OBJECTIVE

Neutrophils promote experimental abdominal aortic aneurysm (AAA) formation via a mechanism that is independent from MMPs (matrix metalloproteinases). Recently, we reported a dominant role of IL (interleukin)-1β in the formation of murine experimental AAAs. Here, the hypothesis that IL-1β-induced neutrophil extracellular trap formation (NETosis) promotes AAA was tested.

UNASSIGNED

NETs were identified through colocalized staining of neutrophil, Cit-H3 (citrullinated histone H3), and DNA, using immunohistochemistry. NETs were detected in human AAAs and were colocalized with IL-1β. In vitro, IL-1RA attenuated IL-1β-induced NETosis in human neutrophils. Mechanistically, IL-1β treatment of isolated neutrophils induced nuclear localization of ceramide synthase 6 and synthesis of C16-ceramide, which was inhibited by IL-1RA or fumonisin B1, an inhibitor of ceramide synthesis. Furthermore, IL-1RA or fumonisin B1 attenuated IL1-β-induced NETosis. In an experimental model of murine AAA, NETs were detected at a very early stage-day 3 of aneurysm induction. IL-1β-knockout mice demonstrated significantly lower infiltration of neutrophils to aorta and were protected from AAA. Adoptive transfer of wild-type neutrophils promoted AAA formation in IL-1β-knockout mice. Moreover, treatment of wild-type mice with Cl-amidine, an inhibitor NETosis, significantly attenuated AAA formation, whereas, treatment with deoxyribonuclease, a DNA digesting enzyme, had no effect on AAA formation.

CONCLUSIONS

Altogether, the results suggest a dominant role of IL-1β-induced NETosis in AAA formation.

BACKGROUND

Prostate cancer (PCa) harbors a myriad of genomic and epigenetic defects. Cytosine methylation of CpG-rich promoter DNA is an important mechanism of epigenetic gene inactivation in PCa. There is considerable amount of data to suggest that DNA methylation-based biomarkers may be useful for the early detection and diagnosis of PCa. In addition, candidate gene-based studies have shown an association between specific gene methylation and alterations and clinicopathologic indicators of poor prognosis in PCa.

METHODS

To more comprehensively identify DNA methylation alterations in PCa initiation and progression, we examined the methylation status of 485 577 CpG sites from regions with a broad spectrum of CpG densities, interrogating both gene-associated and non-associated regions using the recently developed Illumina 450K methylation platform.

RESULTS

In all, we selected 33 promoter-associated novel CpG sites that were differentially methylated in high-grade prostatic intraepithelial neoplasia and PCa in comparison with benign prostate tissue samples (false discovery rate-adjusted P-value <0.05; β-value 0.2; fold change >1.5). Of the 33 genes, hierarchical clustering analysis demonstrated BNC1, FZD1, RPL39L, SYN2, LMX1B, CXXC5, ZNF783 and CYB5R2 as top candidate novel genes that are frequently methylated and whose methylation was associated with inactivation of gene expression in PCa cell lines. Pathway analysis of the genes with altered methylation patterns identified the involvement of a cancer-related network of genes whose activity may be regulated by TP53, MYC, TNF, IL1 and 6, IFN-γ and FOS in prostate pathogenesis.

CONCLUSIONS

Our genome-wide methylation profile shows epigenetic dysregulation of important regulatory signals in prostate carcinogenesis.

Cell proliferation and differentiation are intimately related processes where the proto-oncogenes c-myc and c-myb have been implicated to play a role. Previously, we have shown that both c-myc and c-myb were induced in normal myeloid precursors when the cells were stimulated for growth, were expressed in the autonomously proliferating myeloid leukemic M1 cell line and were rapidly suppressed in both normal and M1 cells following induction of terminal differentiation associated with growth arrest. In order to distinguish molecular events associated with terminal differentiation versus those due to growth inhibition, as well as to increase our understanding of the role of the proto-oncogenes c-myc and c-myb in both of these cellular processes, in this work we have studied the expression of c-myc and c-myb in M1 cells induced for growth inhibition associated with terminal differentiation (via treatment with the physiological inducers IL6 or leukemia inhibitory factor mean value of LIF), partial differentiation (using IL1 or LPS) or no detectable differentiation properties (using IFN beta or IFN gamma). We show that, for all the treatments used in this study, down regulation of the proto-oncogenes c-myc and c-myb occurred only when M1 cells were stimulated to undergo terminal differentiation. In addition, we transfected the M1 cell line with a vector containing the c-myc gene under control of the beta-actin promoter, so that c-myc was no longer down regulated by IL6 or LIF. Previously, we have shown that in the presence of the myeloid differentiation inducers IL6 or LIF, these M1myc cells were blocked at an intermediate stage of myeloid differentiation and continued to proliferate. In sharp contrast to their altered response to IL6 or LIF, M1myc cells were as responsive as the parental M1 cells to growth suppression by the different antiproliferative compounds which do not induce terminal differentiation. Thus, continued expression of c-myc had no effect on growth suppression induced by IL1, IFN beta, IFN gamma and LPS. Taken together, these results indicate that c-myc and c-myb down regulation is not necessary for growth suppression, but down regulation of c-myc is, and c-myb may be, essential for terminal differentiation.

Interleukin-1 (IL1) is a potent endogenous pyrogen and inducer of the acute phase response, and these innate immune responses are an important part of the human host's initial reaction to infection by the malaria parasite. In addition, several single-nucleotide polymorphisms (SNPs) in this region have previously been demonstrated to be associated with susceptibility to infectious disease. Therefore, a possible association with malaria susceptibility was investigated. A total of 13 polymorphic markers were used in a two-stage screening strategy to genotype a Gambian case-control study group by either restriction endonuclease digestion or the Sequenom MassARRAY assay. This involved an initial screen of 188 severe cases and 188 mild controls, and if there was a significant association with a malaria phenotype (P<0.05); this was followed by screening of the remaining 1044 samples. Two markers showed significant association with malaria: interleukin-1 alpha +4845 G ->> T (P=0.035 for mild malaria versus controls) and interleukin-1 beta +3953 C ->> T (P=0.030 for mild malaria versus severe malaria). Haplotypes constructed using the SNPHAP programme were not associated with any of the malaria phenotypes investigated. In summary, if IL1 variants are involved in malaria susceptibility in the Gambia at all, then the effects are small.

Interleukin 1 beta (IL-1 beta) is produced as a biologically inactive 31 kD precursor, which is converted to the active 18 kD form by proteolytic processing. Keratinocytes express IL-1 beta but not the active form of the specific IL-1 beta converting enzyme (ICE). We have recently presented evidence that IL-1 beta activation in human epidermis occurs via an alternative mechanism involving hitherto unknown proteases. We asked whether stratum corneum chymotryptic enzyme (SCCE), which is a serine protease specifically expressed in keratinizing squamous epithelia, can act as an IL-1 beta activator in vitro. Recombinant human pro-IL-1 beta was incubated with recombinant SCCE, and the reaction products were characterized as regards molecular size and ability to induce expression of E-Selectin in human umbilical cord endothelial cells. SCCE caused degradation of pro-IL-1 beta and the accumulation of a component with electrophoretic mobility slightly lower than recombination mature IL-1 beta. Whereas incubation mixtures with pro-IL-1 beta which had been incubated in the absence of SCCE, or with SCCE, which had been incubated in the absence of pro-Il-1 beta, did not induce expression above baseline levels of E-Selectin, pro-Il1 beta which had been incubated with SCCE induced a significant increase in E-Selectin expression. This effect could be abolished by neutralizing antibodies to IL-1 beta, but not by antibodies to IL-1 alpha. In addition to IL-1 beta activation, SCCE also prepared to be able to catalyze a further degradation of IL-1 beta, leading to a loss of biological activity. We conclude that SCCE is a potential candidate for being responsible for IL-1 beta activation in human epidermis.

Vascular endothelial cells (EC) play a key role in viral tropism in vivo. Since conflicting reports have been published on the capability of HIV to infect EC in vitro, we analyzed some factors potentially capable of influencing the susceptibility of human umbilical vein endothelial cells (HUVEC) to HIV-1. Both primary cultures and differentiated immortalized HUVEC lines were used. HUVEC were negative for the expression of CD4, but weakly CD26- and galactosylceramide-positive. Although binding of HIV to EC was substantial, the virus was apparently incapable of replicating in nonproliferating cultures. In resting cultures, the content of cell-associated HIV disappeared 4-6 days after infection without production of p24 and infectious progency. In contrast, infection of proliferating EC cultures led to the transient release of p24 and infectious virus (10(2.5)-10(3.5) SFU/ml) peaking 2-6 days postinfection. Antibody neutralization of cytokines that may be produced by EC (IL1, IL6, IL8, TNF, IFN-beta) failed to modify virus adsorption and replication, whereas treatment with IL1-beta plus TNF-alpha stimulated both virus binding and virus release. As seen by gag polymerase chain reaction (PCR), the viral genome persisted up to 15 days in untreated EC cultures, but over 20 days in cultures exposed to IL1-beta plus TNF-alpha. This study shows that: (a) CD4-negative HUVEC are capable of binding substantial amounts of HIV-1; (b) binding is enhanced by proinflammatory cytokines; (c) the establishment of productive infection is favored by cell proliferation; and (d) exposure to IL1-beta plus TNF-alpha enhances virus replication.

OBJECTIVE

The P2X(7) receptor exhibits a high degree of plasticity with agonist potency increasing after prolonged receptor activation. In this study we investigated the ability of lipids to modulate agonist potency at P2X(7) receptors.

METHODS

A variety of lipids, including lysophosphatidylcholine, sphingosylphosphorylcholine and hexadecylphosphorylcholine were studied for their effect on P2X(7) receptor-stimulated ethidium bromide accumulation in cells expressing human recombinant P2X(7) receptors and on P2X(7) receptor-stimulated interleukin-1 beta (IL1 beta) release from THP-1 cells. The effects of the lipids were also assessed in radioligand binding studies on human P2X(7) receptors.

RESULTS

At concentrations (3-30 microM) below the threshold to cause cell lysis, the lipids increased agonist potency and/or maximal effects at P2X(7) receptors in both ethidium accumulation and IL1 beta release studies. There was little structure activity relationship (SAR) for this effect and sub-lytic concentrations of Triton X-100 partially mimicked the effects of the lipids. The lipids caused cell lysis and increased intracellular calcium at higher concentrations (30-100 microM) which complicated interpretation of their effects in functional studies. However, the lipids (3-100 microM) also increased agonist potency 30-100 fold in radioligand binding studies.

CONCLUSIONS

This study demonstrates that a diverse range of lipids increase agonist potency at the P2X(7) receptor in functional and binding studies. The broad SAR, including the effect of Triton X-100, suggests this may reflect changes in membrane properties rather than a direct effect on the P2X(7) receptor. Since many of the lipids studied accumulate in disease states they may enhance P2X(7) receptor function under pathophysiological conditions.

It has been described that exercise can modulate both inflammatory response and epigenetic modifications, although the effect of exercise on these parameters during the normal brain aging process yet remains poorly understood. Here, we investigated the effect of aging and treadmill exercise on inflammatory and epigenetic parameters specifically pro and anti-inflammatory cytokines levels, activation of NF-kB and histone H4 acetylation levels in hippocampus from Wistar rats. Additionally, we evaluated aversive memory through inhibitory avoidance task. Rats of 3 and 20 months of age were assigned to non-exercised (sedentary) and exercised (running daily for 20 min for 2 weeks) groups. The effect of daily forced exercise in the treadmill was assessed. The levels of inflammatory and epigenetic parameters were determined 1h, 18 h, 3 days or 7 days after the last training session of exercise. It was observed an age-related decline on aversive memory, as well as aged rats showed increased hippocampal levels of inflammatory markers, such as TNFα, IL1-β and NF-kB and decreased IL-4 levels, an anti-inflammatory cytokine. Moreover, lower levels of global histone H4 acetylation were also observed in hippocampi from aged rats. Interestingly, there was a significant correlation between the biochemical markers and the inhibitory avoidance test performance. The forced exercise protocol ameliorated aging-related memory decline, decreased pro-inflammatory markers and increased histone H4 acetylation levels in hippocampi 20-months-old rats, while increased acutely IL-4 levels in hippocampi from young adult rats. Together, these results suggest that an imbalance of inflammatory markers might be involved to the aging-related aversive memory impairment. Additionally, our exercise protocol may reverse aging-related memory decline through improving cytokine profile.

The cause of Parkinson's disease remains unknown although some evidence suggests that an inflammatory reaction, mediated by cytokines such as TNF-alpha and IL-1beta, is related with dopaminergic degeneration in the brain. In the present work we measured the plasma levels of TNF-alpha and IL-1beta in parkinsonian monkeys one year after MPTP administration. TNF-alpha levels were seen to have increased in parkinsonian monkeys reflecting the clinical symptoms observed, while IL-1beta levels remained unchanged. These results suggest that TNF-alpha plays a role in sustaining of dopaminergic degeneration in chronic parkinsonism.

Skimmin is one of the major pharmacologically active molecules present in Hydrangea paniculata, a medical herb used in the traditional Chinese medicine as an anti-inflammatory agent. In the current study, we attempted to investigate its renoprotective activity and underlying mechanisms in a rat model of membranous glomerulonephritis induced by cationic bovine serum albumin (c-BSA). Sprague-Dawley (SD) rats were divided into five groups, including normal control, model control, Mycophenolate Mofetil-treated group, and two skimming-treated groups (15 mg/kg and 30 mg/kg). Our research showed that treatment with skimmin significantly reduced the levels of blood urea nitrogen (BUN), urinary albumin excretion (UAE), and serum creatinine (Scr) as compared with model control after experimental induction of membranous glomerulonephritis (P < 0.01). Moreover, glomerular hypercellularity, tubulointerstitial injury, and glomerular deposition of IgG were less intense after skimmin treatment. By immunochemistry analysis, we demonstrated that skimmin could significantly inhibit interleukin-1 β (IL1 β ) and IL-6 expression (P < 0.05), reduce the loss of nephrin and podocin, and suppress the infiltration of renal interstitium by CD3-positive T cell and CD20-positive B cell. These results suggest that treatment with skimmin can significantly improve renal function and suppress the IgG deposition as well as the development of glomerular lesions in a rat model of membranous glomerulonephritis.

BACKGROUND

Prolonged psychosocial stress is a condition assessed through self-reports. Here we aimed to identify biochemical markers for screening and early intervention in women.

METHODS

Plasma concentrations of interleukin (IL) 1-alpha, IL1-beta, IL-2, IL-4, IL-6, IL-8, IL-10, interferon-gamma (INF-gamma), tumor necrosis factor-alpha (TNF-alpha), monocyte chemotactic protein-1 (MCP-1), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), thyroid stimulating hormone (TSH), total tri-iodothyronine (TT3), total thyroxine (TT4), prolactin, and testosterone were measured in: 195 women on long-term sick-leave for a stress-related affective disorder, 45 women at risk for professional burnout, and 84 healthy women.

RESULTS

We found significantly increased levels of MCP-1, VEGF and EGF in women exposed to prolonged psychosocial stress. Statistical analysis indicates that they independently associate with a significant risk for being classified as ill.

CONCLUSIONS

MCP-1, EGF, and VEGF are potential markers for screening and early intervention in women under prolonged psychosocial stress.

To assess whether NLR pyrin domain-containing protein 3 (NLRP3) inflammasome, a multiprotein complex that mediates the activation of caspase-1 (CASP-1) and pro-inflammatory cytokines IL-18 and IL-1β, could be involved in the chronic inflammatory state observed in chronic kidney disease patients undergoing hemodialysis treatment (CKD-HD), we employed several biomolecular techniques including RT-PCR, western blot, FACS analysis, confocal microscopy and microarray. Interestingly, peripheral blood mononuclear cells from 15 CKD-HD patients showed higher mRNA levels of NLRP3, CASP-1, ASC, IL-1β, IL-18 and P2X7 receptor compared to 15 healthy subjects. Western blotting analysis confirmed the above results. In particular, active forms of CASP-1, IL1-β and IL-18 resulted significantly up-regulated in CKD-HD versus controls. Additionally, elevated mitochondrial ROS level, colocalization of NLRP3/ASC/mitochondria in peripheral blood mononuclear cells from CKD-HD patients and down-regulation of CASP-1, IL1-β and IL-18 protein levels in immune-cells of CKD-HD patients stimulated with LPS/ATP in presence of mitoTEMPO, inhibitor of mitochondrial ROS production, suggested a possible role of this organelle in the aforementioned CKD-associated inflammasome activation. Then, microarray analysis confirmed, in an independent microarray study cohort, that NLRP3 and CASP-1, along with other inflammasome-related genes, were up-regulated in 17 CKD-HD patients and they were able to clearly discriminate these patients from 5 healthy subjects. All together these data showed, for the first time, that NLRP3 inflammasome was activated in uremic patients undergoing dialysis treatment and they suggested that this unphysiological condition could be possibly induced by mitochondrial dysfunction.

BACKGROUND

Interleukin-1 beta (IL-1 beta) is a potent inflammatory mediator and an important polymorphism in the locus +3954 (C/T) of the human IL1 B gene has been shown to affect the levels of this cytokine. This functional polymorphism has been associated with the establishment of inflammatory diseases, including periodontal disease, in European, Asian and North American populations.

OBJECTIVE

The aim of this study was to investigate the association between the IL1 B (+3954) gene polymorphism and the occurrence of different clinical forms of periodontitis in a sample of Brazilian individuals.

METHODS

This study employed a cross-sectional design involving individuals from the State of Minas Gerais in the south-eastern region of Brazil. Genomic DNA was obtained from oral swabs of 129 individuals and amplified using the polymerase chain reaction (PCR) with specific primers flanking the locus +3954 of IL1 B. PCR products were submitted to restriction endonuclease digestion and analyzed by polyacrylamide gel electrophoresis, to distinguish alleles T and C of the IL1 B gene, allowing for the determination of the genotypes and detection of the polymorphism.

CONCLUSIONS

The chronic periodontitis group displayed a higher percentage of the T allele (28%) when compared to the aggressive periodontitis group (10.7%, chi(2)=5.24, p=0.02, OR=0.31, CI=0.11--0.88) and to control group (8.7%, chi(2)=7.11, p=0.007, OR=0.24, CI=0.08--0.73). Our data suggested that the polymorphism in the locus +3954 of IL1 B gene could be a risk factor for chronic periodontitis in a sample of Brazilian individuals.

OBJECTIVE

To compare respiratory symptoms and upper airway inflammation in domestic waste collectors and controls, and to find the association between measures of upper airway inflammation on the one hand and exposure concentrations of organic dust or respiratory symptoms on the other hand.

METHODS

In a cross sectional study among 47 waste collectors and 15 controls, questionnaire data on respiratory symptoms were collected. Nasal lavage (NAL), to assess upper airway inflammation, was performed before and after a work shift at the beginning and at the end of the working week. In NAL fluid, cells were counted and differentiated and concentrations of interleukin 6 (IL6), IL8, tumour necrosis factor-alpha (TNF alpha), and IL1 beta were measured. In collectors, inhalable dust samples were collected in which bacterial endotoxin and mould beta(1-->3)-glucan were assessed.

RESULTS

Prevalence of respiratory symptoms was higher in waste collectors than in controls. Geometric mean exposure concentrations were 0.58 mg/m(3) for dust, 39 EU/m(3) for endotoxin, and 1.3 microg/m(3) for beta(1-->3)-glucan. At the end of the week collectors had higher concentrations of total cells and IL8 in NAL before and after a shift than controls (cells, before 1.9-fold p<0.10, after 3.3-fold p<0.01; IL8, before and after 1.8-fold p<0.05), and after/before work shift ratios of total cells were also higher (2.3-fold p=0.06) in collectors than in controls. Cells in NAL fluid consisted predominantly of neutrophils and epithelial cells, whereas eosinophils and mononuclear cells were rarely found. Exposure to dust and endotoxin was associated with concentrations of IL8 after the shift (p<0.05). Increased concentrations of IL8 (p<0.05) and total cells (p<0.10) after the shift were associated with respiratory symptoms. Concentrations of IL6, TNF alpha, and IL1 beta were not associated with waste collecting, symptoms, or exposure.

CONCLUSIONS

Waste collectors show signs of increased upper airway inflammation and respiratory symptoms compared with controls. Exposure to organic dust probably underlies the inflammation mediated by neutrophils that result in respiratory symptoms.

To investigate the effect of the heme moiety of malaria pigment, hemozoin, on phagocyte functions, mouse macrophages were fed with insoluble beta-hematin, the synthetic heme-polymer chemically identical to the native pigment, or the soluble monomer, hematin. Production of inflammatory cytokines, interleukin 1 (IL1), tumor necrosis factor alpha (TNF alpha), and nitric oxide (NO) was assayed in the supernatants after stimulation with lipopolysaccharide. The results indicate that both beta-hematin and hematin induce a dose-dependent inhibition of macrophage production of TNF alpha and NO, but not of IL1. One-hour pretreatment with soluble hematin inhibited production of cytotoxic mediators by more than 50% compared to controls, while 6-hr exposure was necessary for insoluble beta-hematin to induce the same level of inhibition. However, the same treatment did not modify the production of TNF alpha and NO by mouse microglia cell lines. The inhibition was partially counterbalanced by adding sulphydryl group donors such as 2-mercaptoethanol, glutathione, or N-acetyl-cysteine during the preincubation time. The results of the present study confirm the inhibitory role of malaria pigment and show that such effect is due to the heme moiety and may be selective for the production of cytotoxic mediators by specific phagocytes. The implications of these findings in the control of malaria infection and disease and in the pathogenesis of severe malaria are discussed.

OBJECTIVE

To evaluate the effect of the 4G/5G PAI-1 gene polymorphism on the development of organ failure and outcome in critically ill patients with septic syndromes.

METHODS

Prospective, observational study in a medical intensive care unit of a university hospital.

METHODS

224 consecutively admitted patients.

METHODS

Epidemiological data, severity scores, and the primary site of infection were recorded. DNA genotyping of the PAI-1, TNF-beta, and IL1-ra genes, and measurement of plasma PAI-1 antigen and D-dimer were carried out.

METHODS

The primary outcome variables were organ dysfunction and mortality.

RESULTS

Eighty-eight subjects had septic shock at ICU entry or within 48 h from admission. Homozygotes for the 4G allele exhibited higher plasma concentrations of PAI-1 antigen and D-dimer than 4G/5G and 5G/5G subjects). ICU mortality was 44.0% in patients with 4G/4G, 23.4% in 4G/5G and 12.5% in 5G/5G, mainly due to multiorgan failure. After adjusting for SAPS II at admission the genotypes independently associated with ICU mortality in septic shock were TNF-B2/B2 (OR 2.83, 1.04-7.67) and 4G/4G of PAI-1 (OR 2.23, 1.02-4.85). The PAI-1 genotype did not determine susceptibility to infection or the outcome in nonseptic systemic inflammatory response syndrome, sepsis, severe sepsis, and nosocomial septic shock.

CONCLUSIONS

Homozygosity for 4G of the PAI-1 gene confers an increase in the risk of mortality in adult patients with septic shock due to a greater organ failure.

Previously we reported that several microRNAs (miRNA) are upregulated following experimentally induced traumatic brain injury (TBI) using both in vivo and in vitro approaches. Specific miRNAs were found to be sensitive to therapeutic hypothermia and may therefore be important targets for neuroprotective strategies. In this study we developed plasmid constructs that overexpress temperature sensitive miRNAs: miR-34a, miR-451, and miR-874. These constructs were transfected into cultured cortical neurons that were subjected to stretch injury using a cell injury controller device. Levels of expression of genes associated with stress, inflammation, apoptosis and transcriptional regulation were measured by qRT-PCR. mRNA levels of cytokines interleukin 1-β (IL1-β) and tumor necrosis factor alpha (TNF-α) as well as heat shock protein 70 (HSP70) and Caspase 11 were found to be increased up to 24 fold higher than controls in cells overexpressing these miRNAs. After moderate stretch injury, the expression of IL1-β, TNF-α, HSP70 and Caspase 11 all increased over control levels found in uninjured cells suggesting that overexpression of these miRNAs increases cellular vulnerability. miR-34a directly inhibits Bcl2 and XIAP, both anti-apoptotic proteins. The observed increase in Caspase 11 with over-expression of miR-34a indicates that miR-34a may be inducing apoptosis by reducing the levels of anti-apoptotic proteins. miR-34a is predicted to inhibit Jun, which was seen to decrease in cells overexpressing this miRNA along with Fos. Over expression of several miRNAs found to be induced by TBI in vivo (miR-34a, miR-451 and miR-874) leads to increased vulnerability in transfected neurons. Therapeutic hypothermia blunts the expression of these miRNAs in vivo and antisense silencing could be a potential therapeutic approach to targeting the consequences of TBI.

We report here defined culture conditions that allow reproducibly the growth of the majority of immature thymocytes from both fetal (14-15 days of gestation) and adult mice. The combination of phorbol myristate acetate (PMA), ionomycin and recombinant interleukin 2 (IL2) is both sufficient and necessary to induce growth of about 1/6.2 (range 1/3-1/9) and 1/4.3 (range 1/2-1/7) immature thymocytes from adult and fetal mice, respectively, in serum-free cultures. Several other combinations tested (e.g. PMA + IL2, concanavalin A + IL2) were poorly or not active. None of the agents tested alone (PMA, ionomycin, concanavalin A, pokeweed mitogen, IL2) had any effect. We found no evidence for a role of IL1 and IL3 on growth of these cells. The growth of activated immature thymocytes from either fetal or adult mice was inhibited by a monoclonal antibody against mouse IL2 receptors. Under the same conditions that stimulated growth of most immature thymocytes, they did not mature into cells expressing Lyt-2, L3T4 or T cell antigen receptor (KJ16) after 7 to 15 days of continuous proliferation in culture. Nor did they give rise to cells with cytolytic activity after 7-9 days of culture. In some but not all experiments cultures of immature thymocytes from adult mice but not from fetal mice generated cells (1 out of 120-310) with helper function for B lymphocytes. While we confirmed here that approximately 50-70% freshly isolated immature thymocytes express receptors for IL2, our results indicate that these cells need to be activated (by e.g. PMA + ionomycin) to respond to IL2. A possible mechanism to account for the expression of nonfunctionally competent IL2 receptors is proposed and our results concerning the maturation of immature thymocytes in vitro are discussed.

Alternate colorectal cancer (CRC) screening and surveillance strategies are needed to pre-select candidates for invasive methods. We compared systemic inflammatory profiles in CRC (n=99), health (n=98), high CRC-risk conditions (n=48) and overt inflammation (n=69) by multiplexed analysis of IL-1β, IL-6, IL-8, FGF-2, G-CSF, GM-CSF, MCP-1, MIP-1α, TNF-α, VEGF-A, and PDGF-B and CEA. Cytokines corresponded with CRC advancement. FGF2, GM-CSF, IL-1β, IL-6, MIP-1α, PDGF-BB, TNF-α, and VEGF-A were higher than in controls already in stage I CRC with FGF2, IL1-β, and MIP-1α higher than in high CRC-risk individuals as well. Cytokine panels devised to differentiate early CRC from controls, adenomas, or inflammatory bowel disease patients (IBD) had good accuracy but only IBD panel had promising specificity at 95% sensitivity.

Tobacco smoking is the main risk factor for lung cancer. Only 10-15% of smokers develop lung cancer, suggesting that genetic factors are of importance in determining individual susceptibility to the disease. Several studies in recent years indicate that chronic inflammation is a cofactor in lung carcinogenesis. We have previously reported an association of interleukin 1 beta gene (IL1B) polymorphisms with lung cancer risk. Interleukin-1 receptor antagonist (IL-1Ra) has been implicated in carcinogenesis of different cancer types. IL-1Ra binds competitively to the same membrane receptor as interleukin-1beta (IL-1beta) and thereby acts as an antagonist to the pro-inflammatory actions of IL-1beta. The aim of the study was to examine whether a common VNTR polymorphism in the interleukin 1 receptor antagonist gene (IL1RN) is associated with lung cancer risk. Due to the tight relationship between IL1RN and IL1B, we also explored the possibility of an interaction between the two genes. The study population comprised of 340 non-small cell lung cancer cases and 412 healthy controls of Norwegian origin. Our results indicate that individuals homozygous for the IL1RN*1 allele and carrying the IL1B-31T allele had increased risk of non-small cell lung cancer (odds ratio C/T 3.08; 1.10-8.62 and T/T 5.87; 2.15-16.05). Furthermore, IL1RN*1 carriers had nearly two-fold higher levels of bulky/hydrophobic DNA adducts in the lung. Our findings support the significance of IL1 gene cluster polymorphisms and risk of lung cancer.

Fibroblasts of the periodontium may be involved in extracellular matrix degradation in response to inflammatory cytokines produced by mononuclear phagocytes. Interleukin 1 (IL1), one of these biologically-active agents, is produced by such cells when stimulated by lipopolysaccharide (LPS). Periodontal-ligament (PLF) and gingival fibroblasts responded to recombinant human IL1 beta and to media conditioned by LPS-stimulated mononuclear phagocytes by secreting prostaglandin E (PGE). This response was dose- and time-dependent. Stimulated gingival fibroblasts also produced about five- to ten-fold as much collagenolytic activity when compared to controls but PLF produced no more activity. On mixing the conditioned media from both fibroblast types, inhibitory activity was found in the PLF-culture medium. Thus gingival fibroblasts in particular may be involved in the pathogenesis of periodontal disease by responding to factors produced by inflammatory phagocytes.

OBJECTIVE

To investigate if a difference exists between young and old mice in the response of articular cartilage to interleukin 1 (IL1) and transforming growth factor beta (TGFbeta) alone or in combination.

METHODS

The interaction of IL1 and TGFbeta was studied in cartilage of young (three months) and old mice (18 months) both in vivo and in vitro. Therefore, IL1, TGFbeta, or IL1 together with TGFbeta was injected into the knee joints of mice on days 1, 3, and 5 before harvest of the patellae on day 6. Alternatively, isolated patellae were stimulated with IL1, TGFbeta, or IL1 together with TGFbeta in culture for 48 hours. Proteoglycan (PG) synthesis and nitric oxide (NO) production were measured.

RESULTS

IL1 inhibited PG synthesis and increased NO production in cartilage of both young and old mice. On the other hand, TGFbeta stimulated PG synthesis and reduced NO production in both age groups. Importantly, TGFbeta was able to counteract IL1 mediated effects on PG synthesis and NO production in young but not in old mice.

CONCLUSIONS

Contrary to the findings in young mice, the cartilage of old animals does not antagonise IL1 effects via TGFbeta. This loss of responsiveness to the pivotal cytokine TGFbeta on effects of IL1 can be important in the initiation and progression of osteoarthritis (OA).

Rheumatoid arthritis (RA) is characterized by a pre-vascular seriously inflammatory phase, followed by a vascular phase with high increase in vessel growth. Since angiogenesis has been considered as an essential event in perpetuating inflammatory and immune responses, as well as supporting pannus growth and development of RA, inhibition of angiogenesis has been proposed as a novel therapeutic strategy for RA. Triptolide, a diterpenoid triepoxide from Tripterygium wilfordii Hook F, has been extensively used in treatment of RA patients. It also acts as a small molecule inhibitor of tumor angiogenesis in several cancer types. However, it is unclear whether triptolide possesses an anti-angiogenic effect in RA. To address this problem, we constructed collagen-induced arthritis (CIA) model using DA rats by the injection of bovine type II collagen. Then, CIA rats were treated with triptolide (11-45 µg/kg/day) starting on the day 1 after first immunization. The arthritis scores (P<0.05) and the arthritis incidence (P<0.05) of inflamed joints were both significantly decreased in triptolide-treated CIA rats compared to vehicle CIA rats. More interestingly, doses of 11~45 µg/kg triptolide could markedly reduce the capillaries, small, medium and large vessel density in synovial membrane tissues of inflamed joints (all P<0.05). Moreover, triptolide inhibited matrigel-induced cell adhesion of HFLS-RA and HUVEC. It also disrupted tube formation of HUVEC on matrigel and suppressed the VEGF-induced chemotactic migration of HFLS-RA and HUVEC, respectively. Furthermore, triptolide significantly reduced the expression of angiogenic activators including TNF-α, IL-17, VEGF, VEGFR, Ang-1, Ang-2 and Tie2, as well as suppressed the IL1-β-induced phosphorylated of ERK, p38 and JNK at protein levels. In conclusion, our data suggest for the first time that triptolide may possess anti-angiogenic effect in RA both in vivo and in vitro assay systems by downregulating the angiogenic activators and inhibiting the activation of mitogen-activated protein kinase downstream signal pathway.

OBJECTIVE

Ellagic acid (EA) has shown antinociceptive and anti-inflammatory effects. Inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2) enzymes and also cytokines play a key role in many inflammatory conditions. This study was aimed to investigate the mechanisms involved in the anti-inflammatory effect of EA.

METHODS

Carrageenan-induced mouse paw edema model was used for induction of inflammation.

RESULTS

The results showed that intraplantar injection of carrageenan led to time-dependent development of peripheral inflammation, which resulted in a significant increase in the levels of tumor necrosis factor α (TNF-α) and interleukin 1 (IL-1) β, nitric oxide (NO) and prostaglandin E2 (PGE2) and also iNOS and COX-2 protein expression in inflamed paw. However, systemic administration of EA (1-30 mg/kg, intraperitoneal [i.p.]) could reduce edema in a dose-dependent fashion in inflamed rat paws with ED50 value 8.41 (5.26-14.76) mg/kg. It decreased the serum concentration of NO, PGE2, aspartate aminotransferase and alanine aminotransferase, and suppress the protein expression of iNOS, COX-2 enzymes, and attenuated the formation of PGE2, TNF-α and IL-1 β in inflamed paw tissue. We also demonstrated that EA significantly decreased the malondialdehyde (MDA) level in liver at 5 h after carrageenan injection. Moreover, histopathological studies indicated that EA significantly diminished migration of polymorphonuclear leukocytes into site of inflammation, as did indomethacin.

CONCLUSIONS

Collectively, the anti-inflammatory mechanisms of EA might be related to the decrease in the level of MDA, iNOS, and COX-2 in the edema paw via the suppression of pro-inflammatory cytokines (TNFα, IL1 β), NO and PGE2 overproduction.