CCR2 Deficiency Results in Increased Osteolysis in Experimental Periapical Lesions in Mice

Introduction: Periapical lesions are chronic inflammatory disorders of periradicular tissues caused by etiologic agents of endodontic origin. The inflammatory chemokines are thought to be involved in the latter observed osteolysis. With a murine model of experimental periapical lesion, the objective of this study was to evaluate the role of the chemokine receptor CCR2 in the lesion progression, osteoclast differentiation and activation, and expression of inflammatory osteolysis-related mediators. Methods: For lesion induction, right mandibular first molars were opened surgically with a (1)/(4) carbine bur, and 4 bacterial strains were inoculated in the exposed dental pulp; left mandibular first molars were used as controls. Animals were killed at 3, 7, 14, and 21 days after surgeries to evaluate the kinetics of lesion development. Results: CCR2 KO mice showed wider lesions than WT mice. CCR2 KO mice also expressed higher levels of the osteoclastogenic and osteolytic factors, receptor activator of nuclear factor kappa B ligand (RANKL) and cathepsin K, of the proinflammatory cytokine tumor necrosis factor alpha, and of the neutrophil migration related chemokine, KC. Conclusions: These results suggest that CCR2 is important in host protection to periapical osteolysis. (J Endod 2010;36:244-250)

Slower rescue of ER homeostasis by the unfolded protein response pathway associated with common variable immunodeficiency

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinemia and recurrent infections. Herein we addressed the role of unfolded protein response (UPR) in the pathogenesis of the disease. Augmented unspliced X-box binding protein 1 (XBP-1) mRNA concurrent with co-localization of IgM and BiP/GRP78 were found in one CVID patient. At confocal microscopy analysis this patient`s cells were enlarged and failed to present the typical surface distribution of IgM, which accumulated within an abnormally expanded endoplasmic reticulum. Sequencing did not reveal any mutation on XBP-1, neither on IRE-1 alpha that could potentially prevent the splicing to occur. Analysis of spliced XBP-1, IRE-1 alpha and BiP messages after LPS or Brefeldin A treatment showed that, unlike healthy controls that respond to these endoplasmic reticulum (ER) stressors by presenting waves of transcription of these three genes, this patient`s cells presented lower rates of transcription, not reaching the same level of response of healthy subjects even after 48 h of ER stress. Treatment with DMSO rescued IgM and IgG secretion as well as the expression of spliced XBP-1. Our findings associate diminished splicing of XBP-1 mRNA with accumulation of IgM within the ER and lower rates of chaperone transcription, therefore providing a mechanism to explain the observed hypogammaglobulinemia. (C) 2008 Elsevier Ltd. All rights reserved.

Endurance training activates AMP-activated protein kinase, increases expression of uncoupling protein 2 and reduces insulin secretion from rat pancreatic islets

Endurance exercise is known to enhance peripheral insulin sensitivity and reduce insulin secretion. However, it is unknown whether the latter effect is due to the reduction in plasma substrate availability or alterations in beta-cell secretory machinery. Here, we tested the hypothesis that endurance exercise reduces insulin secretion by altering the intracellular energy-sensitive AMP-activated kinase (AMPK) signaling pathway. Male Wistar rats were submitted to endurance protocol training one, three, or five times per week, over 8 weeks. After that, pancreatic islets were isolated, and glucose-induced insulin secretion (GIIS), glucose transporter 2 (GLUT2) protein content, total and phosphorylated calmodulin kinase kinase (CaMKII), and AMPK levels as well as peroxisome proliferator-activated receptor-gamma coactivator-1-alpha (PGC-1 alpha) and uncoupling protein 2 (UCP2) content were measured. After 8 weeks, chronic endurance exercise reduced GIIS in a dose-response manner proportionally to weekly exercise frequency. Contrariwise, increases in GLUT2 protein content, CaMKII and AMPK phosphorylation levels were observed. These alterations were accompanied by an increase in UCP2 content, probably mediated by an enhancement in PGC-1 alpha protein expression. In conclusion, chronic endurance exercise induces adaptations in beta-cells leading to a reduction in GIIS, probably by activating the AMPK signaling pathway. Journal of Endocrinology (2011) 208, 257-264

Bradykinin inducible receptor is essential to lipopolysaccharide-induced acute lung injury in mice

Lipopolysaccharides from gram-negative bacteria are amongst the most common causative agents of acute lung injury, which is characterized by an inflammatory response, with cellular infiltration and the release of mediators/cytokines. There is evidence that bradykinin plays a role in lung inflammation in asthma but in other types of lung inflammation its role is less clear. In the present study we evaluated the role of the bradykinin B(1) receptor in acute lung injury caused by lipopolysaccharide inhalation and the mechanisms behind bradykinin actions participating in the inflammatory response. We found that in C57BI/6 mice, the bradykinin B(1) receptor expression was up-regulated 24 h after lipopolysaccharide inhalation. At this time, the number of cells and protein concentration were significantly increased in the bronchoalveolar lavage fluid and the mice developed airway hyperreactivity to methacholine. In addition, there was an increased expression of tumor necrosis factor-alpha, interleukin-1 beta and interferon-gamma and chemokines (monocytes chemotactic protein-1 and KC) in the bronchoalveolar lavage fluid and in the lung tissue. We then treated the mice with a bradykinin B, receptor antagonist, R-954 (Ac-Orn-[Oic(2), alpha-MePhe(5), D-beta Nal(7), Ile(8)]desArg(9)-bradykinin), 30 min after lipopolysaccharide administration. We observed that this treatment prevented the airway hyperreactivity as well as the increased cellular infiltration and protein content in the bronchoalveolar lavage fluid. Moreover, R-954 inhibited the expression of cytokines/chemokines. These results implicate bradykinin, acting through B(1) receptor, in the development of acute lung injury caused by lipopolysaccharide inhalation. (C) 2010 Elsevier B.V. All rights reserved.

Metabotropic glutamate receptors transduce signals for neurite outgrowth after binding of the prion protein to laminin gamma 1 chain

The prion protein (PrP(C)) is highly expressed in the nervous system, and its abnormal conformer is associated with prion diseases. PrP(C) is anchored to cell membranes by glycosylphosphatidylinositol, and transmembrane proteins are likely required for PrP(C)-mediated intracellular signaling. Binding of laminin (Ln) to PrP(C) modulates neuronal plasticity and memory. We addressed signaling pathways triggered by PrP(C)-Ln interaction in order to identify transmembrane proteins involved in the transduction of PrP(C)-Ln signals. The Ln gamma 1-chain peptide, which contains the Ln binding site for PrP(C), induced neuritogenesis through activation of phospholipase C (PLC), Ca(2+) mobilization from intracellular stores, and protein kinase C and extracellular signal-regulated kinase (ERK1/2) activation in primary cultures of neurons from wild-type, but not PrP(C)-null mice. Phage display, coimmunoprecipitation, and colocalization experiments showed that group I metabotropic glutamate receptors (mGluR1/5) associate with PrP(C). Expression of either mGluR1 or mGluR5 in HEK293 cells reconstituted the signaling pathways mediated by PrP(C)-Ln gamma 1 peptide interaction. Specific inhibitors of these receptors impaired PrP(C)-Ln gamma 1 peptide-induced signaling and neuritogenesis. These data show that group I mGluRs are involved in the transduction of cellular signals triggered by PrP(C)-Ln, and they support the notion that PrP(C) participates in the assembly of multiprotein complexes with physiological functions on neurons.-Beraldo, F. H., Arantes, C. P., Santos, T. G., Machado, C. F., Roffe, M., Hajj, G. N., Lee, K. S., Magalhaes, A. C., Caetano, F. A., Mancini, G. L., Lopes, M. H., Americo, T. A., Magdesian, M. H., Ferguson, S. S. G., Linden, R., Prado, M. A. M., Martins, V. R. Metabotropic glutamate receptors trans-duce signals for neurite outgrowth after binding of the prion protein to laminin gamma 1 chain. FASEB J. 25, 265-279 (2011). www.fasebj.org

Identification of non-zein proteins in BR473 maize protein bodies by LC-nanoESI-MS/MS

The nutritional value of maize seed is limited due to its high content of storage proteins (zeins), which are deficient in essential amino acids such as lysine and tryptophan. In a previous paper, we showed that protein bodies obtained from BR473 maize variety, developed by Embrapa (Brazilian Agricultural Research Corporation), were mainly constituted by Z27 and a smaller quantity of Z50 gamma-zeins. Besides zein proteins, other not identified protein band in the SDS/PAGE was also observed, which could indicate the presence of non-zein proteins additionally to gamma-zeins. In the present paper, we have demonstrated the presence of non-zein proteins in BR473 maize protein bodies by LC-nanoESI-MS/MS and database searching. This fact could be related to the excellent energetic value and higher protein quality of BR473 maize grains, since high lysine concentration in some maize varieties has been related to the presence of cytoskeleton proteins that are non-zeins. We have identified the following proteins: Brittle-1 protein (chloroplast precursor), Legumin-1, glyceroldehyde-3-phosphate dehydrogenase, and elongation factor 1-alpha.

Piroxicam encapsulated in liposomes: Characterization and in vivo evaluation of topical anti-inflammatory effect

Liposomes of soya phosphatidylcholine, cholesterol, and stearylamine (molar ratio 6/3/1) and 0.1% alpha-tocopherol were prepared by the extrusion of multilamellar vesicles through 0.2-mu m polycarbonate membrane. Liposomes were characterized by electron transmission microscopy, and the mean structure diameter was 278 nm. The encapsulation efficiency obtained was 12.73%. The topical anti-inflammatory effect was evaluated in vivo by the cotton pellet granuloma method. We analyzed free piroxicam at 4 mg/kg, piroxicam encapsulated in liposomes added to 1.5% hydroxyethylcellulose (HEC) gel at 1.6 mg/kg, and piroxicam encapsulated in liposomes added to HEC gel at 4 mg/kg; the inhibition of inflammation obtained was 21.1%, 32.8%, and 47.4%, respectively. These results showed that the encapsulation of piroxicam produced an increase of topical anti-inflammatory effect, suggesting that the inhibition of inflammation can be obtained with lower drug concentrations.

Expressão Imuno-histoquímica das proteínas GLUT-1 e HIF-1α em lesões vasculares de mucosa oral

The correct histological diagnosis of vascular lesions in the oral mucosa is critical, especially in defining the treatment and prognosis, as some vascular lesions show spontaneous involution and others do not show such behavior. This study analyzed the expression immunohistochemistry of human glucose transporter protein (GLUT-1), in oral benign vascular tumors and to reclassify such lesions according to with his immunoexpression. In addition, we evaluated the immunohistochemical expression of hypoxia-inducible factor 1 alpha (HIF-1α), the main transcription factor involved in cellular adaptation to hypoxia. We analyzed 60 cases of benign oral vascular tumors: 30 cases with histological diagnosis of HEM and 30 cases of oral pyogenic granuloma (PG). The results of this research showed that of the 30 lesions initially classified as HEM, only 7 showed immuno-positivity for GLUT-1, remaining with the initial diagnosis. The remaining 23 were reclassified as vascular malformation (VM) (13 cases) and PG (10 cases). All cases in the sample with an initial diagnosis of PG were negative for GLUT-1, demonstrating the accuracy of histological diagnosis of these lesions. Concerning to the immunoexpression of HIF-1α, the Mann-Whitney test revealed a statistically significant difference between the cases of GP and MV (p = 0.002), where the median of GP (m=78) was higher than the MV (m=53). Based on these results, this study showed that a histological diagnosis alone is not always sufficient for the correct diagnosis of oral HEM and that HIF-1α participates in the pathogenesis of vascular lesions

Citocinas e proteínas de fase aguda do soro como marcadores de regressão da resposta inflamatória ao tratamento da tuberculose pulmonar

OBJETIVO: Analisar o padrão de citocinas pró- e antiinflamatórias e da resposta de fase aguda (RFA) como marcadores de resposta ao tratamento da tuberculose pulmonar. MÉTODOS: Determinação dos níveis de interferon-gama (IFN-γ), tumor necrosis factor-alpha (TNF-α, fator de necrose tumoral-alfa), interleucina-10 (IL-10) e transforming growth factor-beta (TGF-β, fator transformador de crescimento-beta), pelo método ELISA, em sobrenadante de cultura de células mononucleares do sangue periférico e monócitos, assim como dos níveis de proteínas totais, albumina, globulinas, alfa-1-glicoproteína ácida (AGA), proteína C reativa (PCR) e velocidade de hemossedimentação (VHS) em 28 doentes com tuberculose pulmonar, em três tempos: antes (T0), aos três meses (T3) e aos seis meses (T6) de tratamento, em relação aos controles saudáveis, em um único tempo. RESULTADOS: Os pacientes apresentaram valores maiores de citocinas e RFA que os controles em T0, com diminuição em T3 e diminuição (TNF-α, IL-10, TGF-β, AGA e VHS) ou normalização (IFN-γ e PCR) em T6. CONCLUSÕES: PCR, AGA e VHS são possíveis marcadores para auxiliar no diagnóstico de tuberculose pulmonar e na indicação de tratamento de indivíduos com baciloscopia negativa; PCR (T0 > T3 > T6 = referência) pode também ser marcador de resposta ao tratamento. Antes do tratamento, o perfil Th0 (IFN-γ, IL-10, TNF-α e TGF-β), indutor de e protetor contra inflamação, prevaleceu nos pacientes; em T6, prevaleceu o perfil Th2 (IL-10, TNF-α e TGF-β), protetor contra efeito nocivo pró-inflamatório do TNF-α ainda presente. O comportamento do IFN-γ (T0 > T3 > T6 = controle) sugere sua utilização como marcador de resposta ao tratamento.

An immunocytochemical and in situ hybridization analysis of annexin 1 expression in rat mast cells: Modulation by inflammation and dexamethasone

The presence and localization of the anti-inflammatory protein annexin 1 (also known as lipocortin 1) in perivenular rat mast cells was investigated here. Using the rat mesenteric microvascular bed and a combination of morphologic techniques ranging from immunofluorescence to electron microscopy analyses, we detected the presence of annexin 1 in discrete intracellular sites, both in the nucleus and in the cytoplasm. In resting mast cells, most of the protein pool (approximately 80% of the cytosolic portion) was localized to cytoplasmic granules. In agreement with other cell types, treatment of rats with dexamethasone (0.2 mg/kg, ip) increased annexin 1 expression in mast cells, inducing a remarkable appearance of dusters of protein immunoreactivity. This effect was most likely the result of de novo protein synthesis as determined by an increase in mRNA seen by in situ hybridization. Triggering an ongoing experimental inflammatory response (0.3 mg of carrageenin, ip) increased annexin 1 mRNA and protein levels. In conclusion, we report for the first time the localization of annexin 1 in connective tissue mast cells, and its susceptibility not only to glucocorticoid hormone treatment, but also to an experimental acute inflammatory response.

REGULATION OF BOVINE ENDOMETRIAL SECRETION OF PROSTAGLANDINS AND SYNTHESIS OF 2',5'-OLIGOADENYLATE SYNTHETASE BY INTERFERON-ALPHA MOLECULES

Experiments were performed to (1) verify the inhibitory effect of bovine trophoblast protein-1 (bTP-1) on uterine prostaglandin synthesis, (2) evaluate whether other interferon-alpha (IFN-alpha) molecules also inhibit prostaglandin secretion, and (3) test whether the enzyme 2',5'-oligoadenylate synthetase (2-5A synthetase) can be induced in endometrium by interferon-alpha. In experiment 1, all interferon molecules (bTP-1, oTP-1, bIFN-alpha and hIFN-alpha) equally inhibited secretion of PGF and PGE2 from endometrial explant cultures obtained at day 17 of the estrous cycle. In experiment 2, endometrial explants obtained from day 17 of the cycle were cultured with and without bovine serum albumin (BSA; 50-mu-g/ml) and bIFN-alpha (0, 0.84, 4.2, and 42 nM). Addition of BSA to the culture medium greatly enhanced the accumulation of PGF into the medium. The bIFN-alpha inhibited accumulation of PGF and PGE2 in both the presence or absence of BSA by 12 h. All three concentrations of bIFN-alpha were equally effective in inhibiting prostaglandin accumulation. Additionally, all concentrations of bIFN-alpha increased the amounts of 2-5A synthetase in endometrium. In conclusion, these results confirm the inhibitory effect of bTP-1 on PGF release from endometrium and demonstrate that bTP-1 can also inhibit PGE2 secretion. Furthermore, other interferon-alpha molecules, including bIFN-alpha, hIFN-alpha, and oTP-1, also reduced PGF and PGE2 secretion in culture. It is likely, therefore, that conceptus and other interferon-alpha molecules exert similar effects on endometrium in vitro and that the antiluteolytic effects of bIFN-alpha in vivo are mediated in part by changes in endometrial prostaglandin synthesis.

The effect of bovine interferon-alpha I1 on pregnancy rate in heifers.

Bovine interferon-alpha I1 (bIFN-alpha) may be useful for enhancing fertility in sheep and cattle because it has extensive sequence homology with ovine and bovine trophoblast protein-1 and, like those proteins, extends corpus luteum lifespan. To test the effectiveness of bIFN-alpha to enhance fertility, several experiments were performed in which inseminated heifers were given i.m. injections of bIFN-alpha approximately at the time of embryo-mediated signals that result in maintenance of the corpus luteum. In Exp. 1, heifers given 20 mg of bIFN-alpha daily from d 14 to 17 tended (P less than .07) to have lower pregnancy rates at d 110 to 112 of gestation (36/75; 48% vs 43/72; 60%). Similar results were obtained in Exp. 2 when heifers received a single injection of 40 mg of bIFN-alpha or placebo at d 13 after estrus; pregnancy rates at d 42 were 39/104 (38%) for bIFN-alpha and 47/98 (48%) for placebo. In Exp. 3, heifers were given gradually increasing doses of bIFN-alpha or placebo from d 11 to 19, because such a regimen had been shown to reduce the number of heifers experiencing hyperthermia after bIFN-alpha injection. Pregnancy rates were 42/95 (44%) for bIFN-alpha and 62/111 (56%) for placebo. Across all three experiments, pregnancy rates were lower (P less than .01) for heifers treated with bIFN-alpha (117/274; 43%) than for heifers treated with placebo (152/281; 54%). In conclusion, these results demonstrate that, under the administration systems used, bIFN-alpha does not increase pregnancy rate, but rather tends to reduce it.

Proinflammatory profile of in vitro monocytes in the ageing is affected by lymphocytes presence

Background: Aging is associated with complex and constant remodeling of the immune function, resulting in an increasing susceptibility to infection and others diseases. The infections caused by Gram-negative microorganisms, present in nursing homes and hospitals, constitute one of the most common infections in the elderly, and are mainly combated by innate immune cells. Although the functions of innate immunity seem more preserved during aging than of adaptive immune mechanisms, two systems operate in an integrated way in the body, so that injury in one part of the immune system inevitably affects the other as they are part of a defensive network. The aim of this study was to investigate the in vitro production of proinflammatory (TNF-α, IL-6, IL-1β, CXCL-8 and MCP-1) and anti-inflammatory (TGF-β and IL-10) cytokines by monocytes, stimulated or not (basal) with lipopolysaccharide, from healthy young and elderly subjects. By means of PBMCs, we also studied if cytokine profile is altered in these different patient groups, in the presence of lymphocytes, under the same experimental conditions.Results: The monocytes from elderly presented higher basal production of TNF-α, MCP-1 and lower of TGF-β than young monocytes. PBMC showed similar cytokines production, irrespective age or stimulation presence. In the presence of lymphocytes, the spontaneous production of IL-10 was higher and of TGF-β was lower than monocytes, regardless of age. After LPS-stimulation, the presence of lymphocytes resulted in increased IL-6, IL-1β, MCP-1 and IL-10 and decreased CXCL-8 and TGF-β in comparison to pure culture of monocytes from young patients. With age, the same differences were observed, except for CXCL-8 and TGF-β which production was the same between monocytes and PBMC stimulated with LPS.Conclusion: These findings reinforce the systemic state of inflamm-aging frequently reported in elderly and considered a factor of susceptibility to numerous diseases. Still, the cytokine production from just monocytes of the elderly showed alterations, while in the lymphocyte presence not, suggesting an immunomodulator role of lymphocytes on monocytes. In addition, the differences between the production patterns by LPS-stimulated PBMC between young and elderly volunteers can be related with an imbalance in response against Gram-negative bacteria in throughout life. © 2013 Pinke et al.; licensee BioMed Central Ltd.

Stimulation of MMP-1 and CCL2 by NAMPT in PDL cells

Periodontitis is an inflammatory disease caused by pathogenic microorganisms and characterized by the destruction of the periodontium. Obese individuals have an increased risk of periodontitis, and elevated circulating levels of adipokines, such as nicotinamide phosphoribosyltransferase (NAMPT), may be a pathomechanistic link between both diseases. The aim of this in vitro study was to examine the regulation of periodontal ligament (PDL) cells by NAMPT and its production under inflammatory and infectious conditions. NAMPT caused a significant upregulation of 9 genes and downregulation of 3 genes, as analyzed by microarray analysis. Eight of these genes could be confirmed by real-time PCR: NAMPT induced a significant upregulation of EGR1, MMP-1, SYT7, ITPKA, CCL2, NTM, IGF2BP3, and NRP1. NAMPT also increased significantly the MMP-1 and CCL2 protein synthesis. NAMPT was significantly induced by interleukin-1β and the periodontal microorganism P. gingivalis. NAMPT may contribute to periodontitis through upregulation of MMP-1 and CCL2 in PDL cells. Increased NAMPT levels, as found in obesity, may therefore represent a mechanism whereby obesity could confer an increased risk of periodontitis. Furthermore, microbial and inflammatory signals may enhance the NAMPT synthesis in PDL cells and thereby contribute to the increased gingival and serum levels of this adipokine, as found in periodontitis. © 2013 Marjan Nokhbehsaim et al.

Early endosome antigen 1 (EEA1) decreases in macrophages infected with Paracoccidioides brasiliensis

Paracoccidioidomycosis (PCM) is a chronic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis, endemic in Latin America. P. brasiliensis has been observed in epithelial cells in vivo and in vitro, as well as within the macrophages. The identification of the mechanism by which it survives within the host cell is fertile ground for the discovery of its pathogenesis since this organism has the ability to induce its own endocytosis in epithelial cells and most likely in macrophages. The study of the expression of endocytic proteins pathway and co-localization of microorganisms enable detection of the mechanism by which microorganisms survive within the host cell. The aim of this study was to evaluate the expression of the endocytic protein EEA1 (early endosome antigen 1) in macrophages infected with P. brasiliensis. For detection of EEA1, three different techniques were employed: immunofluorescence, real-time polymerase chain reaction (PCR) and immunoblotting. In the present study, decreased expression of EEA1 as well as the rearrangement of the actin was observed when the fungus was internalized, confirming that the input mechanism of the fungus in macrophages occurs through phagocytosis. © 2013 ISHAM.