A single intra-articular (ia) injection of 2 mg zymosan on D0 led to the production of acute periarticular edema followed by subacute erosive synovitis. The development of the zymosan-induced arthritis was associated with an initial loss of running activity and with an initial decrease of proteoglycan synthesis. Febrile response was present only on D1. In addition, on D20 synovial pannus led to a marked depletion of the proteoglycan content in the articular cartilage. When injected ia, IL1 beta (1 microgram) provoked similar fever and similar changes in cartilage anabolism, but did not affect cartilage proteoglycan content (D20). These results suggest that zymosan-induced synovitis in the rat combines early prostaglandin-dependent processes (edema, pain, fever) with IL1-related effects on cartilage metabolism, thus allowing evaluation of chondroprotective drugs.

The addition of ATP, but not ADP or AMP, to the culture media of bovine nasal cartilage explants caused an acceleration in the rate of proteoglycan loss from the tissue. The ATP-stimulated loss of proteoglycan was not inhibited by the IL1-receptor antagonist protein, but was partially inhibited by the presence of ADP or AMP. The proteolytic events resulting from the presence of ATP were found to be similar to those following treatment with IL1, in that inhibitors of the cysteine-peptidase cathepsin B, serine-proteinases with trypsin-like specificity, and of some of the matrixins, could all prevent proteoglycan loss, which was mediated, at least in part, by the action of 'aggrecanase'. In contrast to its effects on nasal cartilage, ATP inhibited basal and stimulated proteoglycan release from articular cartilage. Both ADP and AMP had no effect on proteoglycan release in articular cartilage but enhanced the response to ATP when added concurrently. We conclude that extracellular ATP, probably acting via P2-purinoceptors, stimulates proteoglycan breakdown from bovine nasal cartilage and thus, may have a role in diseases which primarily involve destruction of non-articular cartilage. Extracellular ATP has, in contrast, a chondroprotective effect on bovine articular cartilage.

Lipopolysaccharides (LPS) activate both the immune and the stress response system. The effects of these bacterial endotoxins involve the release of interleukin 1 (IL1) and other cytokines, which in turn stimulate the hypothalamic-pituitary-adrenal (HPA) axis. We studied the binding properties of the corticosteroid receptor system, which mediates feedback inhibition of the HPA axis, in two brain areas and in the pituitary gland in rats treated with LPS and recombinant murine IL1 beta. The binding properties of the corticosteroid receptors were determined by Scatchard plot analyses of in vitro cytosolic binding of the tritiated mineralocorticoid receptor (MR) radioligand aldosterone and the tritiated glucocorticoid receptor (GR) ligand RU28362. Tissues were collected 48 h after administration of LPS, including a 24-hour period for depletion of endogenous corticosterone. LPS treatment increased the Kd of [3H]aldosterone of the hippocampal MR 4.3-fold and the apparent maximum binding capacity (Bmax) of [3H]aldosterone by 65% during a time interval when the concentration of corticosterone, the endogenous ligand of both hippocampal MR and GR, was elevated in the intact rat. Thereafter, MR binding properties were not different from vehicle-injected controls, at 96 h, when in intact animals the enhanced HPA activity subsided. GRs, determined by binding of [3H]RU28362, were not affected by LPS. IL1 evoked a 2.7-fold increase in the Kd of the hippocampal MR and a 57% increase in Bmax 3 h after injection into the lateral cerebral ventricle. An autoradiographic procedure revealed that the same treatment with IL1 reduced the retention of the tritiated endogenous MR ligand corticosterone by 40-60% in all pyramidal cell layers and in the dentate gyrus of the hippocampus, when a tracer dose of the steroid was administered that gives rise to a concentration around the Kd of the MR. This reduced in vivo retention of corticosterone is predicted in view of the reduced affinity of hippocampal MRs. The data are consistent with the hypothesis that an impaired feedback of the HPA axis via deficient hippocampal MRs contributes to stimulate corticosterone secretion from the adrenals during infection.

Using affinity crosslinking techniques, we have biochemically characterized the interleukin-1 (IL1) receptor and investigated its distribution on a range of murine and human cell lines. We show that two forms of IL1 receptor can be identified on the basis of specific crosslinking with 125I-IL1 alpha and 125I-IL1 beta. The two receptor forms have an approximate molecular mass of approximately 80 and approximately 60 kDa, and were found on both murine and human cells. Their relative distribution shows no clear cell lineage restriction and does not correlate with preferential binding of IL1 alpha or IL1 beta. Some cells, such as the T helper cell line D10.G4.1, express both forms of the receptor. Iodine 125-IL1 was crosslinked to the two receptor forms and a partial peptide map analysis of the two receptor/ligand complexes was performed. Comigration of the major partial peptide fragments suggests that the approximately 80 and approximately 60 kDa forms of the receptor may be differentially processed forms of the same protein. Treatment of the approximately 60 kDa IL1 receptor on Raji cells with N-glycanase reduced its molecular mass by 12 kDa, showing that this lower molecular mass form is a glycoprotein; glycosylation differences alone probably do not account for the difference in mass between the two forms.

Monocytes of healthy donors were infected with HIV1 in vitro: 14-21 days after infection 50-70% of the cells produced p24 HIV1 antigen as detected with anti-p24 immunostaining; infected cultures showed enhanced secretion of interleukin-6 (IL6), interleukin-8 (IL8) and tumour necrosis factor alpha (TNF-alpha). The expression of cytokines on the single-cell level was further analysed by in situ hybridization using nonradioactive digoxigenin for detection. HIV1 (p24+) -producing cells were compared with non-HIV (p24-) -producing cells. All morphological subtypes of macrophages showed HIV production; no difference in cytokine expression was observed. Immunocytochemistry of HIV-infected and uninfected cultures also showed no difference in the pattern of IL1-beta, IL6, IL8 and TNF-alpha protein expression in the cells.

Cystic fibrosis is associated with an cAMP-regulated channel defect, which has been evidenced in many cell types including B lymphocytes. To document a B-cell dysfunction potentially related to this defect, we studied the in vitro IgG production by lymphocytes from 11 cystic fibrosis patients. B lymphocytes were co-cultured with autologous monocytes and stimulated with Staphylococcus aureus Cowan or with Nocardia-delipidated cell mitogen in the presence of low concentrations of IL2. Cystic fibrosis patients' cells produced amounts of IgG comparable with that of normal and control patients' cells. However, dexamethasone (10(-7) mol l-1) had no effect on the response of cystic fibrosis patients' cells, whereas it enhanced that of the latter two groups. This resistance of cystic fibrosis cells was true with concentrations of dexamethasone up to 10(-6) mol l-1, whereas this agent induced a dose-related enhancement from 10(-8) to 10(-6) mol l-1 in cultures of normal cells. Co-culture experiments showed that cystic fibrosis B lymphocytes themselves are resistant to the effect of dexamethasone. In contrast dexamethasone normally suppressed the anti-CD3 antibody-induced response of cystic fibrosis T cells in the presence of IL2 and the IL1 alpha- or beta-induced collagenase production of cystic fibrosis fibroblast cell lines. Thus cystic fibrosis B lymphocytes exhibit a selective defect which may interfere with the normal interactions between the hormonal and immune systems and may participate in the sensitivity of cystic fibrosis patients to bacterial bronchopulmonary infections.

Cyclosporin A (CsA) is a potent immunosuppressive agent that inhibits T-cell proliferation and lymphokine production. There is less information on the direct effect of CsA on B-cells. We investigated the proliferative responses of human tonsillar B-lymphocytes to a "T dependent" mitogen, pokeweed mitogen (PWM), and to a "T independent" mitogen, Staphylococcus aureus (SA). Both responses were strongly inhibited by CsA. Nonspecific cytotoxicity was ruled out, and the inhibition was not reversed by adding IL1, IL2, or BCGF individually or in combination. Maximal inhibition of the PWM response occurred when CsA was added early in the culture period. Cyclosporin A added 18 hours after the start of culture was less effective, and adding CsA after 36 hours resulted in only minimal inhibition. However, with SA as mitogen, addition after 36 hours still affected substantial inhibition. These results, on the time of action and resistance to reversal by exogenous growth factors, suggest that CsA can directly inhibit human B-cells by a mechanism similar to its action on T-lymphocytes, blocking an early event critical to entry into cell cycle, but an additional mechanism of inhibition later in the cell cycle may also operate when the proliferative signal is provided by the T-independent mitogen SA.

We have investigated the effects of interleukin (IL)-1 beta and IL6 on expression and phenobarbital (PB) induction of ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-deethylase (PROD) activities, as well as on mRNA levels of cytochromes P450 (CYP) 1A, 2B, 2C, 2E and 3A, in rat hepatocytes in primary culture. IL6 slightly antagonized PB-induced PROD activity. Strikingly, IL1 beta strongly inhibited basal EROD and PROD activities, and fully blocked their induction by PB in a dose-dependent fashion. Furthermore IL1 beta completely suppressed PB induction of all CYP mRNAs analyzed. Our results demonstrate that IL1 beta can suppress basal CYP activities, as well as PB-inducible expression of five CYP mRNAs in rat hepatocytes in primary culture.

The role of thymic epithelium in T cell development has given rise to a number of studies, but less information is available concerning the factors regulating thymic epithelial cells (TEC) themselves. Several cytokines, natural or recombinant, were investigated for their effects on human TEC proliferation. This study presents evidence for the first time that human recombinant interleukin 1 (IL1) and IL1-containing mixed cytokine preparations induced DNA synthesis of TEC as measured in a 48-hr stimulation assay. The effects of IL1 were dose dependent and sustained in time. The following recombinant cytokines, IL2, IL3, IL4, interferon-gamma (IFN-gamma), IFN-alpha, tumor necrosis factor-alpha (TNF alpha), and TNF beta, as well as thymosin fraction 5 and Escherichia coli lipopolysaccharide (LPS), were not found to modify TEC proliferation but IFN-gamma and TNF alpha enhanced the effects of IL1. We also report that IL1 induced a profound change in the morphology of TEC. Our observations suggest that TEC are targets for the action of cytokines and emphasize the important role played by IL1 within the thymus.

Extraintestinal dissemination of Entamoeba histolytica is frequently manifested by the life-threatening amebic liver abscess (ALA). The hepatic establishment of amebas implies invasion of blood vessels and contact with the endothelium. By means of a fluorescence-based quantitative adhesion assay, we assessed the binding to human endothelial cells of two E. histolytica strains of different virulence. The highly virulent strain (L-A) adhered substantially more strongly to unstimulated endothelium than the non-virulent one (BG3). Attachment of L-A was increased by treatment of endothelial cells with interleukin-1 beta (IL1 beta). Other proinflammatory cytokines such as interferon-gamma (IFN gamma) and tumor necrosis factor-alpha (TNF alpha) did not modify the spontaneous adhesion capacity of amebas. For purposes of comparison we also performed adhesion of the parasites to skin fibroblasts. Adhesion to this cell type was quite low (< 10%). Parasite virulence, differential adhesive capacity to endothelial cells, and modulation of the latter phenomenon by proinflammatory factors (IL1 beta) may influence the evolution and outcome of extraintestinal amebiasis, especially hepatic abscesses.

Inflammatory response plays an important role in the pathogenesis of cerebral injury in bacterial meningitis. In this study, we evaluated the cytokine levels of interleukin 1-beta (IL1 beta), tumour necrosis factor alpha (TNF alpha) and interleukin 6 (IL6) in the cerebrospinal fluid (CSF), and determined their correlation with acute clinical complications and with changes in CSF biochemistry. Interleukin 6, TNF alpha and IL1 beta were present in 9/9, 3/9 and 4/9 patients, respectively. The CSFs with detectable TNF alpha or IL1 beta had higher levels of IL6 (p < 0.02), protein (NS) and lower glucose levels (p < 0.02), compared with those in which TNF alpha and IL1 beta were absent. Tumour necrosis factor alpha and IL1 beta levels also correlated with the presence of prolonged fever, fits, spasticity and death (logTNF alpha: r = 0.70, p < 0.05; logIL1 beta: r = 0.62, p = 0.08). The cytokine levels reflect the degree of inflammatory response and are positively correlated with the severity of acute clinical complications. Modulation of this inflammatory response in bacterial meningitis may improve its morbidity and mortality.

OBJECTIVE

To investigate the regulatory roles of interleukin 1 beta (IL1 beta), tumour necrosis factor alpha (TNF alpha), interferon gamma (IFN gamma) or transforming growth factor beta 1 (TGF beta 1) on hyaluronan (HA) synthesis by human fibroblastic synovial lining cells.

METHODS

Concentrations of HA in culture supernatants of fibroblastic synovial lining cell line (RAMAK-1 cell line) with or without stimulation by IL1 beta, TNF alpha, IFN gamma or TGF beta 1 were measured by sandwich binding protein assay. Levels of HA synthase mRNA of the cells with or without stimulation were detected by reverse transcribed polymerase chain reaction. Molecular weights of HA in the culture supernatants of the cells with or without stimulation were measured using high performance gel permeation liquid chromatography.

RESULTS

HA synthesis by the cells was not significantly augmented by TNF alpha or by IFN gamma. It was significantly stimulated by IL1 beta but inhibited by TGF beta 1. Molecular weights of HA in the culture supernatants of the cells were unchanged by stimulation with TNF alpha. They were remarkably increased by stimulation with IL1 beta and IFN gamma, but reduced with TGF beta 1.

CONCLUSIONS

IL 1 beta is an up regulator of HA synthesis, while TGF beta 1 is a down regulator. HA production in the synovial lining cells of inflamed joints (for example, rheumatoid arthritis) might be regulated by the balance of these cytokines.

Thrombosis and disseminated intravascular coagulation (DIC) are common complications of infections. Abnormal activation of coagulation is due in part of expression of tissue factor on intravascular cells in response to cytokines, including interleukin-1 beta (IL1 beta ) and tumor necrosis factor (TNF). Both TNF and IL1 beta are thought to play significant roles in producing the pathologic manifestations of sepsis. Therefore, we examined the effects of thrombin on TNF and IL1 beta secretion of monocytes, and the ability of monocyte products to promote tissue factor expression by endothelial cells. Human monocytes were treated with thrombin or a thrombin receptor agonist peptide (SFLLRN), and/or bacterial lipopolysaccharide (LPS). The agonists were removed, and monocytes cultured 18 hours. The monocyte-conditioned supernatants were assayed for TNF and IL1 beta antigen, and for their ability to induce tissue factor expression on human umbilical vein endothelial cells and the Ea.hy endothelial cell line. Thrombin alone did not promote monocyte TNF or IL-1 beta secretion. However, thrombin enhanced LPS-induced TNF and IL1 secretion. Supernatants from monocytes exposed to LPS plus thrombin promoted greater tissue factor expression on endothelial cells than supernatants from those treated with LPS only. SFLLRN did not increase TNF secretion in response to LPS, but did enhance LPS-induced IL1 beta secretion and tissue factor-inducing activity. Neither SFLLRN nor active thrombin augmented the level of mRNA for TNF above that induced by LPS alone. However, both increased the LPS-induced level of IL1 beta message. Thus, thrombin enhanced LPS-induced TNF and IL1 beta secretion by monocytes. Unexpectedly, the effects on these two cytokines were mediated by different mechanisms. Enhancement of LPS-induced IL1 beta secretion was largely mediated via the tethered ligand type thrombin receptor and correlated with an increase in the steady state level of mRNA. By contrast, enhanced TNF required proteolytically active thrombin, but was not mediated by the tethered ligand receptor. These data demonstrate that physiologically relevant amounts of thrombin can synergize with endotoxin to stimulate monokine release. Thrombin could thereby play a role in the complex network of mediators involved in the pathophysiology of sepsis. We speculate that limiting thrombin activity during DIC could be a beneficial adjunct in the management of sepsis.

Endotoxemia results in the release of cytokines that exert complex effects on the cardiovascular system. The purpose of this study was to 1) determine if interleukin 1 beta (IL1 beta) and tumor necrosis factor alpha (TNF alpha) elicit the release of endothelium-derived relaxing factor (EDRF) and nitric oxide derived from the constitutive nitric oxide synthase present in vascular endothelium, and 2) determine if these cytokines alter endotoxin-mediated decreases in EDRF bioactivity and nitric oxide production. Cultured bovine aortic endothelial cells were directly exposed to endotoxin, human recombinant TNF alpha, interleukin 1 beta, or a combination of endotoxin and cytokine for 1 h, followed by a second hour without endotoxin. Subsequently, both basal as well as agonist-stimulated (bradykinin) EDRF bioactivity and nitric oxide (NO) content of the effluent were quantitated. In additional experiments, endothelial cells were exposed acutely over a 30-min assay period to either endotoxin alone, cytokine alone, or endotoxin and cytokine. Following the 2-h incubation, endotoxin alone markedly reduced basal EDRF bioactivity and NO production (44 +/- 13% control, 66 +/- 13% control, respectively) and decreased bradykinin-stimulated EDRF bioactivity and NO production (58 +/- 5% control, 55 +/- 4% control, respectively). TNF alpha and IL1 beta did not stimulate EDRF release or NO production either acutely or after prolonged exposure, nor did they alter agonist-stimulated EDRF bioactivity and NO production. Similarly co-incubation of endotoxin with TNF alpha or IL1 beta failed to significantly alter the inhibitory effects of endotoxin on EDRF bioactivity and NO production.(ABSTRACT TRUNCATED AT 250 WORDS)

Using tetradecanoylphorbol 13-acetate (TPA), a known inducer of epithelial cell differentiation, and Northern blot analysis, we investigated in normal versus well-differentiated malignant keratinocytes the modulation of genes implicated in their growth or differentiated function. In normal keratinocytes transient c-fos induction was detected within 30 min after stimulation and was followed by rapid down regulation of the proto-oncogenes c-myc and epidermal growth factor receptor. Within hours after stimulation mRNA levels for three well-characterized differentiated keratinocyte products, involucrin, interleukin-1 beta (IL1-beta), and fibronectin, were induced, as were those for a growth arrest and DNA damage-inducible (GADD 153) gene and a small proline-rich (SPR1) gene, both known to be associated with differentiation but with of as yet unknown functions. Heat shock protein 70 gene was initially down regulated and was induced only after 48 h. The well-differentiated malignant keratinocyte cell line differed in that the c-fos, GADD, SPR1, and IL1-beta genes had several-fold higher induction, but involucrin mRNA was undetectable and fibronectin mRNA was only minimally induced after TPA stimulation. Malignant cells reached terminal differentiation faster than normal keratinocytes as measured by inability to exclude trypan blue dye, and in situ hybridization using a riboprobe for the differentiation-associated SPR1 gene showed that normal keratinocytes constitutively express this transcript while malignant keratinocytes with virtually identical morphology and growth rate do not. These studies greatly expand our understanding of gene activation and down regulation during normal keratinocyte differentiation and imply that a malignant cell line, even when retaining the phenotype of normal cells, differs in its response to outside stimuli, furnishing at best an imperfect model for investigating the molecular mechanisms of cellular differentiation.

Endotoxin, interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) dose-dependently increased the expression of tissue factor and at the same time induced thrombomodulin down-regulation on the surface of cultured bovine aortic endothelial cells. Chelerythrine, a selective protein kinase C inhibitor, strongly reduced endotoxin-, IL1 beta- and TNF alpha-induced tissue factor expression but remained without effect with regard to thrombomodulin down-regulation measured in parallel. On the contrary, staurosporine, a highly potent, non-selective PKC inhibitor, simultaneously abolished tissue factor expression and thrombomodulin down-regulation induced by endotoxin, IL1 beta and TNF alpha. These results show that protein kinase C is deeply involved in the process leading to pyrogen-induced tissue factor expression and suggest that thrombomodulin down-regulation is regulated by a different pathway.

BACKGROUND

There is a relationship between cardiovascular morbidity, inflammatory activity, and changes in the lipid profile in rheumatoid arthritis (RA), although the mechanisms are not fully elaborated. Recent know-ledge that white adipose tissue (WAT) is a producer of immunologically and metabolically active substances gives another perspective to study.

OBJECTIVE

To evaluate the relationship between interleukin-1 receptor antagonist (IL-1Ra) and variables associated with WAT and inflammation in RA.

METHODS

Anthropometric, inflammatory and metabolic variables were assessed in 23 women with RA and 23 matched controls. Spearman, partial correlation and factor analyses were performed.

RESULTS

Inflammatory markers were increased in patients. In both groups, IL-1Ra correlated with leptin independent of age and BMI. IL-1Ra also correlated with haptoglobin and apolipoprotein (Apo) B in patients and with soluble TNF receptor (sTNFR) 1 in controls. In factor analysis, three latent factors were identified among patients. The first loaded on IL-1Ra, leptin, BMI, ApoB and body fat content (BF%), the second loaded on IL1-Ra and sTNF-receptors and the third showed inverse loadings on ApoA-I together with loadings on ESR, haptoglobin, orosomucoid, BF% and BMI.

CONCLUSIONS

IL-1Ra was associated with markers of inflammation and with fat-related factors in RA patients, suggesting a dualistic relationship of IL-1Ra in RA. IL-1Ra correlated independently with leptin in both patients and controls, indicating a relationship between inflammation and leptin.

BACKGROUND

The massive use of Highly-Active Antiretroviral Therapy (HAART) in individuals with human immunodeficiency virus (HIV) coincided with an increase in cardiovascular disease, a major cause of morbidity and mortality in this group.

OBJECTIVE

To determine the frequency of carotid atherosclerosis and the association between biomarker levels and carotid intimal-medial thickening in HIV-positive individuals treated for HIV at referral centers in Pernambuco.

METHODS

This was a cross-sectional study of 122 HIV-positive patients. Subclinical carotid atherosclerosis was considered with the presence of increased intimal-medial thickness of the common carotid artery>> 0.8 mm or plaques in the carotid ultrasound. The following inflammatory biomarkers were analyzed: IL6, IL1-β, TNF-α, high-sensitivity CRP, sVCAM-1 and sICAM-1.

RESULTS

Of the 122 patients analyzed, most were men (60.7%) aged>> 40 years (57.4%) receiving HAART (81.1%). The prevalence of atherosclerosis was 42.6% (52 cases). Patients older than 40 years and intermediate or high Framingham score were more likely to develop atherosclerosis at the univariate analysis. Age older than 40 years (OR = 6.57, 95%CI: 2.66 to 16.2, p = 0.000), male gender (OR = 2.76, 95%CI: 1.12 to 6.79, p = 0.027) and presence of syndrome metabolic (OR = 2.27, 95%CI: 0.94 to 5.50, p = 0.070) were associated with atherosclerosis at the multivariate analysis. Elevated levels of inflammatory cytokines and adhesion molecules were not associated with the presence of atherosclerosis.

CONCLUSIONS

There was no association between inflammatory biomarkers, adhesion molecules and presence of carotid atherosclerosis. However, a higher chance of subclinical atherosclerosis was observed in men, those older than 40 years, with intermediate / high Framingham score or metabolic syndrome.

Macrophages are versatile phagocytic cells in immune system with immunoregulatory functions. However, the removal of apoptotic cells by macrophages is disturbed in systemic lupus erythematosus (SLE). Aryl hydrocarbon receptor (AhR) is a ligand-activated cytoplasmic receptor and transcription factor with diverse effects on immune response. Indole-3-carbinol (I3C) is an AhR agonist which has been implicated as a beneficial factor in regulating inflammation and cytokine expression in murine models of SLE. However, the molecular mechanisms are not thoroughly studied. Here, we aimed to investigate the ex vivo effects of I3C on polarization of monocyte-derived macrophages (MDMs) in SLE patients and the expression of regulatory cytokines upon AhR activation. MDMs from 15 newly diagnosed SLE patients and 10 normal subjects were induced by Jurkat apoptotic bodies (JABs) and treated with I3C. I3C enhanced the nuclear accumulation of AhR among MDMs of SLE patients and altered the expression of AhR target genes including CYP1A1, <em>IL1</em>- <em>β</em>, IDO-1 and MRC-1. The imbalanced expression of pro- and anti- inflammatory cytokines (IL-10, IL-12, TGF<em>β</em>1, TNFα, IL-23, IL-6 and IFN-γ) was compensated in response to I3C. AhR activation was also associated with the overexpression of M2 markers (CD163) and downregulation of M1 markers (CD86). Thus, macrophages are activated alternatively in response to I3C. The obtained data indicate that I3C-mediated AhR activation possess immunoregulatory effects on macrophages of SLE patients by exerting an obvious downregulation in the expression of pro-inflammatory and overexpression of anti-inflammatory cytokines. Therefore, AhR could be targeted and further investigated as a choice of anti-inflammatory therapies for autoimmune disorders such as SLE.

1. The effects of propentofylline (PPF, 25 mg kg(-1) body weight per day) on rat cerebral energy state and cytokine expression as well as on behaviour and histopathology were studied after acute and long-term permanent bilateral common carotid artery occlusion (BCCAO). 2. In the absence of PPF, acute ischaemia led to a decrease in energy-rich phosphates in parietotemporal cortex and hippocampus which correlated with an increase in AMP and adenosine concentrations measured by high-performance liquid chromatography technique. The concentrations of cortical cytokines TNF alpha and IL1 beta were increased 12 and 19 fold, respectively. 3. PPF had a neuroprotective action after 20 min of BCCAO, reducing the deleterious effect of acute ischaemia on rat brain energy state and microglial reaction. Simultaneously, PPF treatment increased cyclic-AMP 3 fold. 4. Three weeks of permanent BCCAO did not significantly disturb brain energy metabolism, microglial reaction or histopathology. However, a significant reduction of 30 -- 50% in rat memory capacities and a locomotor hyperactivity were obtained. 5. Continuous PPF-application, however, led to a marked increase in rat working memory and to reduced locomotor activity, which were returned nearly to control levels by 1 week after permanent BCCAO. In summary, PPF showed a clear neuroprotective effect on cerebral energy state and pro-inflammatory cytokines under conditions of acute global ischaemia. Continuous administration of PPF led to memory improvement during permanent BCCAO. 6. These results underscore the benefit of treatment with PPF in clinical practice, particularly during stroke, but also in cerebrovascular and neurodegenerative disorders.

To determine if mononuclear cells (MNC) infiltrating various types of human solid tumours express genes for cytokines, in situ hybridisation with 35S-labelled cDNA antisense probes for interleukin 2 (IL2), interferon gamma (IFN-gamma), tumour necrosis factor alpha (TNF-alpha), interleukin 1-beta (IL1-beta), transforming growth factor beta (TGF-beta) and interleukin 2-receptors (IL2R) was performed. Fresh-frozen tissue samples of ovarian carcinomas (n = 13), breast carcinomas (n = 12), and squamous cell carcinomas of the head and neck (SCCHN, n = 7) were evaluated for the presence and localization in the tumour of MNC positive for cytokine genes. In ovarian tumours and those breast carcinomas producing little or no mucin, only rare positive MNC were observed. In contrast, breast carcinomas producing mucin and all SCCHN contained numerous MNC expressing gene transcripts for IL2, IFN-gamma, TNF-alpha, IL2R as well as TGF-beta. In tumour-involved lymph nodes of patients with SCCHN, MNC expressing genes for cytokines were found around tumour metastases but not in non-involved areas. These data suggest that tumours expressing immunogenic antigens (e.g. mucin) contain many activated MNC, while other tumours either fail to activate or suppress functions of infiltrating MNC. In SCCHN or tumour-draining lymph nodes, local down-regulation of antitumour responses might be mediated by TGF-beta produced by activated tumour-infiltrating MNC.

Rheumatoid arthritis inflammation process is characterised by the production of soluble mediators with final alteration of cartilage and bone erosions. Etiological factors of RA remain obscure, however intermediate mechanisms are better understood: proinflammatory cytokines belonging to innate as well as adeptive immunity are principal effectors. TNF alpha and IL1 beta are major components of the inflammatory process: TNF alpha is an important stimulus of cells producing inflammatory mediators (cytokines, metalloproteinases, NO, PGE2,...) and IL1 beta mediates cartilage and bone destruction (via secretion of metalloproteinases, decrease synthesis of glycosaminoglycans...). Natural inhibitors of proinflammatory cytokines are also present such as IL1-Ra for IL1 or secreted soluble receptors, sIL1-RI, sIL1-RII, sTNF-RI, sTNF-RII. The level of these inhibitors are increased but not enough to sustain an anti-inflammatory effect. Progress in animal models and in clinical practice has driven to market biological drugs targeted to inhibit TNF alpha and recently IL1 beta. Other cytokines taking place in the inflammatory cascade before TNF alpha and IL1 beta are potential future therapeutic targets such as IL1IL1IL1IL1 beta.

BACKGROUND

Lipocalin 2 (LCN2), a protein primarily produced by hepatocytes, is highly upregulated under various conditions that induce cellular stress, such as intoxication, infection or inflammation. However, the precise biological functions and underlying mechanisms of LCN2 in hepatocytes remains unknown.

METHODS

Hepatocyte stress was successfully induced by treating Huh7 cells with interleukin-1β (IL-1β). Interleukin-6 (IL-6), Tumor Necrosis Factor-α (TNF-α) and LCN2 levels were measured in IL-1β treated Huh7 cells and supernatant. Additionally, microarray analysis was conducted to identify genes differentially expressed in LCN2-silenced and control Huh7 cells.

RESULTS

TNF-α, IL-6 and LCN2 were significantly elevated in Huh7 cells after IL-1β) treatment. In LCN2-silenced Huh7 cells, expression of IL-6 and TNF-α was significantly increased when compared with the expression levels of control Huh7 cells. Furthermore, differentially expressed genes were observed between the LCN2-silenced and control cells. Microarray analysis indicated that LCN2 acted by influencing genes involved in protein metabolism, stress response, cell cycle and proliferation.

CONCLUSIONS

Our results suggest that LCN2 upregulation protects hepatocytes from IL-1β-induced stress. Additionally, our microarray analysis of LCN2-silenced and control cells provides a better understanding of the mechanisms that may be influenced by LCN2 induction.

Cyclophosphamide (CP) is a widely used chemotherapeutic agent but its clinical usefulness is challenged with different forms of toxicities. No studies have evaluated the possible protective effect of nicorandil (NIC) in CP-induced cardiotoxicity. Our study aimed to investigate this effect by using NIC (3 mg/kg/day) orally for 5 days, in the presence or absence of cardiotoxicity induced by intraperitoneal (i.p.) injection of CP (150 mg/kg) on 4th and 5th days. We confirmed the role of ATP-sensitive potassium channel (K_ATP) by coadministration of glibenclamide (GP) (5 mg/kg/day) 2 h before NIC (3 mg/kg/day) for 5 days. Moreover, the role of endothelial nitric oxide synthase (eNOS) was confirmed by coadministration of nitro-ω-L-arginine (L-NNA) (25 mg/kg/day) for 5 days. Results showed that CP succeeded in induction of cardiotoxicity which manifested by a significant increase in heart weights, creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), troponin I, cardiac tissue malondialdehyde (MDA), tumor necrosis factor alpha (TNF-α), interleukin 1β (IL1 β), and caspase-3 levels. Furthermore, CP group showed toxic histopathological changes of marked cardiac damage in addition to a significant decrease in total antioxidant capacity (TAC), superoxide dismutase (SOD), eNOS gene expression, and B cell lymphoma 2 (Bcl2) immunoexpression. NIC succeeded in reversing CP-induced cardiotoxicity by its potassium channel opening effect, stimulating eNOS gene expression, anti-inflammatory, antiapoptotic, and antioxidant properties. Coadministration of GP or L-NNA could diminish the protective effect of NIC. This proves the important role of K_ATP and eNOS in mediating such protection.