The green tea polyphenol (-)-epigallocatechin-3-gallate blocks nuclear factor-kappa B activation by inhibiting I kappa B kinase activity in the intestinal epithelial cell line IEC-6.

The I kappa B kinase complex (IKK) mediates activation of the transcription factor nuclear factor-kappa B (NF-kappa B). We previously showed that green tea polyphenols inhibited endotoxin-mediated tumor necrosis factor-alpha (TNF alpha) production by blocking NF-kappa B activation. In this study, we evaluated whether green tea polyphenols inhibit NF-kappa B by blocking IKK activity. We assessed IKK activity by detecting changes in phosphorylation of an I kappa B alpha-glutathione S-transferase (GST) fusion protein. IEC-6 cells pretreated with an extract of green tea polyphenols (GrTPs; 0--0.4 mg/ml) had diminished TNF alpha-induced IKK and NF-kappa B activity. Of the various GrTPs, (-)-epigallocatechin-3-gallate (EGCG) was the most potent inhibitor. We next examined whether EGCG inhibited activated IKK. In cytosolic extracts of TNF alpha-stimulated cells, EGCG inhibited phosphorylation of I kappa B alpha-GST (IC(50) > 18 microM) consistent with inhibition of IKK activity. Using other polyphenols, we showed that the gallate group was essential for inhibition, and antioxidants were ineffective in blocking activated IKK. Importantly, EGCG decreased IKK activity in cytosolic extracts of NIK transiently transfected cells. This latter finding showed that our findings were not related to nonspecific kinase activity. In conclusion, EGCG is an effective inhibitor of IKK activity. This may explain, at least in part, some of the reported anti-inflammatory and anticancer effects of green tea.

Green tea polyphenol extract attenuates inflammation in interleukin-2-deficient mice, a model of autoimmunity.

Green tea polyphenols (GrTP) have been previously shown to decrease endotoxin-induced tumor necrosis factor-alpha production and lethality in mice. Our present studies demonstrate that GrTP inhibit inflammatory responses and may be useful in treating chronic inflammatory states, such as inflammatory bowel disease. In this preliminary study, we examined whether GrTP decrease disease activity in interleukin-2-deficient (IL-2(-/-) mice. Eight-week old IL-2(-/-) C57BL/6J mice who were fed nonpurified diet were randomly assigned to receive water with GrTP (5 g/L) or water alone (control) for up to 6 wk. After 1 wk, explant colon and lamina propria lymphocyte (LPL) cultures were established from a subgroup of mice and supernatants collected. Culture supernatants from GrTP-treated mice showed decreased spontaneous interferon-gamma and tumor necrosis factor-alpha secretion compared with that of controls. At 6 wk, the GrTP group had less severe colitis as demonstrated by lower histologic scores and wet colon weights. This was associated with lower plasma levels of serum amyloid A, increased weight gain and improved hematocrits. These results show that GrTP attenuated inflammation in IL-2(-/-) mice and suggest a role for GrTP in treating chronic inflammatory diseases such as inflammatory bowel disease.

A rat model for combined Trypanosoma cruzi and Pneumocystis carinii infection.

Dexamethasone treated rats inoculated with Trypanosoma cruzi developed acute parasitemia. In addition, these animals concomitantly developed severe Pneumocystis carinii pneumonia (PCP) and died after 4 weeks of immunosuppression (100%). However, immunocompetent (untreated) rats inoculated with T. cruzi did not acquire P. carinii and recovered from T. cruzi infection. Rats immunosuppressed, but not inoculated with T. cruzi, developed only PCP and died 5-6 weeks later (93%). In contrast, immunocompetent or immunocompromised IRC mice infected with T. cruzi all died of acute parasitemia in only 8-12 days with no detectable PCP infection. In conclusion, rats immunosuppressed and T. cruzi inoculated can serve as a MOPPS model for a single drug evaluation. In addition, T. cruzi infection independently does not provoke P. carinii pneumonia in this model. Finally, patients with Chagas' disease treated with corticosteroids may be at risk for PCP and should be considered for chemoprophylaxis.

Increased serum amyloid a levels reflect colitis severity and precede amyloid formation in IL-2 knockout mice.

The lack of sensitive and relatively non-invasive measures has hampered monitoring the clinical course of spontaneously developing colitis in IL-2-deficient (-/-) mice. We selected (i) to study the correlation of the acute phase plasma proteins serum amyloid A (SAA) and serum amyloid P component (SAP) levels with colonic disease and (ii) to characterize the amyloidosis in the IL-2(-/-)animals. IL-2(-/-)mice exhibited increasing severity of gross intestinal inflammation with age, confined to the distal colon. Histologically, the colonic disease score increased serially in IL-2(-/-)animals. Wild-type mice showed no activity, while 16-week-old IL-2(+/-)animals had minimal colitis with small ulcers and lamina propria inflammatory infiltrate. Periportal hepatitis was present and positive Congo red staining indicated amyloidosis of the liver and spleen in 16 week IL-2(-/-)mice. SAA immunostaining in the liver and spleen was increased in the 8 week and 16 week IL-2(-/-)and 16 week IL-2(+/-)animals indicating AA amyloid deposits. Plasma SAA and SAP levels were markedly elevated, and generally preceded the onset of colitis and reflected its severity. Northern analysis showed markedly increased SAA expression in the liver and intestine of IL-2(-/-)and intestine of IL-2(+/-)16-week-old animals. Increased intestinal expression of SAA3 (lamina propria macrophages) indicates local inflammation in IL-2(+/-)animals at 16 weeks. Treatment of 3-week-old animals with systemic IL-2 or IL-1 receptor antagonist (IL-1ra) delayed inflammation, postponed the increase in SAA levels and minimized disease onset. These results further demonstrate that IL-2 plays a significant role in normal immune responses in the body and that plasma SAA levels both reflect colonic disease severity and may indicate subclinical disease in both IL-2(-/-)and IL-2(+/-)mice. Furthermore. The mechanism of IL-2-deficient induced colitis appears to be mediated in part through the increase in IL-1. In addition, the IL-2(-/-)mouse of spontaneous enterocolitis may provide a unique system for studying spontaneously developing AA amyloidosis.

Cyclooxygenase 2 expression is increased in the stroma of colon carcinomas from IL-10(-/-) mice.

BACKGROUND & AIMS: The pathological and molecular changes associated with colitis-associated colorectal cancer and sporadic colorectal cancer are considered to be distinct. Therefore, we have used a mouse model of ulcerative colitis to determine if expression of the enzyme cyclooxygenase (COX)-2 is increased in colitis-associated tumors. METHODS: Reverse-transcription polymerase chain reaction and Western analysis were used to determine if COX-2 expression is increased in these tumors; in situ hybridization and immunohistochemistry were used to determine the localization of COX-2. RESULTS: Increased levels of COX-2 messenger RNA and protein were detected in interleukin (IL)-10 (-/-) tumors and in an inflamed region of the colon that contained no macroscopically detected tumors. This expression was localized to the inflammatory cells associated with ulcerated regions of the tumor by in situ hybridization and immunohistochemistry. Increased COX-2 expression was also associated with the areas of the tumor expressing alpha-smooth muscle actin, which is a molecular marker for subepithelial myofibroblasts. The association between COX-2 expression and subepithelial myofibroblasts was also noted in tumors derived from the multiple intestinal neoplasia mice (Min/+) and from carcinogen-induced tumors. CONCLUSIONS: These results indicate that COX-2 is expressed very early in the pathogenesis of colitis-associated tumors, and that the expression pattern is similar to that seen in tumors from azoxymethane-treated and Min/+ mice.

Increased nuclear factor-kappaB activation in colitis of interleukin-2-deficient mice.

Recent studies support nuclear factor-kappaB (NF-kappaB) as a critical transcription factor in inflammatory bowel disease. We examined NF-kappaB and its inhibitors, IkappaB-alpha and IkappaB-beta, in the colitis of interleukin-2 deficient (IL-2-/-) mice at the ages of 5, 10, and 15 weeks and compared them with those of age-matched wild-type mice. Colon levels of nuclear NF-kappaB and mRNA for NF-kappaB responsive cytokines interleukin-1beta and tumor necrosis factor-alpha were markedly increased in interleukin-2-/-mice. Colon interleukin-1beta protein levels were significantly elevated, consistent with increased interleukin-1beta mRNA, whereas tumor necrosis factor-alpha protein levels were either lower than those of the control group or did not differ. Protein levels of the immunomodulatory cytokine interleukin-10 were diminished. The NF-kappaB responsive IkappaB-alpha was also increased, mirroring NF-kappaB activation. In contrast, IkappaB-beta levels did not differ from those of wild-type mice in the 5- and 10-week groups and were only mildly increased in the 15-week group. Serum amyloid A, an acute phase protein that also is NF-kappaB-responsive, was dramatically elevated in the serum of interleukin-2-/- mice and correlated with the severity of the colitis. These data support a role for NF-kappaB in the pathogenesis of intestinal inflammation in interleukin-2-/- mice. The measurement of NF-kappaB in colon tissue samples may provide a sensitive means of assessing the state of activation of the mucosal immune response, and serum amyloid A appears to be a reliable biochemical marker of disease activity.

What could be causing chronic abdominal pain? Anything from common peptic ulcers to uncommon pancreatic trauma.

Establishing a reasonable diagnosis and treatment plan in patients with chronic abdominal pain can be difficult and frustrating. Most cases involve a common and readily identified condition (e.g., gallbladder disease, GERD, irritable bowel syndrome). Other cases, however, resist ready diagnosis because they offer no intra-abdominal explanation. In this article, the authors summarize several diverse possibilities as the source of the pain, and they describe how to approach evaluation to avoid unnecessary testing.

Preliminary evaluation of safety and activity of recombinant human interleukin 11 in patients with active Crohn's disease.

BACKGROUND & AIMS: Recombinant human interleukin 11 (rhIL-11) is a cytokine with thrombocytopoietic activity and anti-inflammatory and mucosal protective effects. The objectives of this study were to investigate the safety and tolerability of rhIL-11 in patients with Crohn's disease and to explore the effects of dose and schedule on platelet count and Crohn's disease activity. METHODS: A multicenter, double-masked, placebo-controlled, dose-escalation study of 76 patients with active Crohn's disease was performed. Patients were randomized to receive subcutaneous placebo or rhIL-11 at doses of 5, 16, or 40 microgram. kg-1. wk-1 given 2 or 5 times weekly for 3 weeks. Clinical and laboratory safety data were recorded, and disease activity was measured at each visit. RESULTS: Subcutaneous injection of rhIL-11 generally was well tolerated. Significantly greater increases in platelet counts were found among patients receiving rhIL-11 40 microgram. kg-1. wk-1 as 2 or 5 weekly doses and 16 microgram. kg-1. week-1 as 5 weekly doses compared with patients receiving placebo (P < 0.05). Patients receiving 16 microgram. kg-1. wk-1 had the highest clinical response rates, with a response seen in 42% of patients (5/12) receiving 5 weekly doses and 33% of patients (4/12) receiving 2 weekly doses, compared with 7% of patients (1/15) receiving placebo. CONCLUSIONS: Short-term treatment with rhIL-11 is well tolerated in patients with active Crohn's disease. The thrombocytopoietic effect of rhIL-11 seems to be both dose and schedule dependent and may be minimized with retained clinical benefit in Crohn's disease at 16 microgram. kg-1. wk-1 given in 2 equal doses.

Green tea polyphenols block endotoxin-induced tumor necrosis factor-production and lethality in a murine model.

Green tea polyphenols are potent antioxidants. They have both anti-cancer and anti-inflammatory effects. However, their mechanisms of actions remain unclear. In inflammation, tumor necrosis factor-alpha(TNFalpha) plays a pivotal role. NF-KB, an oxidative stress -sensitive nuclear transcription factor, controls the expression of many genes including the TNFalpha gene. We postulated that green tea polyphenols regulate TNFalpha gene expression by modulating NF-KB activation through their antioxidant properties. In the macrophage cell line, RAW264.7, (-)epigallocatechin gallate (EGCG), the major green tea polyphenol, decreased lipopolysaccharide (LPS)-induced TNFalpha production in a dose-dependent fashion (50% inhibition at 100 mmol/L). EGCG also inhibited LPS-induced TNFalpha mRNA expression and nuclear NF-KB-binding activity in RAW264.7 cells (30-40% inhibition at 100 mmol/L). Similarly, EGCG inhibited LPS-induced TNFalpha production in elicited mouse peritoneal macrophages. In male BALB/c mice, green tea polyphenols (given by oral gavage 2 h prior to an i.p. injection of 40 mg LPS/kg body wt) decreased LPS-induced TNFalpha production in serum in a dose-responsive fashion. At a dose of 0.5 g green tea polyphenols/kg body wt, serum TNFalpha was reduced by 80% of control. Moreover, 0.5 g green tea polyphenols/kg body wt completely inhibited LPS-induced lethality in male BALB/c mice. We conclude that the anti-inflammatory mechanism of green tea polyphenols is mediated at least in part through down-regulation of TNFalpha gene expression by blocking NF-KB activation. These findings suggest that green tea polyphenols may be effective therapy for a variety of inflammatory processes.

Inflammatory diseases of the colon. Narrowing a wide field of symptoms and possible causes.

Symptoms and physical findings may indicate the severity of inflammatory disease of the colon, but detailed history taking is needed to limit the wide spectrum of possible causes. Infectious causes should be ruled out before other disease is assumed to be present. No single test is sufficient to diagnose ulcerative colitis or Crohn's disease. Laboratory testing, histologic assessment, endoscopy, radiology, and bowel studies are often necessary in differential diagnosis. Because of the systemic nature of colitis, manifestations in the musculoskeletal, ocular, dermatologic, hepatobiliary, and other systems may occur and provide clues. Treatment depends on the type and severity of disease. Sulfasalazine (Azulfidine), sulfa-free 5-aminosalicylic acid compounds, and corticosteroids are mainstays of treatment of ulcerative colitis and Crohn's disease. Supportive care and judicious use of antimicrobial therapy are usually effective in colitis due to bacterial, parasitic, and sexually transmitted infections and are useful for symptoms caused by colonic ischemia and vasculitis. Colitis resulting from radiation therapy may present several years after the procedure and can be difficult to diagnose and treat. In many cases of inflammatory colon disease, especially chronic conditions, consultation with a gastroenterologist is highly recommended.

Nerve growth factor synthesis by intestinal epithelial cells.

Nerve growth factor (NGF) exists in the gut of adult rats. The cells responsible for NGF synthesis in the gut remain unknown. IEC-6 and Caco-2 cells, established cell culture models of intestinal epithelial cells, were studied to determine whether intestinal epithelial cells, were studied to determine whether they synthesize and release NGF. Conditioned media from both IEC-6 and Caco-2 cells stimulated neurite outgrowth in both rat pheochromocytoma (PC-12) cells and sensory neurons derived from embryonic chick dorsal root ganglia (DRG). The addition of anti-NGF antibody blocked neurite outgrowth in PC-12 cells and partially blocked outgrowth in DRG cells. An NGF-enzyme-linked immunosorbant assay readily detected immunoreactive NGF in conditioned media from both cell lines, whereas cellular extracts from IEC-6, Caco-2, and isolated rat intestinal epithelial cells had low levels of immunoreactivity. Caco-2 monolayers primarily secreted NGF from the basolateral compartment, and interleukin-1 enhanced its secretion. IEC-6, Caco-2, and isolated rat intestinal epithelial cells expressed NGF mRNA as determined by reverse transcription polymerase chain reaction. These observations suggest that intestinal epithelial cells are capable of NGF synthesis.

Caco-2 cells express type I interleukin-1 receptors: ligand binding enhances proliferation.

We examined the effect of interleukin-1 (IL-1) on the rate of proliferation of the human colon carcinoma Caco-2 and characterized the human intestinal epithelial cell IL-1 receptor (IL-1R). IL-1 dose dependently increased tritiated thymidine uptake in confluent Caco-2 monolayers fed complete growth medium. An anti-IL-1 beta completely blocked the increase in tritiated thymidine uptake, whereas an IL-1 receptor antagonist human recombinant blocked it partially. In long-term culture, IL-1 increased DNA content over control, an effect similar to that of epidermal growth factor (EGF). Unlike EGF, IL-1 did not enhance tritiated thymidine uptake in Caco-2 monolayers grown in serum-free medium, implying that IL-1 needs a cofactor(s) to elicit its proliferative effect. Cross-linking 125I-IL-1 beta to Caco-2 membranes revealed a binding protein of approximately 80 kDa with binding saturated at approximately 2.5 x 10(9) M-1 consistent with that for the type I IL-1R. cDNA transcribed from Caco-2 mRNA and amplified by polymerase chain reaction, using complementary oligonucleotides, resulted in a reaction product matching the sequence of the type I IL-1R. Our results demonstrate that IL-1 enhances proliferation of Caco-2 cells. This effect requires the presence of an unidentified cofactor(s). Also, Caco-2 cells express the type I IL-1R.

Identification and characterization of the rat intestinal epithelial cell (IEC-18) interleukin-1 receptor.

Interleukin-1 (IL-1), an important mediator of inflammation, may act in chronic inflammatory disorders of the intestine such as idiopathic inflammatory bowel diseases. Although the IL-1 receptor (IL-1R) has been studied extensively in many cell lines, including T cells, B cells, and fibroblasts, it has not been demonstrated on intestinal epithelial cells. IL-1 affects intestinal epithelial cell proliferation in vitro, suggesting the presence of IL-1Rs on intestinal epithelium. This paper demonstrates and further characterizes an IL-1R in a rat intestinal epithelial cell. Cells from rat intestinal epithelial cell line IEC-18 were grown to confluence in six-well plates, and association studies with 125I-labeled IL-1 beta to determine specific binding and equilibrium conditions were performed. A competitive-inhibition curve verified the IL-1 concentration required to saturate the IL-1R, and a Scatchard plot revealed a dissociation constant (Kd) of 1.2 x 10(-10) M, with 1,000 receptors per epithelial cell. Binding studies using increasing concentrations of 125I IL-1 beta alone confirmed the receptor density. Cross-linking 125I-IL-1 beta to the IEC-18 IL-1R demonstrated an IL-1R of approximately 80.5 kDa. cDNA transcribed from IEC-18 mRNA was used as a template for amplification of a segment of the IL-1R using complementary oligonucleotides. The resulting sequence of the IL-1R demonstrated a high degree of homology with both the human and mouse type I IL-1R.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolated hepatic granulomas from mice infected with Schistosoma mansoni contain nerve growth factor.

Nerve growth factor (NGF) is a neurotrophic factor essential for the development and maintenance of specific neuronal cell populations. In addition, NGF has biological effects on inflammatory cells. The aim of this study was to determine whether NGF is present in chronic inflammation, using isolated hepatic granulomas from mice infected with Schistosoma mansoni as the model. The schistosome granuloma is a complex T-cell-mediated immune response to the egg. Intact granulomas were isolated from the livers of infected mice and examined for the presence of NGF. In homogenized granuloma samples, radioimmunoassay and immunoblotting analyses detected an immunoreactive NGF that had the same molecular mass as that of purified murine NGF (13 kDa). Isolated granulomas cultured in vitro released soluble factor(s) with NGF-like neurite-promoting activity in a rat pheochromocytoma (PC12) bioassay. This activity was partially inhibited by a blocking anti-NGF antibody. There were two potential sources of this NGF-like neurite-promoting activity, either the schistosome egg or the host inflammatory response. Since neither isolated eggs nor soluble egg antigen had neurite-promoting activity, the inflammation was the source of this activity. The inability of the anti-NGF antibody to inhibit completely the granuloma-induced neurite outgrowth in the bioassay signifies that NGF is not the only neurotrophic factor present in these granulomas. The presence of NGF within the granulomas may indicate that NGF has a role in the granulomatous response.

Alterations of the intestinal innervation in mice infected with Schistosoma mansoni.

Schistosomiasis mansoni is a parasitic disease in which granulomas form around schistosome eggs in the liver and intestines. The purpose of this study was to determine the alterations in the intrinsic innervation of the distal ileum and proximal colon resulting from schistosomiasis. Using murine schistosomiasis mansoni, we examined light microscopic preparations stained with osmium-zinc iodide or the dihydronicotinamide adenine dinucleotide: nitro BT oxidoreductase (NADH) method. We also examined specific populations of peptidergic nerves (vasoactive intestinal polypeptide and substance P) using an avidin-biotin complex (ABC) immunohistochemical technique. We found that granulomas focally destroyed the enteric nerves. Occasionally nerves were found within granulomas, particularly at the periphery of the lesions. Nerve cell bodies close to granulomas had altered staining, which included increased staining for vasoactive intestinal polypeptide. The distribution of nerve injury varied between the 2 enteric segments studied. In the distal ileum, the principal injury was to the myenteric plexus; whereas, the submucous and mucosal plexuses were predominantly damaged in the proximal colon. The physiologic significance of this injury to the enteric nerves requires elucidation.