Polysaccharide fraction of Agaricus brasiliensis avoids tumor-induced IL-10 production and changes the microenvironment of subcutaneous Ehrlich adenocarcinoma

Subcutaneous Ehrlich tumor-bearing mice were treated with in situ inoculation of a P-glucan-rich extract of Agaricus brasiliensis (ATF), which reduced tumor growth. Histopathological analysis showed that the tumor masses of control mice (Ehr) presented giant tumor cells and many mitotic figures whereas the tumor tissue obtained from ATF-treated animals (Ehr-ATF) presented a lower frequency of both mitotic and giant cells, associated with a higher frequency of apoptotic cells than Ehr. Analysis of the lymphoproliferative activity of spleen cells showed that the treatment had a suppressive rather than a stimulatory effect. Spleen cells of the Ehr group produced higher in vitro levels of IL-10 than normal controls and this occurrence was partially avoided by treatment with ATF. Analysis of cytokine production by tumor-infiltrating cells (ELISpot) showed that ATF induced a higher number of IFN-gamma-producing cells at 7 and 14 days as well as reduction of IL-10-secreting cells at the latter time. Confocal microscopy analysis showed higher intensity of labeling of CD4+ and Mac-3+ cells in ATF-treated mice. Analysis of in situ expression of angiogenic growth factors showed a slight decrease of FGF-2 mRNA in Ehr-ATF animals (7th day) but not of VEGF-A or TGF-beta expression. This fraction could not directly lyse either lymphocytes or tumor cells and we speculate that antitumor effect of ATF could be due to induction of a selective migration of immunocompetent cells from the spleen to the tumor site and to the switch of cytokine production. (C) 2009 Elsevier Inc. All rights reserved.

The broad effects of the functional IL-10 promoter-592 polymorphism: modulation of IL-10, TIMP-3, and OPG expression and their association with periodontal disease outcome

Periodontal diseases are infectious diseases, in which periodontopathogens trigger chronic inflammatory and immune responses that lead to tissue destruction. It occurs through the generation of metalloproteinases and the activation of bone resorption mechanisms. Anti-inflammatory cytokines such as IL-10 seem to attenuate periodontal tissue destruction through the induction of tissue inhibitors of metalloproteinases (TIMPs) and the inhibitor of osteoclastogenesis osteoprotegerin (OPG). A high individual variation in levels of IL-10 mRNA is verified in periodontitis patients, which is possibly determined by genetic polymorphisms. In this study, the IL-10 promoter -592C/A single nucleotide polymorphism ( SNP), which is associated with a decrease in IL-10 production, was analyzed by RFLP in 116 chronic periodontitis (CP) patients and 173 control (C) subjects, and the IL-10, TIMPs, and OPG mRNA expression levels in diseased gingival tissues were determined by real-time-PCR. The IL-10-592 SNP CA (P=0.0012/OR=2.4/CI:1.4-4.1), AA (P=0.0458/OR=2.3/CI:1.1-4.9), and CA+AA (P=0.0006/OR=2.4/CI: 1.4-3.4) genotypes and the allele A (P=0.0036/OR=1.7/CI:1.2-2.4) were found to be significantly more prevalent in the CP group when compared with control subjects. Both CA and AA genotypes were associated with lower levels of IL-10, TIMP-3, and OPG mRNA expression in diseased periodontal tissues and were also associated with disease severity as mean pocket depth. Taken together, the results presented here demonstrate that IL10-592 SNP is functional in CP, being associated with lower levels of IL-10 mRNA expression, which is supposed to consequently decrease the expression of the downstream genes TIMP-3 and OPG, and influence periodontal disease outcome. J. Leukoc. Biol. 84: 1565-1573; 2008.

The Potential Role of Suppressors of Cytokine Signaling in the Attenuation of Inflammatory Reaction and Alveolar Bone Loss Associated with Apical Periodontitis

Inflammatory cytokines contribute to periapical tissue destruction. Their activity is potentially regulated by suppressors of cytokine signaling (SOCS), which down-regulate signal transduction as part of an inhibitory feedback loop. We investigated the expression of the cytokines tumor necrosis factor alpha (TNF-alpha); interleukin (IL)-10 and RANKL; and SOCS-1, -2, and -3 by real-time polymerase chain reaction in 57 periapical granulomas and 38 healthy periapical tissues. Periapical granulomas exhibited significantly higher SOCS-1, -2, and -3, TNF-alpha, IL-10, and RANKL messenger RNA levels when compared with healthy controls. Significant positive correlations were found between SOCS1 and IL-10 and between SOCS3 and IL-10. Significant inverse correlations were observed between SOCS1 and TNF-alpha, SOCS1 and RANKL, and SOCS3 and TNF-alpha. Increased SOCS-1, -2, and -3 messenger RNA levels in periapical granulomas may be related to the downregulation of inflammatory cytokines in these lesions; therefore, SOCS molecules may play a role in the dynamics of periapical granulomas development. (J Endod 2008;34:1480-1484)

Deficiency of IL-12p40 subunit determines severe paracoccidioidomycosis in mice

Paracoccidioidomycosis, the major systemic mycosis in Latin America, is caused by the thermally dimorphic fungus Paracoccidioides brasiliensis. To investigate the role of interleukin (IL)-12 in this disease, IL-12p40(-/-) deficient mice (IL-12p40(-/-)) and wild type mice (WT) were infected intravenously with viable yeast cells of P. brasiliensis 18 isolate. We found that, unlike WT mice, IL-12p40(-/-) mice did not control fungal proliferation and dissemination and succumbed to infection by day 21 after inoculation. Additionally, IL-12p40(-/-) mice presented a higher number of granulomas/mm(2) in lung tissue than WT mice, and showed unorganized granulomas containing high numbers of yeast cells. Moreover, IL-12p40(-/-) mice did not release detectable levels of IFN-gamma, but they produced high levels of IL-10, as well as IgG1 antibody. Additionally, splenocytes from both infected IL-12p40(-/-) and WT mice exhibited a suppressed Con-A-induced T cell proliferative response. Our findings suggest that the IL-12p40 subunit mediates resistance in paracoccidioidomycosis by inducting IFN-gamma production and a Th1 immune response

Genetic variation in the recognition of Porphyromonas gingivalis antigens in mice

T-cell cytokine profiles, anti Porphyromonas gingivalis antibodies and Western blot analysis of antibody responses were examined in BALB/c, CBA/CaH, C57BL6 and DBA/2J mice immunized intraperitoneally with different doses of P. gingivalis outer membrane antigens, Splenic CD4 and CD8 cells were examined for intracytoplasmic interleukin (IL)-4, interferon (IFN)-gamma and IL-LD by FAGS analysis and levels of anti-P. gingivalis antibodies in the serum samples determined by enzyme-linked immunosorbent assay. Western blot analysis was performed on the sera from mice immunized with 100 mug of P. gingivalis antigens. The four strains of mice demonstrated varying degrees of T-cell immunity although the T-cell cytokine profiles exhibited by each strain were not affected by different immunizing doses. While BALB/c and DBA/2J mice exhibited responses that peaked at immunizing doses of 100-200 mug of P. gingivalis antigens, CBA/CaH and C57BL6 demonstrated weak T-cell responsiveness compared with control mice. Like the T-cell responses, serum antibody levels were not dose dependent. DBA/23 exhibited the lowest levels of anti-P. gingivalis antibodies followed by BALB/c with CBA/CaH and C57BL6 mice demonstrating the highest levels. Western blot analysis showed that there were differences in reactivity between the strains to a group of 13 antigens ranging in molecular weight from 15 to 43 kDa. Antibody responses to a number of these bands in BALB/c mice were of low density, whereas CBA/CaH and C57BL6 mice demonstrated high-density bands and DBA/2J mice showed medium to high responses. In conclusion, different immunizing doses of P. gingivalis outer membrane antigens had little effect on the T-cell cytokine responses and serum anti-P. gingivalis antibody levels. Western blot analysis, however, indicated that the four strains of mice exhibited different reactivity to some lower-molecular-weight antigens. Future studies are required to determine the significance of these differences, which may affect the outcome of P. gingivalis infection.

Histological and molecular temporomandibular joint analyses after mandibular advancement surgery: study in minipigs

Objectives. The objective of this study was to elucidate the changes occurring in the temporomandibular joint (TMJ) after surgical mandibular advancement with different fixation techniques: bicortical screws (rigid fixation) and miniplates (semi-rigid fixation). Study design. Eighteen minipigs were equally and randomly divided into 3 groups: Group I (control), nonoperated animals; Group II, animals submitted to surgical advancement surgery and osteosynthesis by bicortical screws; and Group III, animals submitted to surgical advancement surgery and osteosynthesis by miniplates. Four months after the surgeries, the presence of interleukin (IL)-6 and IL-10 in synovial fluid samples was assessed in ELISA experiments. TMJs were histologically prepared. Results. Higher levels of IL-10 (P = .0436) were found for Group II. Descriptive histological analysis was compatible with the ELISA findings. Conclusions. Rigid fixation evokes more pronounced signs of bone remodeling in the TMJ, whereas malleable fixation promotes a more intense inflammatory activity. Therefore, rigid fixation seems to transmit a higher impact of postoperative masticatory forces to the TMJ.

Differential Production of Macrophage Inflammatory Protein-1 alpha, Stromal-Derived Factor-1, and IL-6 by Human Cultured Periodontal Ligament and Gingival Fibroblasts Challenged With Lipopolysaccharide From P. gingivalis

Background: Fibroblasts are considered important cells in periodontitis. When challenged by different agents, they respond through the release of cytokines that participate in the inflammatory process. The aim of this study is to evaluate and compare the expression and production of macrophage inflammatory protein (MIP)-1 alpha, stromal-derived factor (SDF)-1, and interleukin (IL)-6 by human cultured periodontal ligament and gingival fibroblasts challenged with lipopolysaccharide (LPS) from Porphyromonas gingivalis. Methods: Fibroblasts were cultured from biopsies of gingival tissue and periodontal ligament of the same donors and used on the fourth passage. After confluence in 24-well plates, the culture medium alone (control) or with 0.1 to 10 mu g/ml of LPS from P. gingivalis was added to the wells, and after 1, 6, and 24 hours, the supernatant and the cells were collected and analyzed by enzyme-linked immunosorbent assay and real-time polymerase chain reaction, respectively. Results: MIP-1 alpha, SDF-1, and IL-6 protein production was significantly greater in gingival fibroblasts compared to periodontal ligament fibroblasts. IL-6 was upregulated in a time-dependent manner, mainly in gingival fibroblasts (P<0.05), which secreted more MIP-1 alpha in the lowest concentration of LPS used (0.1 mu g/ml). In contrast, a basal production of SDF-1 that was inhibited with the increase of LPS concentration was detected, especially after 24 hours (P<0.05). Conclusion: The distinct ability of the gingival and periodontal ligament fibroblasts to secrete MIP-1 alpha, SDF-1, and IL-6 emphasizes that these cells may differently contribute to the balance of cytokines in the LPS-challenged periodontium. J Periodontol 2010;81:310-317.

Influence of Antiplatelet Drugs in the Pathogenesis of Experimental Periodontitis and Periodontal Repair in Rats

Background: Platelets contain an array of biologic mediators that can modulate inflammation and repair processes including proinflammatory mediators and growth factors. Previous studies have shown that periodontitis and periodontal repair are associated with platelet activation. We hypothesized that drug-induced platelet inactivation may interfere in the processes of inflammation and repair in experimental periodontitis in rats by suppressing the release of biologic mediators from platelets to the site of injury. Methods: To measure the effects on periodontitis, ligatures were placed around first molars, and aspirin (Asp, 30 mg/kg) or clopidogrel (Clo, 75 mg/kg) was given intragastrically once daily for 15 days. Interleukin-6 (IL-6), tumor necrosis factor-a (TNF-alpha), and thromboxane A(2) levels were measured by enzyme-linked immunosorbent assay. To evaluate the effects of antiplatelet drugs on periodontal repair, ligatures were removed after 15 days of periodontitis induction, and Asp or Clo were administered beginning the following day for 15 days. Periodontal repair was assessed by microcomputed tomography. Results: On periodontitis phase, Asp and Clo significantly reduced levels of TNF-alpha and II-6 (P < 0.05), but only Asp decreased thromboxane A(2) (P < 0.05). Asp and Clo decreased inflammatory infiltration; however, this reduction was more pronounced with Clo treatment (P < 0.05). Histometric analysis showed that Asp and Clo impaired alveolar bone resorption. During the repair phase and after removal of the ligatures, microcomputed tomography analysis demonstrated that treatment with Asp and Clo did not impair alveolar bone repair. Conclusion: Systemic administration of Asp and Clo attenuates the inflammation associated with periodontitis without affecting the repair process when stimulus is removed. J Periodontol 2011;82:767-777.

Increased circulating levels of matrix metalloproteinase (MMP)-8, MMP-9, and pro-inflammatory markers in patients with metabolic syndrome

Background: Metabolic syndrome (MetS) predisposes to cardiovascular complications. Increased concentrations of pro-inflammatory mediators and imbalanced concentrations of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) may reflect the pathophysiology of MetS. We compared the circulating levels of MMPs, TIMPs, and inflammatory mediators in MetS patients with those found in healthy controls. Methods: We studied 25 healthy subjects and 25 MetS patients. The plasma levels of pro-MMP-2 and pro-MMP-9 were determined by gelatin zymography. The plasma concentrations of MMP-8, MMP-3, TIMP-1, TIMP-2, monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), intercellular adhesion molecule (sICAM-1), and sP-selectin were measured by ELISA kits. Results: We found higher sP-selectin, sICAM-1, MCP-1, and IL-6 (all P<0.05) concentrations in MetS patients compared with healthy controls. No differences in pro-MMP-2, MMP-3, and TIMP-2 levels were found (all P>0.05). However, we found higher pro-MMP-9, MMP-8. and TIMP-1 levels in MetS patients compared with healthy controls (all P<0.05). Conclusions: Patients with MetS have increased circulating concentrations of pro-MMP-9, MMP-8, and TIMP-1 that are associated with increased concentrations of pro-inflammatory mediators and adhesion molecules. These findings suggest that MMPs may have a role in the increased cardiovascular risk of MetS patients. Pharmacological interventions targeting MMPs, especially MMP-9 and MMP-8 deserve further investigation in MetS patients. (C) 2009 Elsevier B.V. All rights reserved.

Comprehensive gene expression profiling in lungs of mice infected with Mycobacterium tuberculosis following DNAhsp65 immunotherapy

Background The continued increase in tuberculosis (TB) rates and the appearance of extremely resistant Mycobacterium tuberculosis strains (XDR-TB) worldwide are some of the great problems of public health. In this context, DNA immunotherapy has been proposed as an effective alternative that could circumvent the limitations of conventional drugs. Nonetheless, the molecular events underlying these therapeutic effects are poorly understood. Methods We characterized the transcriptional signature of lungs from mice infected with M. tuberculosis and treated with heat shock protein 65 as a genetic vaccine (DNAhsp65) combining microarray and real-time polymerase chain reaction analysis. The gene expression data were correlated with the histopathological analysis of lungs. Results The differential modulation of a high number of genes allowed us to distinguish DNAhsp65-treated from nontreated animals (saline and vector-injected mice). Functional analysis of this group of genes suggests that DNAhsp65 therapy could not only boost the T helper (Th)1 immune response, but also could inhibit Th2 cytokines and regulate the intensity of inflammation through fine tuning of gene expression of various genes, including those of interleukin-17, lymphotoxin A, tumour necrosis factor-cl, interleukin-6, transforming growth factor-beta, inducible nitric oxide synthase and Foxp3. In addition, a large number of genes and expressed sequence tags previously unrelated to DNA-therapy were identified. All these findings were well correlated with the histopathological lesions presented in the lungs. Conclusions The effects of DNA therapy are reflected in gene expression modulation; therefore, the genes identified as differentially expressed could be considered as transcriptional biomarkers of DNAhsp65 immunotherapy against TB. The data have important implications for achieving a better understanding of gene-based therapies. Copyright (C) 2008 John Wiley & Sons, Ltd.

Medullary neurones regulate hypothalamic corticotropin-releasing factor cell responses to an emotional stressor

Hypothalamic-pituitary-adrenal axis activation is a hallmark of the stress response. In the case of physical stressors, there is considerable evidence that medullary catecholamine neurones are critical to the activation of the paraventricular nucleus corticotropin-releasing factor cells that constitute the apex of the hypothalamic-pituitary-adrenal axis. In contrast, it has been thought that hypothalamic-pituitary-adrenal axis responses to emotional stressors do not involve brainstem neurones. To investigate this issue we have mapped patterns of restraint-induced neuronal c fos expression in intact animals and in animals prepared with either paraventricular nucleus-directed injections of a retrograde tracer, lesions of paraventricular nucleus catecholamine terminals, or lesions of the medulla corresponding to the A1 or A2 noradrenergic cell groups. Restraint-induced patterns of neuronal activation within the medulla of intact animals were very similar to those previously reported in response to physical stressors, including the fact that most stressor-responsive, paraventricular nucleus-projecting cells were certainly catecholaminergic and probably noradrenergic. Despite this, the destruction of paraventricular nucleus catecholamine terminals with 6-hydroxydopamine did not alter corticotropin-releasing factor cell responses to restraint. However, animals with ibotenic acid lesions encompassing either the A1 or A2 noradrenergic cell groups displayed significantly suppressed corticotropin-releasing factor cell responses to restraint. Notably, these medullary lesions also suppressed neuronal responses in the medial amygdala, an area that is now considered critical to hypothalamic-pituitary-adrenal axis responses to emotional stressors and that is also known to display a significant increase in noradrenaline turnover during restraint. We conclude that medullary neurones influence corticotropin-releasing factor cell responses to emotional stressors via a multisynaptic pathway that may involve a noradrenergic input to the medial amygdala. These results overturn the idea that hypothalamic-pituitary-adrenal axis response to emotional stressors can occur independently of the brainstem. (C) 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved.

Stressor categorization: acute physical and psychological stressors elicit distinctive recruitment patterns in the amygdala and in medullary noradrenergic cell groups

It has been hypothesized that the brain categorizes stressors and utilizes neural response pathways that vary in accordance with the assigned category. If this is true, stressors should elicit patterns of neuronal activation within the brain that are category-specific. Data from previous Immediate-early gene expression mapping studies have hinted that this is the case, but interstudy differences in methodology render conclusions tenuous. In the present study, immunolabelling for the expression of c-fos was used as a marker of neuronal activity elicited in the rat brain by haemorrhage, immune challenge, noise, restraint and forced swim. All stressors elicited c-fos expression in 25-30% of hypothalamic paraventricular nucleus corticotrophin-releasing-factor cells, suggesting that these stimuli were of comparable strength, at least with regard to their ability to activate the hypothalamic-pituitary-ad renal axis. In the amygdala, haemorrhage and immune challenge both elicited c-fos expression in a large number of neurons in the central nucleus of the amygdala, whereas noise, restraint and forced swim primarily elicited recruitment of cells within the medial nucleus of the amygdala. In the medulla, all stressors recruited similar numbers of noradrenergic (A1 and A2) and adrenergic (C1 and C2) cells. However, haemorrhage and immune challenge elicited c-fos expression In subpopulations of A1 and A2 noradrenergic cells that were significantly more rostral than those recruited by noise, restraint or forced swim. The present data support the suggestion that the brain recognizes at least two major categories of stressor, which we have referred to as 'physical' and 'psychological'. Moreover, the present data suggest that the neural activation footprint that is left in the brain by stressors can be used to determine the category to which they have been assigned by the brain.

Role of circumventricular organs in pro-inflammatory cytokine-induced activation of the hypothalamic-pituitary-adrenal axis

1. The past 15 years has seen the emergence of a new field of neuroscience research based primarily on how the immune system and the central nervous system can interact. A notable example of this interaction occurs when peripheral inflammation, infection or tissue injury activates the hypothalamic- pituitary-adrenal axis (HPA). 2. During such assaults, immune cells release the pro- inflammatory cytokines interleukin (IL)-1, IL-6 and tumour necrosis factor-alpha into the general circulation. 3. These cytokines are believed to act as mediators for HPA axis activation. However, physical limitations of cytokines impede their movement across the blood-brain barrier and, consequently, it has been unclear as to precisely how and where IL-1beta signals cross into the brain to trigger HPA axis activation. 4. Evidence from recent anatomical and functional studies suggests two neuronal networks may be involved in triggering HPA axis activity in response to circulating cytokines. These are catecholamine cells of the medulla oblongata and the circumventricular organs (CVO). 5. The present paper examines the role of CVO in generating HPA axis responses to pro-inflammatory cytokines and culminates with a proposed model based on cytokine signalling primarily involving the area postrema and catecholamine cells in the ventrolateral and dorsal medulla.

Association of clinical, radiological and synovial immunopathological responses to anti-rheumatic treatment in rheumatoid arthritis

Objectives. To compare immunohistochemical scoring with clinical scoring and radiology for the assessment of rheumatoid arthritis (RA) disease activity, synovial tissue (ST) biopsied arthroscopically was assessed from 18 patients before and after commencement of disease-modifying anti-rheumatic drug (DMARD) therapy. Methods. Lymphocytes, macrophages, differentiated dendritic cells (DC), vascularity, tumour necrosis factor (TNF)alpha and interleukin-1 beta levels were scored. Clinical status was scored using the American College of Rheumatology (ACR) core set and serial radiographs were scored using the Larsen and Sharp methods. Histopathological evidence of activity included infiltration by lymphocytes, DC, macrophages. tissue vascularity, and expression of lining and sublining TNF alpha. These indices co-varied across the set of ST biopsies and were combined as a synovial activity score for each biopsy. Results. The change in synovial activity with treatment correlated with the ACR clinical response and with decreased radiological progression by the Larsen score, The ACR response to DMARD therapy. the change in synovial activity score and the slowing of radiological progression were each greatest in patients with high initial synovial vascularity. Conclusions. The data demonstrate an association between clinical, radiological and synovial immunopathological responses to anti-rheumatic treatment in RA. High ST vascularity may predict favourable clinical and radiological responses to treatment.

The chemistry and biological effects of malondialdehyde-acetaldehyde adducts

This article represents the proceedings of a workshop at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Geoffrey M. Thiele and Simon Worrall. The presentations were (1) The chemistry of malondialdehyde-acetaldehyde (MAA) adducts, by Dean J. Tuma; (2) The formation and clearance of MAA adducts in ethanol-fed rats, by Simon Worrall; (3) Immune responses to MAA adducts may play a role in the development of alcoholic liver disease, by Lynell W. Klassen; (4) Unique biological responses to MAA-modifled proteins that may play a role in the development and/or progression of alcoholic liver disease, by Geoffrey M. Thiele; (5) MAA-adducted bovine serum albumin activates protein kinase C and stimulates interleukin-8 release in bovine bronchial epithelial cells, by Todd A. Wyatt; and (6) An enzyme immune assay for serum antiacetaldehyde adduct antibody using low-density lipoprotein-adduct and its significance in alcoholic liver injury and ALDH2 heterozygotes, by Naruhiko Nagata.