Ebselen and cytokine-induced nitric oxide synthase expression in insulin-producing cells

Interleukin-1 (IL-1) may be a mediator of β-cell damage in insulin-dependent diabetes mellitus (IDDM). The IL-1 mechanism of action on insulin-producing cells probably includes activation of the transcription nuclear factor κB (NF-κB), increased transcription of the inducible form of nitric oxide synthase (iNOS) and the subsequent production of nitric oxide (NO). Reactive oxygen intermediates, particularly H2O2, have been proposed as second messengers for NF-κB activation. In the present study, we tested whether ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a glutathione peroxidase mimicking compound, could counteract the effects of IL-1β, H2O2 and alloxan in rat pancreatic islets and in the rat insulinoma cell line RINm5F (RIN cells). Some of these experiments were also reproduced in human pancreatic islets. Ebselen (20 μM) prevented the increase in nitrite production by rat islets exposed to IL-1β for 6 hr and induced significant protection against the acute inhibitory effects of alloxan or H2O2 exposure, as judged by the preserved glucose oxidation rates. However, ebselen failed to prevent the increase in nitrite production and the decrease in glucose oxidation and insulin release by rat islets exposed to IL-1β for 24 hr. Ebselen prevented the increase in nitrite production by human islets exposed for 14 hr to a combination of cytokines (IL-1β, tumor necrosis factor-α and interferon-γ). In RIN cells, ebselen counteracted both the expression of iNOS mRNA and the increase in nitrite production induced by 6 hr exposure to IL-β but failed to block IL-1β-induced iNOS expression following 24 hr exposure to the cytokine. Moreover, ebselen did not prevent IL-1β-induced NF-κB activation. As a whole, these data indicate that ebselen partially counteracts cytokine-induced NOS activation in pancreatic β-cells, an effect not associated with inhibition of NF-κB activation.

SOCS3 expression correlates with severity of inflammation, expression of proinflammatory cytokines, and activation of STAT3 and p38 MAPK in LPS-induced inflammation in vivo

SOCS3 is an inducible endogenous negative regulator of JAK/STAT pathway, which is relevant in inflammatory conditions. We used a model of LPS-induced periodontal disease in rats to correlate SOCS3 expression with the inflammatory status. In vitro we used a murine macrophage cell line to assess the physical interaction between SOCS3 and STAT3 by coimmunoprecipitation. 30 ug of LPS from Escherichia coli were injected in the gingival tissues on the palatal aspect of first molars of the animals 3x/week for up to 4 weeks. Control animals were injected with the vehicle (PBS). The rats were sacrificed at 7, 15, and 30 days. Inflammation and gene expression were assessed by stereometric analysis, immunohistochemistry, RT-qPCR, and western blot. LPS injections increased inflammation, paralleled by an upregulation of SOCS3, of the proinflammatory cytokines IL-1β, IL-6, and TNF-and increased phosphorylation of STAT3 and p38 MAPK. SOCS3 expression accompanied the severity of inflammation and the expression of proinflammatory cytokines, as well as the activation status of STAT3 and p38 MAPK. LPS stimulation in a macrophage cell line in vitro induced transient STAT3 activation, which was inversely correlated with a dynamic physical interaction with SOCS3, suggesting that this may be a mechanism for SOCS3 regulatory function. © 2013 João Antônio Chaves de Souza et al.

The immunomodulatory effect of propolis on receptors expression, cytokine production and fungicidal activity of human monocytes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of Toll-Like Receptor 2 and 4 RNA Expression and the Cytokine Profile in Postmenopausal Women with Metabolic Syndrome

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Nitric oxide and brazilian propolis combined accelerates tissue repair by modulating cell migration, cytokine production and collagen deposition in experimental leishmaniasis

The fact that drugs currently used in the treatment of Leishmania are highly toxic and associated with acquired resistance has promoted the search for new therapies for treating American tegumentary leishmaniasis (ATL). In this study, BALB/c mice were injected in the hind paw with Leishmania (Leishmania) amazonensis and subsequently treated with a combination of nitric oxide (NO) donor (cis-[Ru(bpy)(2)imN(NO)](PF6)(3)) (Ru-NO), given by intraperitoneal injection, and oral Brazilian propolis for 30 days. Ru-NO reached the center of the lesion and increased the NO level in the injured hind paw without lesion exacerbation. Histological and immunological parameters of chronic inflammation showed that this combined treatment increased the efficacy of macrophages, determined by the decrease in the number of parasitized cells, leading to reduced expression of proinflammatory and tissue damage markers. In addition, these drugs in combination fostered wound healing, enhanced the number of fibroblasts, pro-healing cytokines and induced collagen synthesis at the lesion site. Overall, our findings suggest that the combination of the NO donor Ru-NO and Brazilian propolis alleviates experimental ATL lesions, highlighting a new therapeutic option that can be considered for further in vivo investigations as a candidate for the treatment of cutaneous leishmaniasis.

Influence of functional polymorphisms in TNF-α, IL-8, and IL-10 cytokine genes on mRNA expression levels and risk of gastric cancer

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Up-regulation of chemokine C-C ligand 2 (CCL2) and C-X-C chemokine 8 (CXCL8) expression by monocytes in chronic idiopathic urticaria

The disturbed cytokinechemokine network could play an important role in the onset of diseases with inflammatory processes such as chronic idiopathic urticaria (CIU). Our main objectives were to evaluate the relation between proinflammatory chemokine serum levels from CIU patients and their response to autologous skin test (ASST) and basophil histamine release (BHR). We also aimed to assess the chemokine secretion by peripheral blood mononuclear cells (PBMC) upon polyclonal stimulus and to evaluate chemokine CC ligand 2/C-X-C chemokine 8 (CCL2/CXCL8) and Toll-like receptor-4 (TLR-4) expression in monocytes. We observed significantly higher serum levels of the CXCL8, CXCL9, CXCL10 and CCL2 in CIU patients compared to the healthy group, regardless of the BHR or ASST response. The basal secretion of CCL2 by PBMC or induced by Staphylococcus aureus enterotoxin A (SEA) was higher in CIU patients than in the control group, as well as for CXCL8 and CCL5 secretions upon phytohaemagglutinin stimulation. Also, up-regulation of CCL2 and CXCL8 mRNA expression was found in monocytes of patients upon SEA stimulation. The findings showed a high responsiveness of monocytes through CCL2/CXCL8 expression, contributing to the creation of a proinflammatory environment in CIU.

Proinflammatory and Antiinflammatory Cytokine Levels in Complicated and Noncomplicated Parapneumonic Pleural Effusions

Objectives: This study aimed to evaluate a panel of proinflammatory and antiinflammatory cytokines in noncomplicated and complicated parapneumonic pleural effusions and to correlate their levels with pleural fluid biochemical parameters. Methods: Serum and pleural effusion were collected from 60 patients with noncomplicated (n = 26) or complicated (n = 34) parapneumonic effusions and assayed for cytologic, biochemical, and proinflammatory and antiinflammatory cytokines. Student t test was used to compare serum and pleural fluid values, Spearman correlation to analyze the relationship between pleural fluid cytokines and biochemical parameters, and accuracy of pleural fluid cytokine levels to determine the optimal cutoff value for identification of complicated effusions. Corrections for multiple comparisons were applied and a P value < .05 was accepted as significant. Results: Serum and pleural fluid cytokine levels of IL-8, vascular endothelial growth factor (VEGF), IL-10, and tumor necrosis factor (TNF) soluble receptor (sR) II were similar between groups. In contrast, complicated effusions had higher levels of pleural fluid IL-1 beta, IL-1 receptor antagonist (ra), and TNF sRI. Negative correlations were found between pleural fluid glucose with IL-1 beta and TNF sRI and positive correlations between lactic dehydrogenate (LDH) with IL-1 beta, IL-8, and VEGF. Pleural fluid levels of IL-1 beta, IL-1ra, and TNF sRI were more accurate than IL-8, VEGF, IL-10, and TNF sRII in discriminating complicated effusions. Conclusions: Both proinflammatory and antiinflammatory cytokine levels in pleural fluid are elevated in complicated in comparison with noncomplicated parapneumonic pleural effusions, and they correlate with both pleural fluid glucose and LDH levels. IL-1 beta, IL-1ra, and TNF sRI had higher sensitivity and specificity than IL-8, VEGF, IL-10, and TNF sRII in discriminating complicated effusions. CHEST 2012; 141( 1):183-189

Glucocorticoid Sensitivity and Proinflammatory Cytokines Pattern in Pemphigus

Glucocorticoids (GC) represent the main treatment for pemphigus; however, some patients show GC resistance. GC sensitivity was evaluated in 19 pemphigus patients and 41 controls by the number of binding sites [B-max (fmol/mg protein)] and the affinity of GC receptor [Kd (nM)] to dexamethasone (DEX) as well as by the pattern of cytokine by DEX-mediated inhibition of concanavalin-A (Con-A)-stimulated PBMC proliferation. The Kd (15.7 +/- 2.8 vs.8.1 +/- 1.3) and Bmax (6.5 +/- 0.9 vs. 3.9 +/- 0.3) were higher in pemphigus than controls (p = 0.002). Considering the values above the 95th percentile of normal group as a cut-off (K-d > 24.9 nM and B-max > 8.1 fmol/mg protein), elevated K-d and B-max were observed in 9.8% and 2.4% of controls and 15.8% and 36.8% of patients (p = 0.02). PBMC proliferation was stimulated by Con-A and inhibited by DEX (p < 0.001) in both pemphigus and control groups. IL-6 and TNF alpha (pg/mL) basal production were higher in patients than controls. There was an increment of these cytokines after Con-A stimulation, and they were inhibited by DEX (p = 0.002) in controls and remained elevated in pemphigus (p < 0.02). Patients and controls showed no difference in basal and stimulated production of IL-8 and IL-10. There is an alteration on GC sensitivity in pemphigus patients and a higher production of proinflammatory cytokines. Therefore, in pemphigus patients, proinflammatory cytokines might be involved in the mechanism of GC resistance and/or in its maintenance.

Insulin modulates cytokine release and selectin expression in the early phase of allergic airway inflammation in diabetic rats

Abstract Background Clinical and experimental data suggest that the inflammatory response is impaired in diabetics and can be modulated by insulin. The present study was undertaken to investigate the role of insulin on the early phase of allergic airway inflammation. Methods Diabetic male Wistar rats (alloxan, 42 mg/Kg, i.v., 10 days) and controls were sensitized by s.c. injection of ovalbumin (OA) in aluminium hydroxide 14 days before OA (1 mg/0.4 mL) or saline intratracheal challenge. The following analyses were performed 6 hours thereafter: a) quantification of interleukin (IL)-1β, tumor necrosis factor (TNF)-α and cytokine-induced neutrophil chemoattractant (CINC)-1 in the bronchoalveolar lavage fluid (BALF) by Enzyme-Linked Immunosorbent Assay, b) expression of E- and P- selectins on lung vessels by immunohistochemistry, and c) inflammatory cell infiltration into the airways and lung parenchyma. NPH insulin (4 IU, s.c.) was given i.v. 2 hours before antigen challenge. Results Diabetic rats exhibited significant reduction in the BALF concentrations of IL-1β (30%) and TNF-α (45%), and in the lung expression of P-selectin (30%) compared to non-diabetic animals. This was accompanied by reduced number of neutrophils into the airways and around bronchi and blood vessels. There were no differences in the CINC-1 levels in BALF, and E-selectin expression. Treatment of diabetic rats with NPH insulin, 2 hours before antigen challenge, restored the reduced levels of IL-1β, TNF-α and P-selectin, and neutrophil migration. Conclusion Data presented suggest that insulin modulates the production/release of TNF-α and IL-1β, the expression of P- and E-selectin, and the associated neutrophil migration into the lungs during the early phase of the allergic inflammatory reaction.

Viral load, organ distribution, histopathological lesions, and cytokine mRNA expression in goats infected with a molecular clone of the caprine arthritis encephalitis virus

Caprine arthritis encephalitis virus (CAEV) is a lentivirus of goats that causes persistent infection characterized by the appearance of inflammatory lesions in various organs. To define the sites of persistence, 5 goats were infected with a molecular clone of CAEV, and the viral load was monitored by real-time-PCR and RT-PCR in different sites 8 years after infection. The lymph nodes proved to be an important virus reservoir, with moderate virus replication relative to what is reported for lentiviruses of primates. Mammary gland and milk cells were preferred sites of viral replication. The viral load varied significantly between animals, which points to an important role of the genetic background. We found a clear association between occurrence of histopathological lesions and viral load in specific sites. The mRNA expression analysis of several cytokines did not reveal differences between animals that could explain the considerable individual variations in viral load observed.

Stress-induced modulation of NF-κB activation, inflammation-associated gene expression, and cytokine levels in blood of healthy men

Acute psychosocial stress stimulates transient increases in circulating pro-inflammatory plasma cytokines, but little is known about stress effects on anti-inflammatory cytokines or underlying mechanisms. We investigated the stress kinetics and interrelations of pro- and anti-inflammatory measures on the transcriptional and protein level. Forty-five healthy men were randomly assigned to either a stress or control group. While the stress group underwent an acute psychosocial stress task, the second group participated in a non-stress control condition. We repeatedly measured before and up to 120min after stress DNA binding activity of the pro-inflammatory transcription factor NF-κB (NF-κB-BA) in peripheral blood mononuclear cells, whole-blood mRNA levels of NF-κB, its inhibitor IκBα, and of the pro-inflammatory cytokines interleukin (IL)-1ß and IL-6, and the anti-inflammatory cytokine IL-10. We also repeatedly measured plasma levels of IL-1ß, IL-6, and IL-10. Compared to non-stress, acute stress induced significant and rapid increases in NF-κB-BA and delayed increases in plasma IL-6 and mRNA of IL-1ß, IL-6, and IκBα (p's<.045). In the stress group, significant increases over time were also observed for NF-κB mRNA and plasma IL-1ß and IL-10 (p's<.055). NF-κB-BA correlated significantly with mRNA of IL-1β (r=.52, p=.002), NF-κB (r=.48, p=.004), and IκBα (r=.42, p=.013), and marginally with IL-6 mRNA (r=.31, p=.11). Plasma cytokines did not relate to NF-κB-BA or mRNA levels of the respective cytokines. Our data suggest that stress induces increases in NF-κB-BA that relate to subsequent mRNA expression of pro-inflammatory, but not anti-inflammatory cytokines, and of regulatory-cytoplasmic-proteins. The stress-induced increases in plasma cytokines do not seem to derive from de novo synthesis in circulating blood cells.

NFkappaB and STAT binding elements participate in regulation of MUC1 expression in normal and transformed mammary epithelial cells

The MUC1 gene encodes a transmembrane mucin glycoprotein that is overexpressed in several cancers of epithelial origin, including those of breast, pancreas, lung, ovary, and colon. Functions of MUC1 include protection of mucosal epithelium, modulation of cellular adhesion, and signal transduction. Aberrantly increased expression of MUC1 in cancer cells promotes tumor progression through adaptation of these functions. Some regulatory elements participating in MUC1 transcription have been described, but the mechanisms responsible for overexpression are largely unknown. A region of MUC1 5′ flanking sequence containing two conserved potential cytokine response elements, an NFκB site at −589/−580 and a STAT binding element (SBE) at −503/−495, has been implicated in high level expression in breast and pancreatic cancer cell lines. Persistent stimulation by proinflammatory cytokines may contribute to increased MUC1 transcription by tumor cells. ^ T47D breast cancer cells and normal human mammary epithelial cells (HMEC) were used to determine the roles of the κB site and SBE in basal and stimulated expression of MUC1. Treatment of T47D cells and HMEC with interferon-γ (IFNγ) alone enhanced MUC1 expression at the level of transcription, and the effect of IFNγ was further stimulated by tumor necrosis factor-α (TNFα). MUC1 responsiveness to these cytokines was modest in T47D cells but clearly evident in HMEC. Transient transfection of T47D cells with mutant MUC1 promoter constructs revealed that the κB site at −589/−580 and the SBE at −503/−495 and were required for cooperative stimulation by TNFα and IFNγ. Electrophoretic mobility shift assays (EMSA) revealed that the synergy was mediated not by cooperative binding of transcription factors but by the independent actions of STAT1α and NFκB p65 on their respective binding sites. Independent mutations in the κB site and SBE abrogated cytokine responsiveness and reduced basal MUC1 promoter activity by 45–50%. However, only the κB site appeared to be constitutively activated in T47D cells, in part by NFκB p65. These findings implicate two cytokine response elements in the 5 ′ flanking region of MUC1, specifically a κB site and a STAT binding element, in overexpression of MUC1 in breast cancer cells. ^