Association of-308G/A polymorphism in the tumor necrosis factor-alpha gene promoter with susceptibility to development of hantavirus cardiopulmonary syndrome in the Ribeiro Preto region, Brazil

Activation of the immune response in hantavirus cardiopulmonary syndrome (HCPS) leads to a high TNF production, probably contributing to the disease. The polymorphic TNF2 allele (TNF -308G/A) has been associated with increased cytokine production. We investigated the association of the TNF2 allele with the outcome of hantavirus infection in Brazilian patients. A total of 122 hantavirus-exposed individuals (26 presenting HCPS and 96 only hantavirus seroconversion) were studied. The TNF2 allele was more frequently found in HCPS patients than in individuals with positive serology for hantavirus but without a history of HCPS illness, suggesting that the TNF2 allele could represent a risk factor for developing HCPS.

Crucial Role of TNF Receptors 1 and 2 in the Control of Polymicrobial Sepsis

Sepsis is still a major cause of mortality in the intensive critical care unit and results from an overwhelming immune response to the infection. TNF signaling pathway plays a central role in the activation of innate immunity in response to pathogens. Using a model of polymicrobial sepsis by i.p. injection of cecal microflora, we demonstrate a critical role of TNFR1 and R2 activation in the deregulated immune responses and death associated with sepsis. A large and persistent production of TNF was found in wild-type (B6) mice. TNFR1/R2-deficient mice, compared with B6 mice, survive lethal polymicrobial infection with enhanced neutrophil recruitment and bacterial clearance in the peritoneal cavity. Absence of TNFR signaling leads to a decreased local and systemic inflammatory response with diminished organ injury. Furthermore, using TNFR1/R2-deficient mice, TNF was found to be responsible for a decrease in CXCR2 expression, explaining reduced neutrophil extravasation and migration to the infectious site, and in neutrophil apoptosis. In line with the clinical experience, administration of Enbrel, a TNF-neutralizing protein, induced however only a partial protection in B6 mice, with no improvement of clinical settings, suggesting that future TNF immunomodulatory strategies should target TNFR1 and R2. In conclusion, the present data suggest that the endogenous TNFR1/R2 signaling pathway in polymicrobial sepsis reduces neutrophil recruitment contributing to mortality and as opposed to pan-TNF blockade is an important therapeutic target for the treatment of polymicrobial sepsis. The Journal of Immunology, 2009, 182: 7855-7864.

Factors involved in the T helper type 1 and type 2 cell commitment and osteoclast regulation in inflammatory apical diseases

Periapical chronic lesion formation involves activation of the immune response and alveolar bone resorption around the tooth apex. However, the overall roles of T helper type 1 (Th1), Th2, and T-regulatory cell (Treg) responses and osteoclast regulatory factors in periapical cysts and granulomas have not been fully determined. This study aimed to investigate whether different forms of apical periodontitis, namely cysts and granulomas, show different balances of Th1, Th2 regulators, Treg markers, and factors involved in osteoclast chemotaxis and activation. Gene expression of these factors was assessed using quantitative real-time polymerase chain reaction, in samples obtained from healthy gingiva (n = 8), periapical granulomas (n = 20), and cysts (n = 10). Periapical cysts exhibited a greater expression of GATA-3, while a greater expression of T-bet, Foxp3, and interleukin-10 (IL-10) was seen in granulomas. The expression of interferon-gamma, IL-4, and transforming growth factor-beta was similar in both lesions. Regarding osteoclastic factors, while the expression of SDF-1 alpha/CXCL12 and CCR1 was higher in cysts, the expression of RANKL was significantly higher in granulomas. Both lesions exhibited similar expression of CXCR4, CK beta 8/CCL23, and osteoprotegerin, which were significantly higher than in control. Our results showed a predominance of osteoclast activity in granulomas that was correlated with the Th1 response. The concomitant expression of Treg cell markers suggests a possible suppression of the Th1 response in granulomas. On the other hand, in cysts the Th2 activity is augmented. The mechanisms of periradicular lesion development are still not fully understood but the imbalance of immune and osteoclastic cell activity in cysts and granulomas seems to be critically regulated by Treg cells.

Role of cytokines in mediating mechanical hypernociception in a model of delayed-type hypersensitivity in mice

In the present study, we used the electronic version of the von Frey test to investigate the role of cytokines (TNF-alpha and IL-1 beta) and chemokines (KC/CXCL-1) in the genesis of mechanical hypernociception during antigen-induced inflammation in mice. The nociceptive test consisted of evoking a hindpaw flexion reflex with a hand-held force transducer (electronic anesthesiometer) adapted with a 0.5 mm(2) polypropylene tip. The intraplantar administration of methylated bovine serum albumin (mBSA) in previously immunized (IM), but not in sham-immunized (SI) mice, induced mechanical hypernociception in a dose-dependant manner. Hypernociception induced by antigen was reduced in animals pretreated with IL-lra and reparixin (a non-competitive allosteric inhibitor of CXCR2), and in TNF receptor type 1 deficient (TNFR1-/-) mice. Consistently, antigen challenge induced a time-dependent release of TNF-alpha, IL-1 beta and KC/CXCL-1 in IM, but not in SI, mice. Consistently, antigen challenge induced a time-dependent release of TNF-alpha, IL-1 beta and KC/CXCL-1 in IM, but not in SI, mice. The increase in TNF-alpha levels preceded the increase in IL-1 beta and KC/CXCL1. Antigen-induced release of IL-1 beta and KC/CXCL1 was reduced in TNFR1-/- mice, and TNF-alpha induced hypernociception was inhibited by IL-lra and reparixin. Hypernociception induced by IL-1 beta in immunized mice was inhibited by indomethacin, whereas KC/CXCL1-induced hypernociception was inhibited by indomethacin and guanethidine, Antigen-induced hypernociception was reduced by indomethacin and guanethidine and abolished by the two drugs combined. Together, these results suggest that inflammation associated with an adaptive immune response induces hypernociception that is mediated by an initial release of TNF-alpha, which triggers that subsequent release of IL-1 beta and KC/CXCL1. The latter cytokines in turn stimulate the release of the direct-acting final mediator, prostanoids and sympathetic amines. (C) 2008 European Federation of Chapters of the International Association for the Study of Pain. Published by Elsevier Ltd. All rights reserved.

Local profile of cytokines and nitric oxide in patients with bacterial vaginosis and cervical intraepithelial neoplasia

Objective: To evaluate the local immune response in patients with bacterial vaginosis (BV) and cervical intraepithelial neoplasia (CIN), as assessed by cytokine and nitric oxide (NO) concentrations. Study design: Patients attending for routine gynaecological examination were prospectively enrolled in groups: BV (n = 25) diagnosed by clinical criteria, CIN graded I to III (n = 35, 6 CIN 1, 8 CIN 11 and 21 CIN 111) by histological analysis, and controls (n = 15) without clinical and cytological findings. Randomly selected patients within CIN group at grades 11 or III (n = 15) were re-evaluated at 60 days after surgical treatment. Endocervical (EC) and vaginal secretion samples were collected by cytobrush and the levels of cytokines (ELISA) and NO metabolite (Griess reaction) were assayed. Results: NO was assessed in all subjects, and cytokines in all controls, 15 BV and 30 CIN patients. Interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) and nitrite levels were higher in EC than in vaginal secretions in BV and CIN groups. In CIN group, IL-8, IL-10 and nitrite concentrations were greater in EC and/or vaginal secretions than in BV or controls. Surgical treatment reduced IL-8 levels in EC and vaginal secretions. Conclusion: A similar local immune profile was found in BV and CIN groups. The increased local production of IL-8, IL-10 and NO in CIN suggests a role for these mediators in the immune response against tumour or tumour development. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

Detrimental role of endogenous nitric oxide in host defence against Sporothrix schenckii

We earlier demonstrated that nitric oxide (NO) is a fungicidal molecule against Sporothrix schenckii in vitro. In the present study we used mice deficient in inducible nitric oxide synthase (iNOS(-/-)) and C57BL/6 wild-type (WT) mice treated with N omega-nitro-arginine (Nitro-Arg-treated mice), an NOS inhibitor, both defective in the production of reactive nitrogen intermediates, to investigate the role of endogenous NO during systemic sporotrichosis. When inoculated with yeast cells of S. schenckii, WT mice presented T-cell suppression and high tissue fungal dissemination, succumbing to infection. Furthermore, susceptibility of mice seems to be related to apoptosis and high interleukin-10 and tumour necrosis factor-alpha production by spleen cells. In addition, fungicidal activity and NO production by interferon-gamma (IFN-gamma) and lipopolysaccharide-activated macrophages from WT mice were abolished after fungal infection. Strikingly, iNOS(-/-) and Nitro-Arg-treated mice presented fungal resistance, controlling fungal load in tissues and restoring T-cell activity, as well as producing high amounts of IFN-gamma Interestingly, macrophages from these groups of mice presented fungicidal activity after in vitro stimulation with higher doses of IFN-gamma. Herein, these results suggest that although NO was an essential mediator to the in vitro killing of S. schenckii by macrophages, the activation of NO system in vivo contributes to the immunosuppression and cytokine balance during early phases of infection with S. schenckii.

The influence of glutathione modulators on the course of Leishmania major infection in susceptible and resistant mice

Glutathione (GSH) has an important dual role in parasite-host relationship in Leishmania major infection. Our previous studies showed that both antioxidant systems, glutathione and trypanothione/trypanothione reductase, participate in the protection of Leishmania against the toxic effect of nitrogen-derived reactive species. On the other hand, GSH also is very important to the modulation of the effective immune response, inducting NO production and leishmanicidal activity of macrophages. In the present study, we investigated the role of host GSH during the course of L. major infection, analysing the size of footpad lesions and parasite load from mice treated with two GSH modulators, N-acethyl-L-cysteine (NAC) and buthionine sulphoximine (BSO). Resistant mice treated with BSO, which depletes GSH develop exacerbated lesions, but only harbour higher parasite load in their lesions 2 weeks post-infection. Although the NAC treatment does not affect the footpad lesions development in susceptible BALB/c mice, it significantly reduced the tissue parasitism in the lesions throughout the course of infection. Interestingly, the treatment with BSO did not change the course of L. major infection on susceptible mice when compared with nontreated mice. These results suggest that GSH is an important antioxidant modulator during anti-Leishmania immune response in vivo.

Modulation of mucosal immunity in a murine model of food-induced intestinal inflammation

Background Hypersensitivity or uncontrolled responses against dietary antigens can lead to inflammatory disorders like food allergy and current models reflect a variety of causes but do not reveal the detailed modulation of gut immunity in response to food antigens after breakdown in mucosal tolerance. Objective To develop and characterize a murine model for food-induced intestinal inflammation and to demonstrate the modulation of gut immune response by dietary allergenic antigens. Methods C57BL/6 mice were sensitized with peanut proteins, challenged with peanut seeds and their sera and gut segments were collected for subsequent analyses. Results Sensitization and challenged with peanut seeds led to alterations in gut architecture with inflammatory response characterized by oedema in lamina propria and cell infiltrate composed mainly by eosinophils, mast cells, phagocytes, natural killer and plasma cells, together with low percentage of gamma delta(+) and CD4(+)CD25(+)Foxp3(+) cells in Peyer`s patches. These animals also presented high levels of specific IgE and IgG1 in sera and modulation of mucosal immunity was mediated by increased expression of GATA-3, IL-4, IL-13 and TNF-alpha in contrast to low IFN-gamma in the gut. Conclusion A murine model for food-induced intestinal inflammation was characterized in which modulation of gut immunity occurs by peanut antigens in consequence of T-helper type 2 (Th2) allergic response and failure of regulatory mechanisms necessary for mucosa homeostasis, resembling food allergy. This work shed some light on the understanding of the pathogenesis of gastrointestinal disorders and intolerance in the gut and supports the development of therapies for food-related enteropathies like food allergy, focusing on gut-specific immune response.

The role of neutrophils in severe sepsis

Neutrophils are key effectors of the innate immune response. Reduction of neutrophil migration to infection sites is associated with a poor outcome in sepsis. We have demonstrated a failure of neutrophil migration in lethal sepsis. Together with this failure, we observed more bacteria in both peritoneal exudates and blood, followed by a reduction in survival rate. Furthermore, neutrophils obtained from severe septic patients displayed a marked reduction in chemotactic response compared with neutrophils from healthy subjects. The mechanisms of neutrophil migration failure are not completely understood. However, it is known that they involve systemic Toll-like receptor activation by bacteria and/or their products and result in excessive levels of circulating cytokines/chemokines. These mediators acting together with LPS stimulate expression of iNOS that produces high amounts of NO, which in turn mediates the failure of neutrophil migration. NO reduced expression of CXCR2 on neutrophils and the levels of adhesion molecules on both endothelial cells and neutrophils. These events culminate in decreased endothelium-leukocyte interactions, diminished neutrophil chemotactic response, and neutrophil migration failure. Additionally, the NO effect, at least in part, is mediated by peroxynitrite. In this review, we summarize what is known regarding the mechanisms of neutrophil migration impairment in severe sepsis.

Hematopoietic SCT modulates gut inflammation in experimental inflammatory bowel disease

Hematopoietic SCT (HSCT) and high-dose chemotherapy are being explored as therapy for various human refractory immune-mediated conditions, including inflammatory bowel diseases (IBD). Nevertheless, the exact immunological mechanisms by which the BM cells (BMCs) or immunosuppression provide remission from these diseases is not yet clear. In this work, we investigated the role of these therapies in the modulation of gut mucosal inflammation in an experimental model of IBD. Colitis was induced in mice by 2,4,6-trinitrobenzenesulfonic acid and after CY was administered (200 mg/kg) alone (CY group) or followed by BMCs infusion (HSCT group). Animals were followed for 60 days. Both HSCT and CY reduced the histopathological features of colitis significantly. Infused cells were localized in the gut, and a marked decrease of CD4(+) leukocytes in the inflammatory infiltrate on days +7 and +14 and of CD8(+) cells on day +7 was found in both treatments allied to impressive reduction of proinflammatory Th1 and Th17 cytokines. Although chemotherapy alone was the best treatment regarding the induction of immunosuppressive molecules, only HSCT resulted in increased survival rates compared with the control group. Our findings indicate that high-dose CY followed by HSCT is effective in the modulation of mucosal immunity and in accelerating immune reconstitution after BMT, thus providing valuable tools to support the development and understanding of novel therapeutic strategies for IBD. Bone Marrow Transplantation (2010) 45, 1562-1571; doi:10.1038/bmt.2010.6; published online 15 March 2010

Experimental Chemotherapy in Paracoccidioidomycosis Using Ruthenium NO Donor

Paracoccidioidomycosis (PCM) is a granulomatous disease caused by a dimorphic fungus, Paracoccidioides brasiliensis (Pb). To determine the influence of nitric oxide (NO) on this disease, we tested cis-[Ru(bpy)2(NO)SO3](PF6), ruthenium nitrosyl, which releases NO when activated by biological reducing agents, in BALB/c mice infected intravenously with Pb 18 isolate. In a previous study by our group, the fungicidal activity of ruthenium nitrosyl was evaluated in a mouse model of acute PCM, by measuring the immune cellular response (DTH), histopathological characteristics of the granulomatous lesions (and numbers), cytokines, and NO production. We found that cis-[Ru(bpy)2(NO)SO3](PF6)-treated mice were more resistant to infection, since they exhibited higher survival when compared with the control group. Furthermore, we observed a decreased influx of inflammatory cells in the lung and liver tissue of treated mice, possibly because of a minor reduction in fungal cell numbers. Moreover, an increased production of IL-10 and a decrease in TNF-alpha levels were detected in lung tissues of infected mice treated with cis-[Ru(bpy)2(NO)SO3](PF6). Immunohistochemistry showed that there was no difference in the number of VEGF- expressing cells. The animals treated with cis-[Ru(bpy)2(NO)SO3](PF6) showed high NO levels at 40 days after infection. These results show that NO is effectively involved in the mechanism that regulates the immune response in lung of Pb-infected mice. These data suggest that NO is a resistance factor during paracoccidioidomycosis by controlling fungal proliferation, influencing cytokine production, and consequently moderating the development of a strong inflammatory response.

Leptin resistance and desensitization of hypophagia during prolonged inflammatory challenge

Leptin resistance and desensitization of hypophagia during prolonged inflammatory challenge. Am J Physiol Endocrinol Metab 300: E858-E869, 2011. First published February 22, 2011; doi: 10.1152/ajpendo.00558.2010.-Acute exposure to bacterial lipopolysaccharide (LPS) is a potent inducer of immune response as well as hypophagia. Nevertheless, desensitization of responses to LPS occurs during long-term exposure to endotoxin. We induced endotoxin tolerance, injecting repeated (6LPS) LPS doses compared with single (1LPS) treatment. 1LPS, but not 6LPS group, showed decreased food intake and body weight, which was associated with an increased plasma leptin and higher mRNA expression of OB-Rb, MC4R, and SOCS3 in the hypothalamus. Hypophagia induced by 1LPS was associated with lower levels of 2-arachidonoylglycerol (2-AG), increased number of p-STAT3 neurons, and decreased AMP-activated protein kinase (AMPK) activity. Desensitization of hypophagia in the 6LPS group was related to high 2-AG, with no changes in p-STAT3 or increased p-AMPK. Leptin decreased food intake, body weight, 2-AG levels, and AMPK activity and enhanced p-STAT3 in control rats. However, leptin had no effects on 2-AG, p-STAT3, or p-AMPK in the 1LPS and 6LPS groups. Rats treated with HFD to induce leptin resistance showed neither hypophagia nor changes in p-STAT3 after 1LPS, suggesting that leptin and LPS recruit a common signaling pathway in the hypothalamus to modulate food intake reduction. Desensitization of hypophagia in response to repeated exposure to endotoxin is related to an inability of leptin to inhibit AMPK phosphorylation and 2-AG production and activate STAT3. SOCS3 is unlikely to underlie this resistance to leptin signaling in the endotoxin tolerance. The present model of prolonged inflammatory challenge may contribute to further investigations on mechanisms of leptin resistance.

Effect of BCG stimulus on proinflammatory cytokine production in laryngeal cancer

Background Evaluate the production of TNF and IL-6 in the supernatant of peripheral blood mononuclear cell (PBMC) cultures of patients with supraglottic laryngeal cancer before and after surgical treatment. Materials and methods Adherent cell cultures were stimulated with LPS and BCG. Fourteen patients with advanced supraglottic laryngeal cancer were studied. Cytokine concentration was determined by ELISA in supernatants of mononuclear cell cultures. Results In non-stimulated cultures, lower TNF cytokine levels were detected during the late postoperative (LP) period compared to control (P = 0.02). LP TNF and IL-6 levels were high in cultures stimulated with LPS compared with the preoperative period (PREOP) (P = 0.007; P = 0.008, respectively). Stimulation with BCG led to increased levels of TNF and IL-6 during the LP period compared to control (P = 0.001; P = 0.04, respectively). Conclusions BCG is able to modulate the immune response of patients with advanced supraglottic laryngeal cancer in vitro, increasing the secretion of TNF and IL-6 by macrophages during the postoperative period.

Development of chronic cardiomyopathy in canine Chagas disease correlates with high IFN-gamma, TNF-alpha, and low IL-10 production during the acute infection phase

When infected with Trypanosoma cruzi, Beagle dogs develop symptoms similar to those of Chagas disease in human beings, and could be an important experimental model for a better understanding of the immunopathogenic mechanisms involved in chronic chagasic infection. This study evaluates IL-10, IFN-gamma and TNF-alpha production in the sera, culture supernatant, heart and cervical lymph nodes and their correlation with cardiomegaly, cardiac inflammation and fibrosis in Beagle dogs infected with T. cruzi. Pathological analysis showed severe splenomegaly, lymphadenopathy and myocarditis in all infected dogs during the acute phase of the disease, with cardiomegaly, inflammation and fibrosis observed in 83% of the animals infected by T. cruzi during the chronic phase. The data indicate that infected animals producing IL-10 in the heart during the chronic phase and showing high IL-10 production in the culture supernatant and serum during the acute phase had lower cardiac alterations (myocarditis, fibrosis and cardiomegaly) than those with high IFN-gamma and TNF-alpha levels. These animals produced low IL-10 levels in the culture supernatant and serum during the acute phase and did not produce IL-10 in the heart during the chronic phase of the disease. Our findings showed that Beagle dogs are a good model for studying the immunopathogenic mechanism of Chagas disease, since they reproduce the clinical and immunological findings described in chagasic patients. The data suggest that the development of the chronic cardiac form of the disease is related to a strong Th1 response during the acute phase of the disease, while the development of the indeterminate form results from a blend of Th1 and Th2 responses soon after infection, suggesting that the acute phase immune response is important for the genesis of chronic cardiac lesions. Crown Copyright (C) 2009 Published by Elsevier B.V. All rights reserved.

Evidence of the presence of T helper type 17 cells in chronic lesions of human periodontal disease

Periodontal disease is a chronic inflammation of the attachment structures of the teeth, triggered by potentially hazardous microorganisms and the consequent immune-inflammatory responses. In humans, the T helper type 17 (Th17) lineage, characterized by interleukin-17 (IL-17) production, develops under transforming growth factor-beta (TGF-beta), IL-1 beta, and IL-6 signaling, while its pool is maintained by IL-23. Although this subset of cells has been implicated in various autoimmune, inflammatory, and bone-destructive conditions, the exact role of T lymphocytes in chronic periodontitis is still controversial. Therefore, in this study we investigated the presence of Th17 cells in human periodontal disease. Gingival and alveolar bone samples from healthy patients and patients with chronic periodontitis were collected and used for the subsequent assays. The messenger RNA expression for the cytokines IL-17, TGF-beta, IL-1 beta, IL-6, and IL-23 in gingiva or IL-17 and receptor activator for nuclear factor-kappa B ligand in alveolar bone was evaluated by real-time polymerase chain reaction. The production of IL-17, TGF-beta, IL-1 beta, IL-6, and IL-23 proteins was evaluated by immunohistochemistry and the presence of Th17 cells in the inflamed gingiva was confirmed by immunofluorescence confocal microscopy for CD4 and IL-17 colocalization. Our data demonstrated elevated levels of IL-17, TGF-beta, IL-1 beta, IL-6, and IL-23 messenger RNA and protein in diseased tissues as well as the presence of Th17 cells in gingiva from patients with periodontitis. Moreover, IL-17 and the bone resorption factor RANKL were abundantly expressed in the alveolar bone of diseased patients, in contrast to low detection in controls. These results provided strong evidence for the presence of Th17 cells in the sites of chronic inflammation in human periodontal disease.