GM-CSF Priming Drives Bone Marrow-Derived Macrophages to a Pro-Inflammatory Pattern and Downmodulates PGE(2) in Response to TLR2 Ligands

In response to pathogen recognition by Toll-like receptors (TLRs) on their cell surface, macrophages release lipid mediators and cytokines that are widely distributed throughout the body and play essential roles in host responses. Granulocyte macrophage colony-stimulating factor (GM-CSF) is important for the immune response during infections to improve the clearance of microorganisms. In this study, we examined the release of mediators in response to TLR2 ligands by bone marrow-derived macrophages (BMDMs) primed with GM-CSF. We demonstrated that when stimulated with TLR2 ligands, non-primed BMDMs preferentially produced PGE(2) in greater amounts than LTB4. However, GM-CSF priming shifted the release of lipid mediators by BMDMs, resulting in a significant decrease of PGE(2) production in response to the same stimuli. The decrease of PGE(2) production from primed BMDMs was accompanied by a decrease in PGE-synthase mRNA expression and an increase in TNF-alpha and nitric oxide (NO) production. Moreover, some GM-CSF effects were potentiated by the addition of IFN-gamma. Using a variety of TLR2 ligands, we established that PGE(2) release by GM-CSF-primed BMDMs was dependent on TLR2 co-receptors (TLR1, TLR6), CD14, MyD88 and the nuclear translocation of NF kappa B but was not dependent on peroxisome proliferator-activated receptor-gamma (PPAR-gamma) activation. Indeed, GM-CSF priming enhanced TLR2, TLR4 and MyD88 mRNA expression and phospho-I kappa B alpha formation. These findings demonstrate that GM-CSF drives BMDMs to present a profile relevant to the host during infections.

Influence of N-methyl-D-aspartate receptors on ouabain activation of nuclear factor-kappa B in the rat hippocampus

It has been shown that ouabain (OUA) can activate the Na,K-ATPase complex and mediate intracellular signaling in the central nervous system (CNS). Inflammatory stimulus increases glutamatergic transmission, especially at N-methyl-D-aspartate (NMDA) receptors, which are usually coupled to the activation of nitric oxide synthase (NOS). Nuclear factor-kappa B (NF-kappa B) activation modulates the expression of genes involved in development, plasticity, and inflammation. The present work investigated the effects of OUA on NF-kappa B binding activity in rat hippocampus and the influence of this OUA-Na,K-ATPase signaling cascade in NMDA-mediated NF-kappa B activation. The findings presented here are the first report indicating that intrahippocampal administration of OUA, in a concentration that did not alter Na,K-ATPase or NOS activity, induced an activation of NF-kappa B, leading to increases in brain-derived neurotrophic factor (Bdnf), inducible NOS (iNos), tumor necrosis factor-alpha (Tnf-alpha), and B-cell leukemia/lymphoma 2 (Bcl2) mRNA levels. This response was not linked to any significant signs of neurodegeneration as showed via Fluoro-Jade B and Nissl stain. Intrahippocampal administration of NMDA induced NF alpha B activation and increased NOS and alpha 2/3-Na,K-ATPase activities. NMDA treatment further increased OUA-induced NF-kappa B activation, which was partially blocked by MK-801, an antagonist of NMDA receptor. These results suggest that OUA-induced NF-kappa B activation is at least in part dependent on Na,K-ATPase modulatory action of NMDA receptor in hippocampus. The interaction of these signaling pathways could be associated with biological mechanisms that may underlie the basal homeostatic state linked to the inflammatory signaling cascade in the brain. (c) 2011 Wiley Periodicals, Inc.

Evaluation of sun protection factor of cosmetic formulations by a simple visual in vitro method mimicking the in vivo method

A new concept for in vitro visual evaluation of sun protection factor (SPF) of cosmetic formulations based on a supramolecular ultraviolet (UV) dosimeter was clearly demonstrated. The method closely parallels the method validated for in vivo evaluation and relies on the determination of the slightest perceptible bleaching of an iron-complex dye/nanocrystallinetitanium dioxide interface (UV dosimeter) in combination with an artificial skin substrate simulating the actual human skin in the presence and absence of a cosmetic formulation. The successful evaluation of SPF was ensured by the similarity of the erythema response of our dosimeter and human skin to UV light irradiation. A good linear correlation of in vitro and in vivo data up to SPF 40 confirmed the effectiveness of such a simple, cheap, and fast method. In short, here we unravel a convenient and accessible visual FPS evaluation method that can help improving the control on cosmetic products contributing to the reduction of skin cancer, one of the critical public health issues nowadays. (C) 2011 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:726732, 2012

Cannabinoid CB1 receptor restrains accentuated activity of hypothalamic corticotropin-releasing factor and brainstem tyrosine hydroxylase neurons in endotoxemia-induced hypophagia in rats

It is well known that endocannabinoids play an important role in the regulation of food intake and body weight. Endocannabinoids and cannabinoid receptors are found in the hypothalamus and brainstem, which are central areas involved in the control of food intake and energy expenditure. Activation of these areas is related to hypophagia observed during inflammatory stimulus. This study investigated the effects of cannabinoid (CB1) receptor blockade on lipopolysaccharide (LPS)-induced hypophagia. Male Wistar rats were pretreated with rimonabant (10 mg/kg, by gavage) or vehicle; 30 min later they received an injection of either LPS (100 mu g/kg, intraperitoneal) or saline. Food intake, body weight, corticosterone response, CRF and CART mRNA expression, Fos-CRF and Fos-alpha-MSH immunoreactivity in the hypothalamus and Fos-tyrosine hydroxylase (TH) immunoreactivity in the brainstem were evaluated. LPS administration decreased food intake and body weight gain and increased plasma corticosterone levels and CRF mRNA expression in the PVN. We also observed an increase in Fos-CRF and Fos-TH double-labeled neurons after LPS injection in vehicle-pretreated rats, with no changes in CART mRNA or Fos-alpha-MSH immunoreactive neurons in the ARC. In saline-treated animals, rimonabant pretreatment decreased food intake and body weight gain but did not modify hormone response or Fos expression in the hypothalamus and brainstem compared with vehicle-pretreated rats. Rimonabant pretreatment potentiated LPS-induced hypophagia, body weight loss and Fos-CRF and Fos-TH expressing neurons. Rimonabant did not modify corticosterone, CRF mRNA or Fos-alpha-MSH responses in rats treated with LPS. These data suggest that the endocannabinoid system, mediated by CB1 receptors, modulates hypothalamic and brainstem circuitry underlying the hypophagic effect during endotoxemia to prevent an exaggerated food intake decrease. This article is part of a Special Issue entitled 'Central Control of Food Intake'. (C) 2011 Elsevier Ltd. All rights reserved.

Hydrothermal pretreatment of sugarcane bagasse using response surface methodology improves digestibility and ethanol production by SSF

Sugarcane bagasse was characterized as a feedstock for the production of ethanol using hydrothermal pretreatment. Reaction temperature and time were varied between 160 and 200A degrees C and 5-20 min, respectively, using a response surface experimental design. The liquid fraction was analyzed for soluble carbohydrates and furan aldehydes. The solid fraction was analyzed for structural carbohydrates and Klason lignin. Pretreatment conditions were evaluated based on enzymatic extraction of glucose and xylose and conversion to ethanol using a simultaneous saccharification and fermentation scheme. SSF experiments were conducted with the washed pretreated biomass. The severity of the pretreatment should be sufficient to drive enzymatic digestion and ethanol yields, however, sugars losses and especially sugar conversion into furans needs to be minimized. As expected, furfural production increased with pretreatment severity and specifically xylose release. However, provided that the severity was kept below a general severity factor of 4.0, production of furfural was below an inhibitory concentration and carbohydrate contents were preserved in the pretreated whole hydrolysate. There were significant interactions between time and temperature for all the responses except cellulose digestion. The models were highly predictive for cellulose digestibility (R (2) = 0.8861) and for ethanol production (R (2) = 0.9581), but less so for xylose extraction. Both cellulose digestion and ethanol production increased with severity, however, high levels of furfural generated under more severe pretreatment conditions favor lower severity pretreatments. The optimal pretreatment condition that gave the highest conversion yield of ethanol, while minimizing furfural production, was judged to be 190A degrees C and 17.2 min. The whole hydrolysate was also converted to ethanol using SSF. To reduce the concentration of inhibitors, the liquid fraction was conditioned prior to fermentation by removing inhibitory chemicals using the fungus Coniochaeta ligniaria.

Febrile response induced by cecal ligation and puncture (CLP) in rats: involvement of prostaglandin E-2 and cytokines

The purpose of the present study was to better understand the events involved in the febrile response induced by cecal ligation and puncture (CLP), a complex infectious process. To this end, we conducted in vivo experiments in rats examining (1) fever development, (2) bacterial number in the infection focus and in blood, (3) peripheral and hypothalamic synthesis of cytokines, (4) hypothalamic and cerebrospinal fluid (CSF) synthesis of prostaglandin E-2 (PGE(2)), (5) the effect of anti-IL-6 antibody on fever, and (6) the effect of celecoxib on fever and hypothalamic synthesis of PGE(2) after CLP induction. We found that CLP promotes fever and animal death depending on the number of punctures. The peak of CLP-induced fever overlapped with the maximal increase in the number of bacteria in the infectious focus and blood, which occurred at 6 and 12 h. The peak of the febrile response also coincided with increased amounts of interleukin (IL)-1 beta, IL-6 and IL-10 in the peritoneal exudate and serum; IL-6 in the hypothalamus and PGE(2) in the CSF and predominantly in the hypothalamus. Moreover, intracerebroventricularly injected anti-IL-6 antibody reduced the febrile response while celecoxib reduced the fever and PGE(2) amount in the hypothalamus induced by CLP. Tumor necrosis factor (TNF)-alpha peaked at 3 h at all sites studied. Conversely, IL-10 concentration decreased in the hypothalamus. These findings show that the peak of CLP-induced fever is accompanied by an increase of bacteria in peritoneal fluid (local infection) and blood; local synthesis of pyrogenic (IL-1 beta, IL-6) and antipyretic (IL-10) cytokines and central production of IL-6 and PGE(2), suggesting that these last are the central mediators of this response.

Expression of PAFR as Part of a Prosurvival Response to Chemotherapy: A Novel Target for Combination Therapy in Melanoma

Melanoma cells express the platelet-activating factor receptor (PAFR) and, thus, respond to PAF, a bioactive lipid produced by both tumour cells and those in the tumour microenvironment such as macrophages. Here, we show that treatment of a human melanoma SKmel37 cell line with cisplatin led to increased expression of PAFR and its accumulation. In the presence of exogenous PAF, melanoma cells were significantly more resistant to cisplatin-induced cell death. Inhibition of PAFR-dependent signalling pathways by a PAFR antagonist (WEB2086) showed chemosensitisation of melanoma cells in vitro. Nude mice were inoculated with SKmel37 cells and treated with cisplatin and WEB2086. Animals treated with both agents showed significantly decreased tumour growth compared to the control group and groups treated with only one agent. PAFR accumulation and signalling are part of a prosurvival program of melanoma cells, therefore constituting a promising target for combination therapy for melanomas.

Plasma corticosterone elevation inhibits the activation of nuclear factor kappa B (NFKB) in the Syrian hamster pineal gland

We evaluated how the mild stress-induced increase in endogenous corticosterone affected the pineal gland in Syrian hamsters (Mesocricetus auratus). The animals were maintained under constant light for 1 day, instead of a cycle of 14:10-h, to increase the circulating corticosterone levels during the daytime. The nuclear translocation of nuclear factor kappa B (NFKB), which is the pivotal transcription factor for stress and injury, presented a daily rhythm in normal animals. NFKB nuclear content increased linearly from the onset of light [Zeitgeber Time 0 (ZT0)] until ZT11 and decreased after ZT12 when the plasma corticosterone peak was detected in normal animals. However, the 24-h profiles of the two curves were different, and they did not clearly support an exclusive relationship between corticosterone levels and NFKB content. Therefore, we tested the effect of increased endogenous corticosterone through inducing mild stress by maintaining daytime illumination for one night. This stressful condition, which increased daytime corticosterone levels, resulted in a daytime decrease in NFKB nuclear content, and this was inhibited by mifepristone. Overall, this study shows that NFKB has a daily rhythm in Syrian hamster pineal glands and, by increasing endogenous corticosterone with a stressful condition, NFKB activity is regulated. Therefore, this study suggests that the pineal gland in the Syrian hamster is a sensor of stressful conditions.

Apoptotic Cells Contribute to Melanoma Progression and This Effect is Partially Mediated by the Platelet-Activating Factor Receptor

There is evidence that the platelet-activating factor receptor (PAFR) is involved in the clearance of apoptotic cells by macrophages, and that this is associated with anti-inflammatory phenotype. Our group has previously shown that coinjection of a large number of apoptotic cells can promote tumor growth from a subtumorigenic dose of melanoma cells. Here, we studied the involvement of the PAFR in the tumor growth promoting effect of apoptotic cells. A sub-tumorigenic dose of melanoma cells (Tm1) was coinjected with apoptotic Tm1 cells, subcutaneously in the flank of C57Bl/6 mice, and the volume was monitored for 30 days. Animals received the PAFR antagonists, WEB2170 or PCA4248 (5 mg/kg body weight) or vehicle, by peritumoral daily injection for 5 days. Results showed that PAFR antagonists significantly inhibited the tumor growth induced by the coinjection of a subtumorigenic dose of melanoma cells together with apoptotic cells. This was accompanied by inhibition of early neutrophil and macrophage infiltration. Addition of (platelet-activating factor) to this system has no significant effect. PAFR antagonists did not affect the promoting effect of carrageenan. We suggest that the recognition of apoptotic cells by phagocytes leads to activation of PAFR pathways, resulting in a microenvironment response favorable to melanoma growth.

Low-Level Laser Therapy Influences Mouse Odontoblast-Like Cell Response in Vitro

Objective: The purpose of this study was to analyze the influence of two different irradiation times with 85mW/cm(2) 830nm laser on the behavior of mouse odontoblast-like cells. Background data: The use of low-level laser therapy (LLLT) to stimulate pulp tissue is a reality, but few reports relate odontoblastic responses to irradiation in in vitro models. Methods: Odontoblast-like cells (MDPC-23) were cultivated and divided into three groups: control/nonirradiated (group 1); or irradiated with 85mW/cm(2), 830nm laser for 10 sec (0.8 J/cm(2)) (group 2); or for 50 sec (4.2 J/cm(2)) (group 3) with a wavelength of 830 nm. After 3, 7, and 10 days, it was analyzed: growth curve and cell viability, total protein content, alkaline phosphatase (ALP) activity, calcified nodules detection and quantification, collagen immunolocalization, vascular endothelial growth factor (VEGF) expression, and real-time polymerase chain reaction (PCR) for DMP1 gene. Data were analyzed by Kruskall-Wallis test (alpha = 0.05). Results: Cell growth was smaller in group 2 (p < 0.01), whereas viability was similar in all groups and at all periods. Total protein content and ALP activity increased on the 10th day with 0.8 J/cm(2) (p < 0.01), as well as the detection and quantification of mineralization nodules (p < 0.05), collagen, and VEGF expression (p < 0.01). The expression of DMP1 increased in all groups (p < 0.05) compared with control at 3 days, except for 0.8 J/cm(2) at 3 days and control at 10 days. Conclusions: LLLT influenced the behavior of odontoblast-like cells; the shorter time/smallest energy density promoted the expression of odontoblastic phenotype in a more significant way.

Proteins of Leishmania (Viannia) shawi confer protection associated with Th1 immune response and memory generation

Background: Leishmania (Viannia) shawi parasite was first characterized in 1989. Recently the protective effects of soluble leishmanial antigen (SLA) from L. (V.) shawi promastigotes were demonstrated using BALB/c mice, the susceptibility model for this parasite. In order to identify protective fractions, SLA was fractionated by reverse phase HPLC and five antigenic fractions were obtained. Methods: F1 fraction was purified from L. (V.) shawi parasite extract by reverse phase HPLC. BALB/c mice were immunized once a week for two consecutive weeks by subcutaneous routes in the rump, using 25 mu g of F1. After 1 and 16 weeks of last immunization, groups were challenged in the footpad with L. (V.) shawi promastigotes. After 2 months, those same mice were sacrificed and parasite burden, cellular and humoral immune responses were evaluated. Results: The F1 fraction induced a high degree of protection associated with an increase in IFN-gamma, a decrease in IL-4, increased cell proliferation and activation of CD8(+)T lymphocytes. Long-term protection was acquired in F1-immunized mice, associated with increased CD4(+) central memory T lymphocytes and activation of both CD4+ and CD8(+) T cells. In addition, F1-immunized groups showed an increase in IgG2a levels. Conclusions: The inductor capability of antigens to generate memory lymphocytes that can proliferate and secrete beneficial cytokines upon infection could be an important factor in the development of vaccine candidates against American Tegumentary Leishmaniasis.

Interleukin-4 Modulates the Inflammatory Response in Ifosfamide-Induced Hemorrhagic Cystitis

We investigated whether interleukin-4 (IL-4) is present and capable of reducing inflammatory changes seen in ifosfamide-induced hemorrhagic cystitis. Male Swiss mice were treated with saline or ifosfamide alone or ifosfamide with the classical protocol with mesna and analyzed by changes in bladder wet weight (BWW), macroscopic and microscopic parameters, exudate, and hemoglobin quantification. In other groups, IL-4 was administered intraperitoneally 1 h before ifosfamide. In other experimental groups, C57BL/6 WT (wild type) and C57BL/6 WT IL-4 (-/-) knockout animals were treated with ifosfamide and analyzed for changes in BWW. Quantification of bladder IL-4 protein by ELISA in control, ifosfamide-, and mesna-treated groups was performed. Immunohistochemistry to tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) as well as protein identification by Western blot assay for inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was carried out on ifosfamide- and IL-4-treated animals. In other experimental groups, antiserum against IL-4 was given 30 min before ifosfamide. In IL-4-treated animals, the severity of hemorrhagic cystitis was significantly milder than in animals treated with ifosfamide only, an effect that was reverted with serum anti-IL-4. Moreover, knockout animals for IL-4 (-/-) exhibit a worse degree of inflammation when compared to C57BL/6 wild type. Exogenous IL-4 also attenuated TNF-alpha, IL-1 beta, iNOS, and COX-2 expressions in ifosfamide-treated bladders. IL-4, an anti-inflammatory cytokine, attenuates the inflammation seen in ifosfamide-induced hemorrhagic cystitis.

Partial Replacement of omega-6 Fatty Acids With Medium-Chain Triglycerides, but Not Olive Oil, Improves Colon Cytokine Response and Damage in Experimental Colitis

Background: Soybean oil is rich in omega-6 fatty acids, which are associated with higher incidence and more severe cases of inflammatory bowel diseases. The authors evaluated whether partial replacement of soybean oil by medium-chain triglycerides (MCTs) or olive oil influenced the incidence and severity of experimental ulcerative colitis by using different parenteral lipid emulsions (LEs). Methods: Wistar rats (n = 40) were randomized to receive parenteral infusion of the following LE: 100% soybean oil (SO), 50% MCT mixed with 50% soybean oil (MCT/SO), 80% olive oil mixed with 20% soybean oil (OO/SO), or saline (CC). After 72 hours of infusion, acetic acid experimental colitis was induced. After 24 hours, colon histology and cytokine expression were analyzed. Results: SO was not significantly associated with overall tissue damage. MCT/SO was not associated with necrosis (P < .005), whereas OO/SO had higher frequencies of ulcer and necrosis (P < .005). SO was associated with increased expression of interferon-gamma (P = .005) and OO/SO with increased interleukin (IL)-6 and decreased tumor necrosis factor-alpha expression (P < .05). MCT/SO appeared to decrease IL-1 (P < .05) and increase IL-4 (P < .001) expression. Conclusions: Parenteral SO with high concentration of omega-6 fatty acids was not associated with greater tissue damage in experimental colitis. SO partial replacement with MCT/SO decreased the frequency of histological necrosis and favorably modulated cytokine expression in the colon; however, replacement with OO/SO had unfavorable effects. (JPEN J Parenter Enteral Nutr. 2012; 36: 442-448)

Angiotensin-(1-7) decreases LPS-induced inflammatory response in macrophages

It has been previously shown that besides its classical role in blood pressure control the reninangiotensin system, mainly by action of angiotensin II on the AT1 receptor, exerts pro-inflammatory effects such as by inducing the production of cytokines. More recently, alternative pathways to this system were described, such as binding of angiotensin-(17) to receptor Mas, which was shown to counteract some of the effects evoked by activation of the angiotensin IIAT1 receptor axis. Here, by means of different molecular approaches we investigated the role of angiotensin-(17) in modulating inflammatory responses triggered in mouse peritoneal macrophages. Our results show that receptor Mas transcripts were up-regulated by eightfold in LPS-induced macrophages. Interestingly, macrophage stimulation with angiotensin-(17), following to LPS exposure, evoked an attenuation in expression of TNF-a and IL-6 pro-inflammatory cytokines; where this event was abolished when the receptor Mas selective antagonist A779 was also included. We then used heterologous expression of the receptor Mas in HEK293T cells to search for the molecular mechanisms underlying the angiotensin-(17)-mediated anti-inflammatory responses by a kinase array; what suggested the involvement of the Src kinase family. In LPS-induced macrophages, this finding was corroborated using the PP2 compound, a specific Src kinase inhibitor; and also by Western blotting when we observed that Ang-(17) attenuated the phosphorylation levels of Lyn, a member of the Src kinase family. Our findings bring evidence for an anti-inflammatory role for angiotensin-(17) at the cellular level, as well as show that its probable mechanism of action includes the modulation of Src kinases activities. J. Cell. Physiol. 227: 21172122, 2012. (C) 2011 Wiley Periodicals, Inc.

Differential Gene Expression Profiles May Differentiate Responder and Nonresponder Patients with Rheumatoid Arthritis for Methotrexate (MTX) Monotherapy and MTX plus Tumor Necrosis Factor Inhibitor Combined Therapy

Objective. We aimed to evaluate whether the differential gene expression profiles of patients with rheumatoid arthritis (RA) could distinguish responders from nonresponders to methotrexate (MTX) and, in the case of MTX nonresponders, responsiveness to MTX plus anti-tumor necrosis factor-alpha (anti-TNF) combined therapy. Methods. We evaluated 25 patients with RA taking MTX 15-20 mg/week as a monotherapy (8 responders and 17 nonresponders). All MTX nonresponders received intliximab and were reassessed after 20 weeks to evaluate their anti-TNF responsiveness using the European League Against Rheumatism response criteria. A differential gene expression analysis from peripheral blood mononuclear cells was performed in terms of hierarchical gene clustering, and an evaluation of differentially expressed genes was performed using the significance analysis of microarrays program. Results. Hierarchical gene expression clustering discriminated MTX responders from nonresponders, and MTX plus anti-TNF responders from nonresponders. The evaluation of only highly modulated genes (fold change > 1.3 or < 0.7) yielded 5 induced (4 antiapoptotic and CCL4) and 4 repressed (4 proapoptotic) genes in MTX nonresponders compared to responders. In MTX plus anti-TNF nonresponders, the CCL4, CD83, and BCL2A1 genes were induced in relation to responders. Conclusion. Study of the gene expression profiles of RA peripheral blood cells permitted differentiation of responders from nonresponders to MTX and anti-TNF. Several candidate genes in MTX non-responders (CCL4, HTRA2, PRKCD, BCL2A1, CAV1, TNIP1 CASP8AP2, MXD1, and BTG2) and 3 genes in MTX plus anti-TNF nonresponders (CCL4, CD83, and BCL2A1) were identified for further study. (First Release July 1 2012; J Rheumatol 2012;39:1524-32; doi:10.3899/jrheum.120092)