Lack of association of tumor necrosis factor-alpha gene polymorphisms with disease susceptibility and severity in Behçet’s disease

Although it has been reported that the MHC class I molecule, HLA-B51, is a risk factor for Behçets disease (BD), contribution of the tumor necrosis factor (TNF) genes, which are located in the vicinity of the HLA-B locus, to the genetic susceptibility for BD has yet to be elucidated. The purpose of this study was to analyze the effect of TNF- promoter polymorphisms at positions –308, –238 and –376 on the susceptibility, severity and clinical features of BD. The TNF- gene sequences from 107 patients with BD and 102 healthy subjects were amplified by the polymerase chain reaction. Sequence analysis of the TNF- gene locus, which contains promoter polymorphisms at positions –376, –308, and –238, was performed with a DNA sequencing kit on automated sequencer. The patients were classified according to disease severity and clinical features. Serum TNF- level in the study groups was measured by sandwich enzyme immunoassay. In patients with BD the frequencies of TNF- –308 (19.4% vs 18.4%), –238 (3.7% vs 5.9%), and –376 (0.9% vs 2.9%) gene polymorphisms were not found to be significantly different from those in healthy subjects. The TNF- gene polymorphisms did not show any association with disease severity or clinical features. Serum TNF- level was significantly higher in patients with BD than in healthy controls (3.10±1.45 pg/ml vs 2.43±1.94 pg/ml, P < 0.01). Serum TNF- level was not found to be significantly associated with disease severity, activity, clinical findings and TNF- genotypes. The results of this study suggest that the TNF- gene polymorphisms are unlikely to play an important role in the pathogenesis and severity of BD.     

New tumor necrosis factor-α-inducing protein released from<Emphasis Type="Italic"> Helicobacter pylori</Emphasis> for gastric cancer progression

Purpose To investigate the association between Helicobacter pylori infection and its inflammatory reaction in gastritis, gastric ulcer, and gastric cancer, a new tumor necrosis factor- (TNF-)-inducing protein of H. pylori was studied.Methods The HP0596 gene of H. pylori was identified as the TNF--inducing protein (Tip) gene from genome sequence of H. pylori strain 26695. Using recombinant Tip (rTip) and deleted Tip (rdel-Tip) proteins, the latter of which lacks six amino acids containing two cysteines in the N-terminal domain, we examined their activities in TNF- gene expression and NF-B activation in both Bhas 42 (v-H-ras transfected BALB/3T3) cells and mouse gastric epithelial cell line MGT-40, and in vitro transformation of Bhas 42 cells.Results Tip protein as a homodimer form (38 kDa) was found in both extracts and culture medium of various H. pylori strains. rTip significantly induced TNF- gene expression and NF-B activation in both Bhas 42 cells and MGT-40, and induced in vitro transformation of Bhas 42 cells. However, rdel-Tip did not. Treatment with MG-132, a proteasome inhibitor, inhibited translocation of NF-B p65, and abrogated TNF- induction induced by Tip protein.Conclusion Tip is a new carcinogenic factor released from H. pylori mediated through NF-B activation.     

Local and Systemic Liberation of Proinflammatory Cytokines in Ulcerative Colitis

Determination of plasma and tissue cytokinelevels in inflammatory bowel disease have frequentlyresulted in conflicting data. In the present study wedetermined in patients with ulcerative colitis (UC), the levels of the proinflammatory cytokinesinterleukin (IL)-1, IL-6, interferon(IFN)-, and tumor-necrosis factor (TNF)-liberated by peripheral blood mononuclear cells (PBMC)and lamina propria mononuclear cells (LPMC) after 48-hrculture with pokeweed mitogen (PWM). IL-1, IL-6,IFN- and TNF- in the supernatant weredetected by ELISA. Results show low basal levels ofIL-1 secretion by PBMC and LPMC, and a considerableincrease after mitogen stimulation. Basal IL-6production by PBMC was higher in UC patients than incontrols [2029 pg/ml, CI₉ (–165 to4223) vs 572 pg/ml (–383 to 1527) respectively, P = 0.05] and also afterPWM activation [14,995 pg/ml (7759 -22230) vs 6598 pg/ml(3240-9956), respectively, P = 0.05]. In LPMC, nodifferences in IL-6 secretion were observed. TNF- in activated PBMC of patients with UC was notsignificantly increased in relation to control (P =0.09). No constitutive secretion of IFN- wasobserved in mononuclear cells. IFN- levelssecreted by activated LPMC were lower in patients withUC than in controls [1571 pg/ml (–108 to 3251) vs7953 pg/ml (3851-12,055), respectively, P = 0.03]. Theseresults suggest that IL-6, IL-1, and TNF- participate as mediators in the inflammatoryphenomena observed in UC. Further studies are necessaryto evaluate the role of IFN- in thiscondition.     

Expression of interferon-γ and tumor necrosis factor-α in bone marrow T cells and their levels in bone marrow plasma in patients with aplastic anemia

Immune-mediated stem cell damage has been postulated to be responsible for disease initiation and progression in aplastic anemia (AA). It is hypothesized that T lymphocytes play a major role in destroying the bone marrow (BM) stem cells of AA patients by infiltrating the BM and secreting excessive levels of anti-hematopoietic cytokines, interferon-gamma (IFN-), and tumor necrosis factor-alpha (TNF-). We undertook this study to assess the pathogenic significance of anti-hematopoietic cytokines such as IFN- and TNF- in BM T cells and plasma of AA patients. Significantly elevated levels of IFN- and TNF- were found in the BM plasma of AA patients compared to controls (p=0.05 and 0.006, respectively). Intracellular IFN- and not TNF- in BM CD3⁺ T cells of AA patients was significantly higher compared to controls (p=0.04 and p=0.2, respectively). A follow-up analysis of expression of these cytokines in BM T cells and their levels in BM plasma in five AA patients before and 180 days (6 months) after antithymocyte globulin (ATG) and cyclosporin A (CsA) therapy showed a decline 180 days after therapy compared to pre-therapy. We thus conclude that increased production of both IFN- and TNF- in the BM may contribute to disease pathogenesis in AA and ATG therapy may induce hematological remission by suppressing the elevated levels of IFN- and TNF- in AA BM.     

Ultrastructural Study of Development of Hepatic Necrosis Induced by TNF-α and D-Galactosamine

Recent studies have suggested an associationbetween tumor necrosis factor- (TNF-) andthe development and progression of acute liver failure.To investigate the role of TNF- in the mechanism of massive hepatic necrosis, we studied a mousemodel of TNF- and D-galactosamine (GalN)-inducedhepatic necrosis by ultrastructural analysis.Administration of GalN caused edema of hepatocellular microvilli and widening of sinusoidalendothelial fenestrae (SEF); administration ofTNF- caused only a widening of the SEF. Massivehepatic necrosis with hemorrhage was seen 6 hr afterconcomitant administration of TNF- and GalN. In theultrastructural analysis, edema of the hepatocellularmicrovilli, widening of the SEF, and transmigration ofred blood cells (RBC) and platelets to the space of Disse without exfoliation and necrosis ofthe sinusoidal endothelial cells were observed. Fibrindeposits were seen in areas adjacent to injuredhepatocytes. The diameter of the SEF was significantly greater than in the nontreated group and thegroups treated with TNF- or GalN alone. Theseresults suggest that as a consequence of the increase indiameter of the SEF, transmigration of RBCs andplatelets to the space of Disse may have resulted inmassive hepatic necrosis due to occlusion of themicrocirculation.     

Tumor necrosis factor-alpha mediates inhibitory effect of lipopolysaccharide on L-leucine intestinal uptake

Tumor necrosis factor- (TNF-) has been proposed as an early proximal mediator of many metabolic and physiologic responses during septic shock. We have previously shown that direct addition to tissue (local effect) or intravenous administration (systemic effect) of lipopolysaccharide (LPS) reduces l-leucine absorption across rabbit jejunum. In the present study, we investigated whether the inhibitory effect of LPS on l-leucine intestinal absorption in rabbit is related to TNF-. The results shown that the addition of TNF- to tissue does not produce any effect on l-leucine uptake by the enterocyte. When TNF- was inoculated by intravenous administration, a strong inhibition on the l-leucine uptake (about 40%), mediated by a secretagogue effect on water and Cl-ions was induced. We also found that the LPS intestinal effect induced by intravenous administration, was blocked by a TNF- antagonist, indicating that TNF- is a mediator of the LPS systemic effect on l-leucine intestinal uptake inhibition. The study of possible mediators involved in the TNF- effect showed that nitric oxide and prostaglandins are implicated in the l-leucine intestinal uptake.     

Overproduction of inhibitory hematopoietic cytokines by lipopolysaccharide-activated peripheral blood mononuclear cells in patients with aplastic anemia

The aim of this study was to measure the level of cytokines produced by peripheral blood mononuclear cells (PBMNC) in patients with aplastic anemia (AA) and to determine their effect on the clonal growth of normal bone marrow (BM) cells. Twenty-one patients with AA and 11 normal controls were enrolled in this study. Medium conditioned by PBMNC of AA patients in the presence of lipopolysaccharide (LPS) was found to be suppressive to the colony growth of normal BM cells. Thus, we further determined the presence in the PBMNC-conditioned medium (CM) of both inhibitory cytokines: macrophage inflammatory protein-1 (MIP-1), tumor necrosis factor- (TNF-), transforming growth factor-2 (TGF-2), and interferon- (IFN-), and stimulatory cytokines: interleukin-3 (IL-3) and stem cell factor (SCF). Spontaneous production of MlP-1 was higher in the AA patients than the normal controls (1887±174 pg/ml vs 1643±93 pg/ml), but the difference was not significant. After LPS stimulation, the production of MIP-1 was markedly increased in the AA patients, and its level was significantly higher than that of the normal controls (2360±149 pg/ml vs 1517±92 pg/ml, p=0.0022). The level of TNF was also higher in the AA patients. However, IFN-, TGF-2, SCF, and IL-3 were not detectable in the PBMNC-CM of either AA patients or normals. The myelopoietic suppressing effect of AA-PBMNC-CM from each AA patient was significantly blocked by pretreatment with anti-TNF-, resulting in a colony-forming enhancement of 174%±12%. A similar effect was noted in six of 11 AA patients by pretreatment with anti-MIP-1. We conclude that TNF and MIP-1 can be overproduced by the PBMNC of some AA patients, which may play a role in the progression of AA.     

Effects of Cytokines on the Binding of Leukocytes to Cultured Rat Hepatocytes and on the Expression of ICAM-1 by Hepatocytes

Adhesions of leukocytes to hepatocytes andsinusoidal endothelial cells mediates the induction andprogression of hepatic injury. However, in contrast toendothelial cells, information regarding the regulation of interactions between leukocytes andhepatocytes is limited. In the present study, weinvestigated the effect of inflammatory mediatorsincluding lipopolysaccharide (LPS), staphylococcalenterotoxin B (SEB), interferon- (IFN-), tumornecrosis factor- (TNF-), andinterleukin-1 (IL-1) on the adhesion ofpolymorphonuclear leukocytes or lymphocytes to primarycultured rat hepatocytes, and on the expression of intercellular adhesionmolecule-1 (ICAM-1) gene in hepatocytes. Bothpolymorphonuclear leukocyte and lymphocyte adhesion tohepatocytes were enhanced after exposure of hepatocytes to IFN- and TNF-, but not afterexposure to LPS, SEB or IL-1. The adhesion inducedby either IFN- or TNF- was inhibited bymonoclonal antibodies against ICAM-1 or lymphocytefunction-associated antigen-1 (LFA-1). Nonstimulated hepatocytesexpressed faintly ICAM-1 mRNA, which increased slightlyduring the culture period. ICAM-1 mRNA expression wasup-regulated to a greater extent by incubating hepatocytes with IFN- or TNF-,and peaked after 12 hr of incubation with TNF-and after 24 hr with IFN-. These results indicatethat IFN- and TNF- induce the expressionof ICAM-1 on parenchymal hepatocytes and that theLFA-1-ICAM-1 pathway plays an important role in theinteraction between hepatocytes and neutrophils orlymphocytes.     

Gene expression of cytokines suppressing hematopoietic progenitor cells in lymphoid malignancies

Summary The expression of cytokine genes for tumor necrosis factor (TNF), lymphotoxin and transforming growth factor (TGF), all of which are known to suppress normal hematopoiesis, was investigated in 32 patients with lymphoid malignancies using Northern blot analysis. Messenger RNA (mRNA) for TNF, lymphotoxin and TGF was detected in 9 cases, 2 cases and 7 cases, respectively. When the relationship between cytokine gene expression and surface phenotype was analyzed, the expression of CD19 correlated significantly with expression of the TNF gene (P<0.05). This suggests that B cell malignancies are likely to produce TNF. When the hematological parameters of patients expressing and not expressing the gene were compared, the expression of TNF mRNA was found to correlate with more profound anemia in acute lymphoblastic leukemia (P<0.05). Both granulocyte and platelet counts were lower in patients expressing TNF mRNA; however, the decreases were not significant. Neither lymphotoxin nor TGF gene expression correlated significantly with any hematological parameter.Abbreviations TNF tumor necrosis factor - TGF transforming growth factor - IL interleukin - ALL acute lymphoblastic leukemia - CLL chronic lymphocytic leukemia - ATLL adult T-cell leukemia/lymphoma - NHL non-Hodgkin's lymphoma - MM multiple myeloma Partly supported by grants-in-aid from the Ministry of Education, Science and Culture of Japan (60770949, 63015063, 02256102, 03670325) and from the Fukuoka Anti-Cancer-Society.     

Regulation of the release of tumour necrosis factor (TNF)α and soluble TNF Receptor by γ irradiation and interferon γ in Ewing's sarcoma/peripheral primitive neuroectodermal tumour cells

This study analyses the production of tumour necrosis factor (TNF) and soluble TNF receptor (sTNF-R) before and after exposure to irradiation and interferon (IFN) in 12 cell lines derived from Ewing's sarcoma (ES)/peripheral primitive neuroectodermal tumours (pPNET). Supernatants from ES/pPNET cell cultures were tested in a TNF-specific amplified enzyme-linked immunosorbent assay (ELISA), a bioassay, and sTNF-R_p55 and sTNF-R_p75 ELISA. The tumour cell lines released minimal amounts of TNF, prominent amounts of sTNF-R_p55 (7/12 cell lines) and no sTNF-R_p75. Exposure to irradiation (5 Gy) either induced (3/12) cell lines) or up-regulated (3/12 cell lines) TNF release without changing sTNF-R_p55 and sTNF-R_p75 levels. Priming of cultures with recombinant human IFN (rhIFN) markedly enhanced TNF secretion in the radiation-responsive cell lines and had no influence on sTNF-R_p55 and sTNF-R_p75 levels. rhIFN affected the magnitude rather than the sensitivity of the radiation response. The TNF secreted was bioactive, as shown by its cytotoxic effect of WEHI-164 cells, and neutralization of its activity by anti-TNF monoclonal antibody. Herbimycin A (a tyrosine-specific protein kinase inhibitor) but not calphostin C (a protein kinase C inhibitor), H89 (a protein kinase A inhibitor), AACOCF3 (a specific inhibitor of phospholipase A₂) and MK-886 (a specific inhibitor of 5-lipoxygenase) abrogated -irradiation-stimulated TNF release. The antioxidantsN-acetylcysteine, nordihydroguaiaretic acid and mepacrine dose-dependently inhibited -irradiation-mediated TNF production. Collectively our findings indicate that IFN priming potentiates the secretion of bioactive TNF by ES/pPNET cells in response to irradiation without affecting sTNF-R release. The data suggest a requirement for protein tyrosine kinase activity and a role for reactive oxygen species in the -irradiation-mediated intracellular signalling pathway leading to TNF production.     

A prospective comparison between treatment with phlebotomy alone and with interferon-alpha in patients with polycythemia vera

Summary Interferon alpha (-IFN) is increasingly used for the treatment of patients affected by polycythemia vera (PV). As prior studies are difficult to interpret in view of the lack of appropriate controls, we undertook a randomized comparison of lymphoblastoid -IFN ( n–1 IFN) treatment against venesection treatment alone. In a crossover trial, we treated 22 PV patients alternatively for 5 months each with 3 MU/day sc of n–1 IFN and phlebotomy alone. During IFN treatment, red blood cell count and hematocrit level were well controlled in both trial groups, reducing or eliminating the need for phlebotomy in all patients; furthermore, platelet number and white blood cell count declined during -IFN therapy. In addition, the number of symptomatic patients was greatly reduced, and in six patients a reduction in splenic size was observed. Finally, the only patient with chromosomal abnormalities showed a complete cytogenetic conversion after 5 months of -IFN therapy. Thus, for the first time, our results provide the unequivocal demonstration that -IFN is superior to phlebotomy in controlling the pathologic expansion of erythroid elements and all the clinical aspects of this disease.     

Influence of COX-2 Inhibition by Rofecoxib on Serum and Tumor Progastrin and Gastrin Levels and Expression of PPARγ and Apoptosis-Related Proteins in Gastric Cancer Patients

Cyclooxygenase-2 (COX-2) expression and certain growth hormones, such as gastrin, have been related to gastric carcinogenesis, but little is known about the factors that enhance this COX-2 expression and whether specific blockade of this enzyme has any influence on tumor growth and progression. Our objective was to determine the influence of a specific COX-2 inhibitor, rofecoxib (Vioxx), on serum and tumor levels of gastrin and its precursor, progastrin, as well as on tumor gene expression of COX-2, peroxisome proliferator-activated receptor gamma (PPAR), and apoptosis-related proteins (Bax and Bcl-2, caspase-3, and survivin). Twenty-four gastric cancer (GC) patients entered this study and were examined twice, once before and then following a 14-day treatment with Vioxx at a dose of 25 mg twice daily. For comparison, 48 age- and sex-matched healthy controls and 24 similarly matched Helicobacter pylori (Hp)-positive subjects were enrolled and treated with Vioxx as GC patients. Serum levels of anti-Hp and anti-CagA antibodies as well as IL-8 and TNF- were measured by enzyme-linked immunosorbent assay (ELISA), while serum and tumor contents of progastrin and amidated gastrin were determined by specific RIA. Tumor gene and protein expressions of COX-2, PPAR, Bax and Bcl-2, caspase-3, and survivin were determined by RT-PCR and western blot. The overall Hp and CagA seropositivity in 24 GC patients was significantly higher (82% and 47%) than in 48 controls (61% and 22%) but not in 24 Hp-infected subjects (100% and 38%). Serum IL-8 and TNF- values were significantly higher in GC patients than in controls without GC or Hp-infected controls. Median serum progastrin and gastrin levels were found to be significantly higher in GC than in controls without GC and in Hp-positive subjects. Treatment of GC patients with Vioxx resulted in a significant decrease in plasma and tumor contents of both progastrin and gastrin, and this was accompanied by the increment in tumor expression of COX-2, PPAR, Bax, and caspase-3 with a concomitant reduction in Bcl-2 and survivin expression. We conclude that: (1) GC patients show significantly higher Hp and CagA seropositivity than age- and sex-matched controls, but not Hp-positive subjects, indicating that infection with cytotoxic Hp is linked to GC. (2) Serum progastrin and gastrin levels are signficantly higher in GC patients than in matched controls, confirming that both gastrins may be implicated in gastric carcinogenesis. (3) GC patients exhibit significantly higher levels of IL-8 and TNF- than non-GC controls and Hp-positive subjects, probably reflecting more widespread gastritis in GC. (4) COX-2, PPAR, Bcl-2, and survivin were overexpressed in gastric tumor, but the inhibition of COX-2 activity by Vioxx resulted in a significant reduction in serum and tumor levels of progastrin and gastrin and serum IL-8 and TNF- levels, suggesting that gastrin and proinflammatory cytokines could mediate the up-regulation of COX-2 in gastric cancerogenesis. (5) Vioxx also enhanced expression of COX-2, PPAR, Bax, and caspase-3, while inhibiting the expression of Bcl-2 and survivin, suggesting that COX-2 blockade might be useful in chemoprevention against cancer possibly due to enhancement of the PPAR- and proapoptotic proteins-dependent apoptosis and the reduction in progastrin/gastrin-induced promotion of tumor growth.     

Selective A2A adenosine agonist ATL-146e attenuates acute lethal liver injury in mice

Background d-Galactosamine (GalN)/lipopolysaccharide (LPS)-induced liver injury is an experimental model of fulminant hepatic failure in which tumor necrosis factor- (TNF-) plays a pivotal role. We examined the effects of a highly selective adenosine A_2A receptor agonist (ATL-146e) on GalN/LPS-induced fulminant hepatic failure.Methods Mice were given an intraperitoneal dose of GalN (800mg/g body weight)/LPS (100ng/g body weight) with and without ATL-146e (0.01µg/kg) treatment. Liver injury was assessed biochemically and histologically. Also, TNF- levels in the serum were determined.Results The serum liver enzyme (ALT) level in vehicle-treated mice was 20960 ± 2800IU/ml and was reduced by 63% to 7800 ± 1670IU/ml by treatment with 0.01µg/kg per minute ATL146e, P < 0.05. Treatment with ATL-146e significantly reduced serum TNF- and greatly reduced inflammation assessed by histopathologic examination compared with control mice treated with GalN/LPS. ATL-146e also reduced lethality at 12h from 65% to 13%.Conclusion The present findings suggest that the highly selective adenosine A_2A receptor agonist (ATL-146e) prevents endotoxin-induced lethal liver injury by suppression of TNF- secretion.     

Increased levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) in tracheal aspirates of newborns with pneumonia

Summary Pneumonia is one of the major sites of infection in ventilated newborns. We investigated whether the cytokines IL-1 and TNF- are detectable in tracheal aspirates of newborns with pneumonia as a diagnostic marker. All 12 infants with pneumonia had elevated levels of IL-1 (range 30–300 pg/ml) and TNF- (range 60–680 pg/ml), whereas control infants (n=21; respiratory distress syndrome, very low birth weight or infants intubated preoperatively) had no detectable levels of IL-1 or TNF-.In vitro investigations with mononuclear cells of umbilical cord blood were performed to rule out that exogenously added surfactant influences IL-1 and TNF- production. It is concluded that IL-1 and TNF- are important and specific mediators of neonatal pneumonia which may be of diagnostic importance.

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Erhöhte Spiegel von TNF- und IL-1 im Trachealsekret von Neugeborenen mit Pneumonie

Zusammenfassung Die Pneumonie ist eine der Hauptlokalisationen von Infektionen bei Neugeborenen. Wir untersuchten, inwieweit die Zytokine IL-1 und TNF- im Trachealsekret von Neugeborenen mit Pneumonie als diagnostischer Marker nachgewiesen werden können. Alle 12 Kinder mit Pneumonie hatten erhöhte Spiegel für IL-1 (30–300 pg/ml) und TNF- (60–680 pg/ml), während die Kontrollen (n=21; Atemnotsyndrom, sehr untergewichtige Neugeborene, präoperativ intubierte Kinder) keine nachweisbaren Spiegel für IL-1 und TNF- hatten. Um auszuschließen, daß exogen appliziertes Surfactant die IL-1 und TNF--Produktion beeinflußt, wurdenIn vitro-Untersuchungen mit mononukleären Zellen von Nabelschnurblut durchgeführt. Wir schließen aus den Ergebnissen, daß IL-1 und TNF- wichtige und spezifische Mediatoren der Neugeborenenpneumonie sind, die von diagnostischer Bedeutung sein können.

     

Effects of tumour necrosis factor α on bone marrow aspirates of patients with acute myelogenous leukemia determined by flow-cytometric cell-cycle analysis

Summary Tumour necrosis factor (TNF) exerts cytotoxic and antiproliferative effects on neoplastic cells. It has been used as a therapeutic agent for solid tumours and haematological malignancies. We report on the ex vivo determination of the effect of recombinant human rhuTNF on bone marrow aspirates by a bromodeoxyuridine/propidium iodide method. Cell samples were drawn after 0.5, 2, 4, 6, 8, 10, 22, and 25 h from shortterm suspension bone marrow cultures from patients with acute myelogenous leukemia (AML). Flow-cytometric cell-cycle analysis was performed after double DNA staining with propidium iodide and anti-BrdU antibodies. By this method the effect of rhuTNF on cell proliferation can be evaluated after only 35 h. In about two-thirds of the bone marrow aspirates of AML an inhibiting effect on rhuTNF can be demonstrated, developing to its full extent after 10 h.Abbreviations TNF tumour necrosis factor - AML acute myelogenous leukemia     

Microheterogeneity of acute phase proteins in patients with clinically active and clinically nonactive osteoarthritis

Summary Microheterogeneity of two acute phase glycoproteins, -1-acid glycoprotein (AGP) and -1-antichymotryspin (ACT), concentrations of AGP, ACT, and C-reactive protein (CRP), and levels of three cytokines: interleukin 1 (IL-1-), interleukin 6 (IL-6), and tumor necrosis factor (TNF-) were determined in 61 serum samples and 7 synovial fluids (SFs) obtained from patients (n=61) with osteoarthritis. Using affinity immunoelectrophoresis with concanavalin A (conA), a significant decrease in the reactivity of AGP and ACT with this lectin was found in patients with clinically active osteoarthritis when compared to those with clinically nonactive disease (p<0.001 and p<0.05, respectively). There was no increase in the concentration of AGP, ACT, and C-reactive protein (CRP) in the sera examined. In particular, no increase in the serum level of these proteins was found in the patients with clinically active disease. Low concentrations of IL-6 and TNF- were found in most sera and SFs examined. In 6 out of 7 SFs available, IL-6 concentrations were higher than in the respective serum samples but for TNF- the same could be shown in one case only. Low concentrations of IL-1- were found in 4 serum samples obtained from patients with clinically active osteoarthritis and in no SF specimen studied. In the entire group, serum level of TNF- correlated weakly with the AGP and ACT reactivity coefficients with conA (r=0.3634, p<0.005 and r=0.3324, p<0.02, respectively).Our findings suggest that there are changes in the microheterogeneity of acute phase glycoproteins in some patients with osteoarthritis similar to those observed in rheumatoid arthritis and other chronic inflammations. Possible mechanisms of the involvement of cytokines in the regulation of glycosylation of acute phase glycoproteins in osteoarthritis are discussed.     

Inhibition of Lipid Peroxidation, NF-κB Activation and IL-8 Production by Rebamipide in Helicobacter pylori-Stimulated Gastric Epithelial Cells

The present study aimed to investigate whether rebamipide, a novel antiulcer agent that has an oxygen radical scavenging activity, would inhibit lipid peroxidation, NF-B activation, and IL-8 production by H. pylori. Human gastric epithelial cells (AGS and KATO III), treated with rebamipide or not were incubated in the absence or the presence of H. pylori. As a result, H. pylori significantly stimulated IL-8 production, which was similar to time course stimulation of lipid peroxidation. Other cytokines (IL-1, IL-1, IL-6, TNF-) were not stimulated by H. pylori. Treatment with H. pylori resulted in the activation of two species of NF-B dimers (a p50/p65 heterodimer and a p50 homodimer). Rebamipide significantly inhibited lipid peroxidation as an indicative of oxidative damage, NF-B complex formation, and IL-8 production by H. pylori. In conclusion, rebamipide may attenuate H. pylori-induced gastric inflammation by inhibiting lipid peroxidation and oxidant-mediated activation of NF-B and thereby decreasing IL-8 production.     

Influence of TNF gene polymorphism in patients with acute and fulminant hepatitis

Background Tumor necrosis factor (TNF) is involved in liver damage, especially in fulminant hepatitis (FH). Our previous data showed that the serum level of TNF- was markedly increased in FH. To investigate the mechanism of the overproduction of TNF in FH patients, polymorphism of the TNF gene was studied.Methods We analyzed 120 healthy subjects (controls), 63 patients with acute hepatitis (AH), and 32 patients with FH. Of the 32 FH patients, 21 died or received liver transplantation (FH-D), and 11 survived with intensive therapy (FH-S). The TNF- promoter region at –1031, –863, –857, –308, and –238, and TNF- Nco1 polymorphism sites were studied.Results (1) The four groups showed no differences in polymorphisms of positions –857, –308, and –238. The allelic frequencies of positions –1031C and –863A in the FH-D patients were significantly higher compared to findings in control subjects. (2) The allelic frequency of B2 in the TNF- gene was significantly higher in FH patients, and particularly in the FH-D patients, compared to control subjects. (3) When the patients were divided into four groups by etiology, hepatitis A virus (HAV), HBV, HCV, and non-A non-B non-C, the allelic frequencies of positions –863A and TNF- B2 in FH patients were increased in the non-A non-B non-C group compared to controls.Conclusions FH patients with a poor prognosis had higher frequencies of positions –1031C and –863A in the TNF- promoter region, and higher frequencies of the B2 allele of the TNF- gene. These data suggest that the genomic background may be associated with the prognosis of acute liver failure.     

Gabexate mesilate, a synthetic protease inhibitor, attenuates carbon tetrachloride-induced liver injury in rats

Background Gabexate mesilate, a synthetic protease inhibitor, is used to treat acute pancreatitis and disseminated intravascular coagulation because it inhibits various serine proteases; however, whether gabexate mesilate prevents acute liver failure has not yet been studied. The aim of the present study was to investigate the effect of gabexate mesilate in carbon tetrachloride (CCl₄)-induced liver injury in rats.Methods Acute hepatic failure was induced by administration of CCl₄ intragastrically to male Sprague–Dawley rats. The effects of gabexate mesilate were examined in terms of serum transaminase levels, liver histology, and the prognosis of rats.Results Gabexate mesilate treatment significantly decreased the elevation of serum transaminase levels and improved liver histology 24h after the administration of CCl₄ (0.2ml/100g rat weight). Plasma tumor necrosis factor- (TNF-) and interleukin-1 (IL-1) decreased significantly in the gabexate mesilate-treated rats compared with saline-treated rats. Gabexate mesilate treatment also significantly improved survival rate after a lethal dose of CCl₄ (0.5ml/100g rat weight) from 0% to 20%.Conclusions Gabexate mesilate treatment attenuated CCl₄-induced liver injury via a suppression of proinflammatory cytokine production. In addition, these investigations suggest that gabexate mesilate treatment may provide therapeutic strategies for human acute liver failure.     

Prospective evaluation of interferon-α in treatment of chronic active Crohn's disease

Several case reports suggested good effects of interferon- in patients with Crohn's disease. In addition, a decreased production of interferon- in Crohn's disease has been shownin vitro. Treatment with interferon- may activate intestinal natural killer cells and down-regulate the overproduction of inflammatory cytokines like interleukin-6 in Crohn's disease. To evaluate the clinical efficacy of interferon-, we treated 12 patients with a chronic active course of Crohn's disease with recombinant human interferon- prospectively for 24 weeks. Prednisolone was continuously tapered and discontinued at week 12. The end point of the study was the prevention of worsening of clinical symptoms defined with the Crohn's disease activity index and was monitored by acute-phase proteins, interleukin-6 serum concentrations, and endoscopy. The biochemical activity of interferon- was measured by 2,5-oligo adenylate serum levels. The end point of the study was reached in four patients (33%). In these patients the final Crohn's disease activity index was above 150, which means that they did not achieve clinical remission. All other patients (66%) did not respond to interferon- and had to be withdrawn prematurely. Interferon- did not show any beneficial effect on interleukin-6 or acute-phase protein concentrations and on endoscopic activity. The 2,5-oligo adenylate levels continuously increased during interferon therapy. Considerable side effects were noted. These results fail to demonstrate a therapeutic role of interferon- in chronic active Crohn's disease.