances and visceral pain (abdominal contractions as an index of visceral pain) induced by rectal distension were investigated. METHODS: Rats were surgically prepared with electrodes implanted on the proximal colon and the abdominal striated muscles. Catheters were implanted in lateral ventricles of the brain. Rectal distension was performed by inflation of a balloon (0.1-1.6 mL) rectally inserted. CP-96,345 and RP-67,580 (neurokinin [NK] 1 antagonists) and SR-48,968 (NK2 antagonist) were injected intraperitoneally (IP) or intracerebroventricularly (ICV) 20 minutes before distension. GR-73,632 and GR-64,639 (NK1, NK2 agonists) were infused intravenously at 0.15 micrograms.kg-1.min-1. RESULTS: Rectal distension evoked a significant inhibition of colonic motility and an increase in abdominal contractions. CP-96,345 injected ICV (0.2-0.8 mg/kg) or IP (5-10 mg/kg) and RP-67,580 (0.2 mg/kg IP) eliminated distension-induced colonic inhibition but did not affect abdominal response. SR-48,968 did not affect colonic response but significantly reduced visceral pain (0.4, 0.8 mg/kg ICV: 5-10 mg/kg IP). GR-73,632 enhanced the rectal distension-induced colonic inhibition, whereas GR-64,349 induced a greater abdominal response. CONCLUSIONS: NK1 receptors mediate the rectocolonic inhibitory reflex, whereas NK2 receptors participate in visceral pain; both responses involve central structures.

Experimental colitis alters visceromotor response to colorectal distension in awake rats.

The influence of intermittent colorectal distension (CRD) on proximal colonic motility and abdominal pain perception was investigated in awake rats equipped with intraparietal electrodes on the cecum, proximal colon, and abdomen, before and three days after rectocolitis induction by trinitrobenzene sulfonic acid (TNB)/ethanol. The normal myoelectrical activities of cecum and proximal colon [5.2 +/- 0.5 and 9.7 +/- 0.7 long spike bursts (LSB) per 5 min, respectively] were significantly (P < 0.05) and gradually decreased by control CRD, at diameters above 9 mm. At the maximum CRD diameter (13.7 mm), 1.6 +/- 0.6 cecal and 3.9 +/- 0.8 colonic spike bursts occurred per 5 min (respectively, 69 and 60% decreases). This upstream inhibition was accompanied by a significant (P < 0.05) and gradual increase in abdominal contractions (0.4 +/- 0.4 per 5 min in control vs 23.4 +/- 1.9 in response to 13.7 mm in diameter). Three days after TNB/ethanol, visceromotor and abdominal responses were significantly (P < 0.05) enhanced at the least CRD diameter of 9 mm (cecum: 3.1 +/- 0.4 after TNB vs 5.0 +/- 0.7 in control; proximal colon: 5.1 +/- 0.9 vs 9.3 +/- 2.2; abdomen: 7.7 +/- 1.5 vs 0.5 +/- 0.4). We conclude that in awake rats, CRD evokes both abdominal contractions in response to pain and inhibition of cecal and proximal colonic motility, which thresholds are both lowered by TNB-induced rectocolitis.

Platelet-activating factor and interleukin 1 are involved in colonic dysmotility in experimental colitis in rats.

BACKGROUND: Intracolonic administration of trinitrobenzene sulfonic acid (TNBS) to rats produces chronic colitis associated with an increased release of eicosanoids, platelet-activating factor (PAF), and interleukins. METHODS: Motor effects of TNBS on proximal colon were evaluated electromyographically in rats. Mediator involvement was investigated using eicosanoids and PAF antagonists. RESULTS: The colonic myoelectrical activity was 59 +/- 17 spike bursts per hour lasting 6.9 +/- 1.3 seconds. Two to eight days after TNBS treatment, spike-burst duration was significantly (P < 0.05) higher, with a maximal 1.5-4-fold enhancement at day 3. These alterations were significantly (P < 0.05) reduced by daily treatment with MK-886, a 5-lipoxygenase inhibitor (10 mg/kg, orally), whereas indomethacin (1 mg/kg per day, intramuscularly) was ineffective. At day 3, RP55778, a PAF antagonist (45, 60 mg/kg, intraperitoneally), and rIRAP, an interleukin 1 antagonist (0.3 mg/kg, intraperitoneally) but not KT1-32, a thromboxane A2 antagonist (30, 60 mg/kg orally), nor SKF104,353, a leukotriene D4 antagonist (2, 4 mg/kg, orally), significantly (P < 0.05) reduced the TNB-induced motor effects. CONCLUSION: TNBS-induced colitis in rats involves a delayed long-lasting dysmotility involving PAF, interleukin 1, and some leukotrienes but not leukotriene D4, thromboxane A2, or other cyclo-oxygenase products.

Specific NF-kappaB blockade selectively inhibits tumour necrosis factor-alpha-induced COX-2 but not constitutive COX-1 gene expression in HT-29 cells.

Cyclo-oxygenase (COX) is the key regulatory enzyme of the prostaglandin/eicosanoid pathway. While COX-1 is mostly constitutively expressed, the COX-2 isoform is inducible by proinflammatory cytokines. We used an adenoviral vector containing an NF-kappaB super-repressor (Ad5IkappaB) to investigate the role of NF-kappaB in tumour necrosis factor-alpha (TNF-alpha)-mediated COX-2 gene expression in a colonic epithelial cell line. COX-1 mRNA and protein were constitutively expressed in uninfected, control Ad5LacZ- or Ad5IkappaB-infected HT-29 cells with no apparent change following TNF-alpha exposure. COX-2 mRNA and protein expression was undetectable in unstimulated cells but was strongly up-regulated after TNF-alpha stimulation in uninfected and Ad5LacZ-infected HT-29 cells. This induction was prevented in Ad5IkappaB cells. TNF-alpha increased prostaglandin E2 production by 20-fold in Ad5LacZ-infected HT-29 cells compared with uninfected cells and was significantly inhibited in Ad5IkappaB-infected cells in agreement with the COX-2 mRNA findings. We conclude that NF-kappaB activation is critical in mediating COX-2, but not COX-1 gene expression in HT-29 cells. Selective inhibition of COX-2 expression with the NF-kappaB super-repressor may be useful in distinguishing the role of inducible versus constitutive prostaglandins in intestinal function and provides greater specificity than pharmacological inhibitors.