Effects of phytoestrogens derived from soy bean on expression of adhesion molecules on HUVEC

Background The risks of hormone replacement therapy have led to a search for new alternatives such as phytoestrogens, plant compounds with estrogen-like biological activity. Isoflavones are the phytoestrogens most extensively studied and can be found in soybean, red clover and other plants. Due to this estrogen-like activity, phytoestrogens can have some effect on atherosclerosis. Human umbilical vein endothelial cells (HUVEC) have been extensively used to study the biology and pathobiology of human endothelial cells and most of the knowledge acquired is due to experiments with cultures of these cells. Objective To evaluate the effects of the phytoestrogen extracts from Glycine max soy bean, genistein, formononetin, biochanin A and daidzein, as well as a mixture of these extracts (Mix), on expression of adhesion molecules, VCAM-1, ICAM-1 and E-selectin, by endothelial cell HUVEC, stimulated with lipopolysaccharide. Methods HUVEC were cultured in medium EBM2, pretreated with isoflavones for 24 and 48 h and then stimulated with lipopolysaccharide; in addition, isoflavones were added, after stimulation by lipopolysaccharide, to HUVEC. We evaluated the production of VCAM-1, ICAM-1 and E-selectin on cell surface, by cell-based enzyme immunoassay, and of sVCAM-1, sICAM-1 and sE-selectin in culture supernatant, by ELISA. Results Genistein, formononetin, biochanin A and daidzein, as well as the Mix were able to reduce VCAM-1, ICAM-1 and E-selectin on cell surface and in culture supernatant. Conclusion Isoflavones extracted from Glycine max soy bean, in vitro, presented antiatherogenic effects, reducing the expression of adhesion molecules and acting as preventive agents as well as therapeutic agents.

BCL2A1a Over-Expression in Murine Hematopoietic Stem and Progenitor Cells Decreases Apoptosis and Results in Hematopoietic Transformation

We previously reported the development of a lethal myeloid sarcoma in a non-human primate model utilizing retroviral vectors to genetically modify hematopoietic stem and progenitor cells. This leukemia was characterized by insertion of the vector provirus into the BCL2A1 gene, with resultant BCL2A1 over-expression. There is little information on the role of this anti-apoptotic member of the BCL2 family in hematopoiesis or leukemia induction. Therefore we studied the impact of Bcl2a1a lentiviral over-expression on murine hematopoietic stem and progenitor cells. We demonstrated the anti-apoptotic function of this protein in hematopoietic cells, but did not detect any impact of Bcl2a1a on in vitro cell growth or cell cycle kinetics. In vivo, we showed a higher propensity of HSCs over-expressing Bcl2a1a to engraft and contribute to hematopoiesis. Mice over-expressing Bcl2a1a in the hematologic compartment eventually developed an aggressive malignant disease characterized as a leukemia/lymphoma of B-cell origin. Secondary transplants carried out to investigate the primitive origin of the disease revealed the leukemia was transplantable. Thus, Bcl2a1 should be considered as a protooncogene with a potential role in both lymphoid and myeloid leukemogenesis, and a concerning site for insertional activation by integrating retroviral vectors utilized in hematopoietic stem cell gene therapy.

Adipose Tissue-Derived Stem Cell Treatment Prevents Renal Disease Progression

Adipose tissue-derived stem cells (ASCs) are an attractive source of stem cells with regenerative properties that are similar to those of bone marrow stem cells. Here, we analyze the role of ASCs in reducing the progression of kidney fibrosis. Progressive renal fibrosis was achieved by unilateral clamping of the renal pedicle in mice for 1 h; after that, the kidney was reperfused immediately. Four hours after the surgery, 2 x 10(5) ASCs were intraperitoneally administered, and mice were followed for 24 h posttreatment and then at some other time interval for the next 6 weeks. Also, animals were treated with 2 x 10(5) ASCs at 6 weeks after reperfusion and sacrificed 4 weeks later to study their effect when interstitial fibrosis is already present. At 24 h after reperfusion, ASC-treated animals showed reduced renal dysfunction and enhanced regenerative tubular processes. Renal mRNA expression of IL-6 and TNF was decreased in ASC-treated animals, whereas IL-4. IL-10, and HO-1 expression increased despite a lack of ASCs in the kidneys as determined by SRY analysis. As expected, untreated kidneys shrank at 6 weeks, whereas the kidneys of ASC-treated animals remained normal in size, showed less collagen deposition, and decreased staining for FSP-1, type I collagen, and Hypoxyprobe. The renal protection seen in ASC-treated animals was followed by reduced serum levels of TNF-alpha, KC, RANTES, and IL-1 alpha. Surprisingly, treatment with ASCs at 6 weeks, when animals already showed installed fibrosis, demonstrated amelioration of functional parameters, with less tissue fibrosis observed and reduced mRNA expression of type I collagen and vimentin. ASC therapy can improve functional parameters and reduce progression of renal fibrosis at early and later times after injury, mostly due to early modulation of the inflammatory response and to less hypoxia, thereby reducing the epithelial-mesenchymal transition.

Augmented nitric oxide production and up-regulation of endothelial nitric oxide synthase during cecal ligation and perforation

Nitric oxide (NO) has been pointed out as being the main mediator involved in the hypotension and tissue injury taking place during sepsis. This study aimed to investigate the cellular mechanisms implicated in the acetylcholine (ACh)-induced relaxation detected in aortic rings isolated from rats submitted to cecal ligation and perforation (CLP group), 6 h post-CLP. The mean arterial pressure was recorded, and the concentration-effect curves for ACh were constructed for endothelium-intact aortic rings in the absence (control) or after incubation with one of the following NO synthase inhibitors: L-NAME (non-selective), L-NNA (more selective for eNOS), 7-nitroindazole (more selective for nNOS), or 1400W (selective for iNOS). The NO concentration was determined by using confocal microscopy. The protein expression of the NOS isoforms was quantified by Western blot analysis. The prostacyclin concentration was indirectly analyzed on the basis of 6-keto-prostaglandin F-1 alpha (6-keto-PGF(1 alpha)) levels measured by enzyme immunoassay. There were no differences between Sham- and CLP-operated rats in terms of the relaxation induced by acetylcholine. However, the NOS inhibitors reduced this relaxation in both groups, but this effect remained more pronounced in the CLP group as compared to the Sham group. The acetylcholine-induced NO production was higher in the rat aortic endothelial cells of the CLP group than in those of the Sham group. eNOS protein expression was larger in the CLP group, but the iNOS protein was not verified in any of the groups. The basal 6-keto-PGF(1 alpha) levels were higher in the CLP group, but the acetylcholine-stimulated levels did not increase in CLP as much as they did in the Sham group. Taken together, our results show that the augmented NO production in sepsis syndrome elicited by cecal ligation and perforation is due to eNOS up-regulation and not to iNOS. (C) 2012 Elsevier Inc. All rights reserved.

Eugenol attenuates pulmonary damage induced by diesel exhaust particles

Zin WA, Silva AG, Magalhaes CB, Carvalho GM, Riva DR, Lima CC, Leal-Cardoso JH, Takiya CM, Valen a SS, Saldiva PH, Faffe DS. Eugenol attenuates pulmonary damage induced by diesel exhaust particles. J Appl Physiol 112: 911-917, 2012. First published December 22, 2011; doi: 10.1152/japplphysiol.00764.2011.-Environmentally relevant doses of inhaled diesel particles elicit pulmonary inflammation and impair lung mechanics. Eugenol, a methoxyphenol component of clove oil, presents in vitro and in vivo anti-inflammatory and antioxidant properties. Our aim was to examine a possible protective role of eugenol against lung injuries induced by diesel particles. Male BALB/c mice were divided into four groups. Mice received saline (10 mu l in; CTRL group) or 15 mu g of diesel particles DEP (15 mu g in; DIE and DEUG groups). After 1 h, mice received saline (10 mu l; CTRL and DIE groups) or eugenol (164 mg/kg; EUG and DEUG group) by gavage. Twenty-four hours after gavage, pulmonary resistive (Delta P1), viscoelastic (Delta P2) and total (Delta Ptot) pressures, static elastance (Est), and viscoelastic component of elastance (Delta E) were measured. We also determined the fraction areas of normal and collapsed alveoli, amounts of polymorpho- (PMN) and mononuclear cells in lung parenchyma, apoptosis, and oxidative stress. Est, Delta P2, Delta Ptot, and Delta E were significantly higher in the DIE than in the other groups. DIE also showed significantly more PMN, airspace collapse, and apoptosis than the other groups. However, no beneficial effect on lipid peroxidation was observed in DEUG group. In conclusion, eugenol avoided changes in lung mechanics, pulmonary inflammation, and alveolar collapse elicited by diesel particles. It attenuated the activation signal of caspase-3 by DEP, but apoptosis evaluated by TUNEL was avoided. Finally, it could not avoid oxidative stress as indicated by malondialdehyde.

Exogenous Administration of 15d-PGJ(2)-Loaded Nanocapsules Inhibits Bone Resorption in a Mouse Periodontitis Model

The 15-deoxy-(Delta 12,14)-PG J(2) (15d-PGJ(2)) has demonstrated excellent anti-inflammatory results in different experimental models. It can be used with a polymeric nanostructure system for modified drug release, which can change the therapeutic properties of the active principle, leading to increased stability and slower/prolonged release. The aim of the current study was to test a nano-technological formulation as a carrier for 15d-PGJ(2), and to investigate the immunomodulatory effects of this formulation in a mouse periodontitis model. Poly (D, L-lactide-coglycolide) nanocapsules (NC) were used to encapsulate 15d-PGJ(2). BALB/c mice were infected on days 0, 2, and 4 with Aggregatibacter actinomycetemcomitans and divided into groups (n = 5) that were treated daily during 15 d with 1, 3, or 10 mu g/kg 15d-PGJ(2)-NC. The animals were sacrificed, the submandibular lymph nodes were removed for FACS analysis, and the jaws were analyzed for bone resorption by morphometry. Immunoinflammatory markers in the gingival tissue were analyzed by reverse transcriptase-quantitative PCR, Western blotting, or ELISA. Infected animals treated with the 15d-PGJ(2)-NC presented lower bone resorption than infected animals without treatment (p < 0.05). Furthermore, infected animals treated with 10 mu g/kg 15d-PGJ(2)-NC had a reduction of CD4(+)CD25(+)FOXP3(+) cells and CD4/CD8 ratio in the submandibular lymph node (p < 0.05). Moreover, CD55 was upregulated, whereas RANKL was downregulated in the gingival tissue of the 10 mu g/kg treated group (p < 0.05). Several proinflammatory cytokines were decreased in the group treated with 10 mu g/kg 15d-PGJ(2)-NC, and high amounts of 15d-PGJ(2) were observed in the gingiva. In conclusion, the 15d-PGJ(2)-NC formulation presented immunomodulatory effects, decreasing bone resorption and inflammatory responses in a periodontitis mouse model. The Journal of Immunology, 2012, 189: 1043-1052.

Joint NOD2/RIPK2 Signaling Regulates IL-17 Axis and Contributes to the Development of Experimental Arthritis

Intracellular pattern recognition receptors such as the nucleotide-binding oligomerization domain (NOD)-like receptors family members are key for innate immune recognition of microbial infection and may play important roles in the development of inflammatory diseases, including rheumatic diseases. In this study, we evaluated the role of NOD1 and NOD2 on development of experimental arthritis. Ag-induced arthritis was generated in wild-type, NOD1(-/-)!, NOD2(-/-), or receptor-interacting serine-threonine kinase 2(-/-) (RIPK2(-/-)) immunized mice challenged intra-articularly with methylated BSA. Nociception was determined by electronic Von Frey test. Neutrophil recruitment and histopathological analysis of proteoglycan lost was evaluated in inflamed joints. Joint levels of inflammatory cytokine/chemokine were measured by ELISA. Cytokine (IL-6 and IL-23) and NOD2 expressions were determined in mice synovial tissue by RT-PCR. The NOD2(-/-) and RIPK2(-/-), but not NOD1(-/-), mice are protected from Ag-induced arthritis, which was characterized by a reduction in neutrophil recruitment, nociception, and cartilage degradation. NOD2/RIPK2 signaling impairment was associated with a reduction in proinflammatory cytokines and chemokines (TNF, IL-1 beta, and CXCL1/KC). IL-17 and IL-17 triggering cytokines (IL-6 and IL-23) were also reduced in the joint, but there is no difference in the percentage of CD4(+) IL-17(+) cells in the lymph node between arthritic wild-type and NOD2(-/-) mice. Altogether, these findings point to a pivotal role of the NOD2/RIPK2 signaling in the onset of experimental arthritis by triggering an IL-17-dependent joint immune response. Therefore, we could propose that NOD2 signaling is a target for the development of new therapies for the control of rheumatoid arthritis. The Journal of Immunology, 2012, 188: 5116-5122.

Expression profile of apoptosis-related genes in childhood adrenocortical tumors: low level of expression of BCL2 and TNF genes suggests a poor prognosis

Background: Impaired apoptosis has been implicated in the development of childhood adrenocortical tumors (ACT), although the expression of apoptosis-related gene expression in such tumors has not been reported. Methods: The mRNA expression levels of the genes CASP3, CASP8, CASP9, FAS, TNF, NFKB, and BCL2 were analyzed by quantitative real-time PCR in consecutive tumor samples obtained at diagnosis from 60 children with a diagnosis of ACT and in 11 non-neoplastic adrenal samples. BCL2 and TNF protein expression was analyzed by immunohistochemistry. Results: A significant association was observed between tumor size >= 100 g and lower expression levels of the BCL2 (P=0.03) and TNF (P=0.05) genes; between stage IV and lower expression levels of CASP3 (P=0.008), CASP9 (P=0.02), BCL2 (P=0.002), TNF (P=0.05), and NFKB (P=0.03); Weiss score >= 3 and lower expression of TNF (P=0.01); unfavorable event and higher expression values of CASP9 (P=0.01) and lower values of TNF (P=0.02); and death and lower expression of BCL2 (P=0.04). Underexpression of TNF was associated with lower event-free survival in uni- and multivariate analyses (P<0.01). Similar results were observed when patients with Weiss score <3 were excluded. Conclusion: This study supports the participation of apoptosis-related genes in the biology and prognosis of childhood ACT and suggests the complex role of these genes in the pathogenesis of this tumor.

Human Leucocytes Response to Viable, Extended Freeze-Drying or Heat-Killed Mycobacterium bovis bacillus Calmette-Guerin

We investigated the effects of viable, extended freeze-drying (EFD) or heat-killed (HK) Mycobacterium bovis bacillus CalmetteGuerin (BCG) in respiratory burst activity, gene expression of CYBB and NCF1 encoding components of the human phagocyte nicotinamide adenine dinucleotide (NADPH) oxidase, TLR2 expression, and in IL-10 and TNF-a cytokine production by human peripheral blood mononuclear cells (PBMCs). Viable BCG significantly inhibited TLR2 and CYBB gene expression, as well as superoxide release by human PBMC. All BCG stimuli augmented IL-10 release, but only HK BCG or viable BCG increased TNF-a release by PBMCs. Our studies show that viable BCG can impair the NADPH oxidase system activation and the TLR2 route in human PBMCs. As well, different BCG preparations can distinctly influence cytokine production by human PBMCs.

11-Oxoaerothionin isolated from the marine sponge Aplysina fistularis shows anti-inflammatory activity in LPS-stimulated macrophages

Marine sponges of the order Verongida are a rich source of biologically active bromotyrosine-derived secondary metabolites. However, none of these compounds are known to display anti-inflammatory activity. In the present investigation, we report the anti-inflammatory effects of 11-oxoaerothionin isolated from the Verongida sponge Aplysina fistularis. When RAW264.7 cells and primary macrophages were preincubated with 11-oxoaerothionin and stimulated with LPS (lipopolysaccharide), a concentration-dependent inhibition of iNOS (inducible nitric oxide synthase) protein and NO2- (Nitrite) production were observed. The same effect was observed when proinflammatory cytokines and PGE(2) (Prostaglandin E2) production was evaluated. In summary, we demonstrated that in the presence of LPS, 11-oxoaerothionin suppresses NO2 and iNOS expression as well as inflammatory cytokines and PGE(2).

Diagnostic methods in sepsis: the need of speed

Objective: Sepsis is a common condition encountered in hospital environments. There is no effective treatment for sepsis, and it remains an important cause of death at intensive care units. This study aimed to discuss some methods that are available in clinics, and tests that have been recently developed for the diagnosis of sepsis. Methods: A systematic review was performed through the analysis of the following descriptors: sepsis, diagnostic methods, biological markers, and cytokines. Results: The deleterious effects of sepsis are caused by an imbalance between the invasiveness of the pathogen and the ability of the host to mount an effective immune response. Consequently, the host's immune surveillance fails to eliminate the pathogen, allowing it to spread. Moreover, there is a pro-inflammatory mediator release, inappropriate activation of the coagulation and complement cascades, leading to dysfunction of multiple organs and systems. The difficulty achieve total recovery of the patient is explainable. There is an increased incidence of sepsis worldwide due to factors such as aging population, larger number of surgeries, and number of microorganisms resistant to existing antibiotics. Conclusion: The search for new diagnostic markers associated with increased risk of sepsis development and molecules that can be correlated to certain steps of sepsis is becoming necessary. This would allow for earlier diagnosis, facilitate patient prognosis characterization, and prediction of possible evolution of each case. All other markers are regrettably constrained to research units.

Ectoplacental Cone Induces Resistance to Apoptosis in High Doses of Interferon (IFN)-gamma-Treated Decidual Cells

Problem In this study, we explored the relationship between decidual cells (DC) and interferon (IFN)-gamma, in the presence or absence of ectoplacental cone (EC) using a coculture system. Method of study Decidual cells and EC were isolated from pregnant mice on gestation day 7.5. DCs were cultured for 48 hr and then treated with fresh EC. After characterization, they were treated with IFN-gamma, and cell death was evaluated. Results Interferon-gamma drastically increased decidual apoptosis, which was partially reverted by the addition of EC to the IFN-gamma-treated decidual culture. Moreover, the addition of EC to non-treated DC cultures was also capable of attenuating death rates. Conclusion Resistance to apoptosis may be induced in DC by the EC. This suggests that EC may participate in the inhibition of IFN-gamma-dependent apoptosis and, therefore, play important role for DC survival in a cytokineenriched placental environment.

Synthesis, biological evaluation and molecular docking studies of 3-(triazolyl)coumarin derivatives: Effect on inducible nitric oxide synthase

A series of 3-(triazolyl)-coumarins were synthesized and tested as anti-inflammatory agents. It was possible to infer that these compounds do not alter the interaction of LPS with TLR-4 or TLR-2, as the intracellular pathways involved in the TNF-alpha secretion and COX-2 activity were not affected. Nevertheless, the compounds inhibited iNOS-derived NO production, without affecting the eNOS activity. The outcome of the docking studies showed that it pi center dot center dot center dot pi interactions with the heme group are important for the iNOS inhibition, thus making compound 3c a promising lead. Moreover, the efficacy of this compound was visualized by the reduced number of neutrophils in the LPS-inflamed subcutaneous tissue. Together, biological and docking data show that triazolyl-substituted coumarins, that can act on iNOS, are a good scaffold to be explored. (C) 2012 Elsevier Masson SAS. All rights reserved.

Advanced glycated albumin impairs HDL anti-inflammatory activity and primes macrophages for inflammatory response that reduces reverse cholesterol transport

Objective: We investigated the effect of advanced glycated albumin (AGE-albumin) on macrophage sensitivity to inflammation elicited by S100B calgranulin and lipopolysaccharide (LPS) and the mechanism by which HDL modulates this response. We also measured the influence of the culture medium, isolated from macrophages treated with AGE-albumin, on reverse cholesterol transport (RCT). Methods and results: Macrophages were incubated with control (C) or AGE-albumin in the presence or absence of HDL, followed by incubations with S100B or LPS. Also, culture medium obtained from cells treated with C- or AGE-albumin, following S100B or LPS stimulation was utilized to treat naive macrophages in order to evaluate cholesterol efflux and the expression of HDL receptors. In comparison with C-albumin, AGE-albumin, promoted a greater secretion of cytokines after stimulation with S100B or LPS. A greater amount of cytokines was also produced by macrophages treated with AGE-albumin even in the presence of HDL Cytokine-enriched medium, drawn from incubations with AGE-albumin and S100B or LPS impaired the cholesterol efflux mediated by apoA-I (23% and 37%, respectively), HDL2 (43% and 47%, respectively) and HDL3 (20% and 8.5%, respectively) and reduced ABCA-1 protein level (16% and 26%, respectively). Conclusions: AGE-albumin primes macrophages for an inflammatory response impairing the RCT. Moreover, AGE-albumin abrogates the anti-inflammatory role of HDL, which may aggravate the development of atherosclerosis in DM. (C) 2012 Elsevier BM. All rights reserved.

Modulation of host cell signaling pathways as a therapeutic approach in periodontal disease

Recently, new treatment approaches have been developed to target the host component of periodontal disease. This review aims at providing updated information on host-modulating therapies, focusing on treatment strategies for inhibiting signal transduction pathways involved in inflammation. Pharmacological inhibitors of MAPK, NFκB and JAK/STAT pathways are being developed to manage rheumatoid arthritis, periodontal disease and other inflammatory diseases. Through these agents, inflammatory mediators can be inhibited at cell signaling level, interfering on transcription factors activation and inflammatory gene expression. Although these drugs offer great potential to modulate host response, their main limitations are lack of specificity and developments of side effects. After overcoming these limitations, adjunctive host modulating drugs will provide new therapeutic strategies for periodontal treatment.