MMP-7 and TIMP-1, news targets in predicting poor wound healing in apical periodontitis

Introduction: Matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) are strongly associated with tissue destruction because of inflammation. In this study, we investigated the expression of MMPs and TIMPs messenger RNA and protein levels in apical periodontitis lesions. Methods: Tissue samples from patients presenting clinical signs of chronic apical abscess (CAA) or asymptomatic apical periodontitis (AAP) were collected postoperatively and used for gene expression analysis of MMP-2, -3, -7, -9, -14, -16, and -25; TIMP-1; and TIMP-2 in real-time polymerase chain reaction. Immunohistochemistry was also performed to detect the expression of MMP-7 and TIMP-1 proteins. Lastly, U-937 cells were induced to terminal differentiation into macrophages, infected with purified Escherichia coli lipopolysaccharide, and assessed for the expression of MMP-7 and TIMP-1 using immunocytochemistry and confocal microscopy. Results: Significantly higher messenger RNA levels were found for all genes in AAP and CAA samples when compared with healthy control samples (P < .001). AAP cases exhibited significantly higher TIMP-1 when compared with CAA cases, whereas CAA cases showed higher MMP-2, MMP-7, and MMP-9 messenger RNA levels (P < .05). We also detected positive the expression of MMP-7 and TIMP-1 proteins in the tissue samples. The expression of both MMP-7 and TIMP-1 were increased in lipopolysaccharide-stimulated cells compared with nonstimulated cells and appear to colocalize in the Golgi apparatus. Conclusions: MMPs appear to have an influential role in CAA cases in which ongoing tissue destruction is observed. TIMPs are preferentially associated with AAP, perhaps as a subsequent defense mechanism against excessive destruction. Taken together, our findings implicate MMP and TIMP molecules in the dynamics of inflammatory periapical lesion development

Advanced Glycation in macrophages induces intracellular accumulation of 7-ketocholesterol and total sterols by decreasing the expression of ABCA-1 and ABCG-1

Abstract Background Advanced glycation end products (AGE) alter lipid metabolism and reduce the macrophage expression of ABCA-1 and ABCG-1 which impairs the reverse cholesterol transport, a system that drives cholesterol from arterial wall macrophages to the liver, allowing its excretion into the bile and feces. Oxysterols favors lipid homeostasis in macrophages and drive the reverse cholesterol transport, although the accumulation of 7-ketocholesterol, 7alpha- hydroxycholesterol and 7beta- hydroxycholesterol is related to atherogenesis and cell death. We evaluated the effect of glycolaldehyde treatment (GAD; oxoaldehyde that induces a fast formation of intracellular AGE) in macrophages overloaded with oxidized LDL and incubated with HDL alone or HDL plus LXR agonist (T0901317) in: 1) the intracellular content of oxysterols and total sterols and 2) the contents of ABCA-1 and ABCG-1. Methods Total cholesterol and oxysterol subspecies were determined by gas chromatography/mass spectrometry and HDL receptors content by immunoblot. Results In control macrophages (C), incubation with HDL or HDL + T0901317 reduced the intracellular content of total sterols (total cholesterol + oxysterols), cholesterol and 7-ketocholesterol, which was not observed in GAD macrophages. In all experimental conditions no changes were found in the intracellular content of other oxysterol subspecies comparing C and GAD macrophages. GAD macrophages presented a 45% reduction in ABCA-1 protein level as compared to C cells, even after the addition of HDL or HDL + T0901317. The content of ABCG-1 was 36.6% reduced in GAD macrophages in the presence of HDL as compared to C macrophages. Conclusion In macrophages overloaded with oxidized LDL, glycolaldehyde treatment reduces the HDL-mediated cholesterol and 7-ketocholesterol efflux which is ascribed to the reduction in ABCA-1 and ABCG-1 protein level. This may contribute to atherosclerosis in diabetes mellitus.

Impairment of the hematological response and interleukin-1β production in protein-energy malnourished mice after endotoxemia with lipopolysaccharide

The objectives of this study were to determine if protein-energy malnutrition (PEM) could affect the hematologic response to lipopolysaccharide (LPS), the interleukin-1β (IL-1β) production, leukocyte migration, and blood leukocyte expression of CD11a/CD18. Two-month-old male Swiss mice were submitted to PEM (N = 30) with a low-protein diet (14 days) containing 4% protein, compared to 20% protein in the control group (N = 30). The total cellularity of blood, bone marrow, spleen, and bronchoalveolar lavage evaluated after the LPS stimulus indicated reduced number of total cells in all compartments studied and different kinetics of migration in malnourished animals. The in vitro migration assay showed reduced capacity of migration after the LPS stimulus in malnourished animals (45.7 ± 17.2 x 10(4) cells/mL) compared to control (69.6 ± 7.1 x 10(4) cells/mL, P ≤ 0.05), but there was no difference in CD11a/CD18 expression on the surface of blood leukocytes. In addition, the production of IL-1β in vivo after the LPS stimulus (180.7 pg·h-1·mL-1), and in vitro by bone marrow and spleen cells (41.6 ± 15.0 and 8.3 ± 4.0 pg/mL) was significantly lower in malnourished animals compared to control (591.1 pg·h-1·mL-1, 67.0 ± 23.0 and 17.5 ± 8.0 pg/mL, respectively, P ≤ 0.05). The reduced expression of IL-1β, together with the lower number of leukocytes in the central and peripheral compartments, different leukocyte kinetics, and reduced leukocyte migration capacity are factors that interfere with the capacity to mount an adequate immune response, being partly responsible for the immunodeficiency observed in PEM.

Morphogenetic and structural characteristics of guinea grass tillers at different ages under intermittent stocking

The objective of this research was to assess morphogenetic and structural characteristics of tillers of guinea grass cv. Tanzania at different ages. The pastures of guinea grass were managed in six pasture conditions related to the combination of three frequencies (90, 95, and 99% light interception) and two post-grazing heights (25 and 50 cm). In these six pastures conditions, three tiller ages were evaluated (young, mature, and old). The design was of completely randomized block with three replications. Young tillers exhibited higher leaf appearance rate and leaf elongation rate and, consequently, higher final leaf length and number of live leaves than mature and old tillers, regardless of the pasture condition. On pastures managed with 90 or 95% light interception associated with a post-grazing height of 25 cm, old tillers presented longer leaf lifespan than young and mature ones. There is a progressive reduction in the vigor of growth of pastures of guinea grass cv. Tanzania with advancing tiller age.

Targeting the non-structural protein 1 from dengue virus to a dendritic cell population confers protective immunity to lethal virus challenge

Dengue is the most prevalent arboviral infection, affecting millions of people every year. Attempts to control such infection are being made, and the development of a vaccine is a World Health Organization priority. Among the proteins being tested as vaccine candidates in preclinical settings is the non-structural protein 1 (NS1). In the present study, we tested the immune responses generated by targeting the NS1 protein to two different dendritic cell populations. Dendritic cells (DCs) are important antigen presenting cells, and targeting proteins to maturing DCs has proved to be an efficient means of immunization. Antigen targeting is accomplished by the use of a monoclonal antibody (mAb) directed against a DC cell surface receptor fused to the protein of interest. We used two mAbs (αDEC205 and αDCIR2) to target two distinct DC populations, expressing either DEC205 or DCIR2 endocytic receptors, respectively, in mice. The fusion mAbs were successfully produced, bound to their respective receptors, and were used to immunize BALB/c mice in the presence of polyriboinosinic: polyribocytidylic acid (poly (I:C)), as a DC maturation stimulus. We observed induction of strong anti-NS1 antibody responses and similar antigen binding affinity irrespectively of the DC population targeted. Nevertheless, the IgG1/IgG2a ratios were different between mouse groups immunized with αDEC-NS1 and αDCIR2-NS1 mAbs. When we tested the induction of cellular immune responses, the number of IFN-γ producing cells was higher in αDEC-NS1 immunized animals. In addition, mice immunized with the αDEC-NS1 mAb were significantly protected from a lethal intracranial challenge with the DENV2 NGC strain when compared to mice immunized with αDCIR2-NS1 mAb. Protection was partially mediated by CD4(+) and CD8(+) T cells as depletion of these populations reduced both survival and morbidity signs. We conclude that targeting the NS1 protein to the DEC205(+) DC population with poly (I:C) opens perspectives for dengue vaccine development.

Palmitoleic acid (n-7) increases white adipocyte lipolysis and lipase content in a PPARα-dependent manner

We investigated whether palmitoleic acid, a fatty acid that enhances whole body glucose disposal and suppresses hepatic steatosis, modulates triacylglycerol (TAG) metabolism in adipocytes. For this, both differentiated 3T3-L1 cells treated with either palmitoleic acid (16:1n7, 200 μM) or palmitic acid (16:0, 200 μM) for 24 h and primary adipocytes from wild-type or PPARα-deficient mice treated with 16:1n7 (300 mg•kg(-1)•day(-1)) or oleic acid (18:1n9, 300 mg•kg(-1)•day(-1)) by gavage for 10 days were evaluated for lipolysis, TAG, and glycerol 3-phosphate synthesis and gene and protein expression profile. Treatment of differentiated 3T3-L1 cells with 16:1n7, but not 16:0, increased basal and isoproterenol-stimulated lipolysis, mRNA levels of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) and protein content of ATGL and pSer(660)-HSL. Such increase in lipolysis induced by 16:1n7, which can be prevented by pharmacological inhibition of PPARα, was associated with higher rates of PPARα binding to DNA. In contrast to lipolysis, both 16:1n7 and 16:0 increased fatty acid incorporation into TAG and glycerol 3-phosphate synthesis from glucose without affecting glyceroneogenesis and glycerokinase expression. Corroborating in vitro findings, treatment of wild-type but not PPARα-deficient mice with 16:1n7 increased primary adipocyte basal and stimulated lipolysis and ATGL and HSL mRNA levels. In contrast to lipolysis, however, 16:1n7 treatment increased fatty acid incorporation into TAG and glycerol 3-phosphate synthesis from glucose in both wild-type and PPARα-deficient mice. In conclusion, palmitoleic acid increases adipocyte lipolysis and lipases by a mechanism that requires a functional PPARα

Loss of the anorexic response to systemic 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside administration despite reducing hypothalamic AMP-activated protein kinase phosphorylation in insulin-deficient rats

This study tested whether chronic systemic administration of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) could attenuate hyperphagia, reduce lean and fat mass losses, and improve whole-body energy homeostasis in insulin-deficient rats. Male Wistar rats were first rendered diabetic through streptozotocin (STZ) administration and then intraperitoneally injected with AICAR for 7 consecutive days. Food and water intake, ambulatory activity, and energy expenditure were assessed at the end of the AICAR-treatment period. Blood was collected for circulating leptin measurement and the hypothalami were extracted for the determination of suppressor of cytokine signaling 3 (SOCS3) content, as well as the content and phosphorylation of AMP-kinase (AMPK), acetyl-CoA carboxylase (ACC), and the signal transducer and activator of transcription 3 (STAT3). Rats were thoroughly dissected for adiposity and lean body mass (LBM) determinations. In non-diabetic rats, despite reducing adiposity, AICAR increased (∼1.7-fold) circulating leptin and reduced hypothalamic SOCS3 content and food intake by 67% and 25%, respectively. The anorexic effect of AICAR was lost in diabetic rats, even though hypothalamic AMPK and ACC phosphorylation markedly decreased in these animals. Importantly, hypothalamic SOCS3 and STAT3 levels remained elevated and reduced, respectively, after treatment of insulin-deficient rats with AICAR. Diabetic rats were lethargic and displayed marked losses of fat and LBM. AICAR treatment increased ambulatory activity and whole-body energy expenditure while also attenuating diabetes-induced fat and LBM losses. In conclusion, AICAR did not reverse hyperphagia, but it promoted anti-catabolic effects on skeletal muscle and fat, enhanced spontaneous physical activity, and improved the ability of rats to cope with the diabetes-induced dysfunctional alterations in glucose metabolism and whole-body energy homeostasis.

On the three-finger protein domain fold and CD59-like proteins in Schistosoma mansoni

Background: It is believed that schistosomes evade complement-mediated killing by expressing regulatory proteins on their surface. Recently, six homologues of human CD59, an important inhibitor of the complement system membrane attack complex, were identified in the schistosome genome. Therefore, it is important to investigate whether these molecules could act as CD59-like complement inhibitors in schistosomes as part of an immune evasion strategy. Methodology/Principal Findings: Herein, we describe the molecular characterization of seven putative SmCD59-like genes and attempt to address the putative biological function of two isoforms. Superimposition analysis of the 3D structure of hCD59 and schistosome sequences revealed that they contain the three-fingered protein domain (TFPD). However, the conserved amino acid residues involved in complement recognition in mammals could not be identified. Real-time RT-PCR and Western blot analysis determined that most of these genes are up-regulated in the transition from free-living cercaria to adult worm stage. Immunolocalization experiments and tegument preparations confirm that at least some of the SmCD59-like proteins are surface-localized; however, significant expression was also detected in internal tissues of adult worms. Finally, the involvement of two SmCD59 proteins in complement inhibition was evaluated by three different approaches: (i) a hemolytic assay using recombinant soluble forms expressed in Pichia pastoris and E. coli; (ii) complement-resistance of CHO cells expressing the respective membrane-anchored proteins; and (iii) the complement killing of schistosomula after gene suppression by RNAi. Our data indicated that these proteins are not involved in the regulation of complement activation. Conclusions: Our results suggest that this group of proteins belongs to the TFPD superfamily. Their expression is associated to intra-host stages, present in the tegument surface, and also in intra-parasite tissues. Three distinct approaches using SmCD59 proteins to inhibit complement strongly suggested that these proteins are not complement inhibitors and their function in schistosomes remains to be determined.