Phlebotomine salivas inhibit immune inflammation-induced neutrophil migration via an autocrine DC-derived PGE(2)/IL-10 sequential pathway

In the present study, we investigated whether saliva from Phlebotomus papatasi and Phlebotomus duboscqi inhibited antigen-induced neutrophil migration and the mechanisms involved in these effects. The pretreatment of immunized mice with salivary gland extracts (SGE) of both phlebotomines inhibited OVA challenge-induced neutrophil migration and release of the neutrophil chemotactic mediators, MIP-1 alpha, TNF-alpha, and leukotriene B-4 (LTB4). Furthermore, SGE treatment enhanced the production of anti-inflammatory mediators, IL-10 and PGE(2). SGE treatments failed to inhibit neutrophil migration and MIP-1 alpha and LTB4 production in IL-10(-/-) mice, also failing in mice treated with nonselective (indomethacin) or selective (rofecoxibe) cyclooxygenase (COX) inhibitors. COX inhibition resulted in diminished SGE-induced IL-10 production, and PGE(2) release triggered by SGE remained increased in IL-10(-/-) mice, suggesting that prostanoids are acting through an IL-10-dependent mechanism. SGE treatments in vivo reduced the OVA-induced lymphoproliferation of spleen-derived cells. Further, the in vitro incubation of bone marrow-derived dendritic cells (DC) with SGE inhibited the proliferation of CD4(+) T cells from OVA-immunized mice, which was reversed by indomethacin and anti-IL-10 antibody treatments. Supporting these results, SGE induced the production of PGE(2) and IL-10 by DC, which were blocked by COX inhibition. These effects were associated with the reduction of DC-membrane expression of MHC-II and CD86 by SGE treatment. Altogether, the results showed that Phlebotomine saliva inhibits immune inflammation-induced neutrophil migration by an autocrine DC sequential production of PGE(2)/IL-10, suggesting that the saliva constituents might be promising therapeutic molecules to target immune inflammatory diseases.

Peroxisome proliferator-activated receptor-gamma ligand, 15-deoxy-Delta A(12,14)-prostaglandin J(2), reduces neutrophil migration via a nitric oxide pathway

Ligands for peroxisome proliferator-activated receptor gamma (PPAR-gamma), such as 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) have been implicated as a new class of anti-inflammatory compounds with possible clinical applications. Based on this concept, this investigation was designed to determine the effect of 15d-PGJ(2)-mediated activation of PPAR-gamma ligand on neutrophil migration after an inflammatory stimulus and clarify the underlying molecular mechanisms using a mouse model of peritonitis. Our results demonstrated that 15d-PGJ(2) administration decreases leukocyte rolling and adhesion to the inflammated mesenteric tissues by a mechanism dependent on NO. Specifically, pharmacological inhibitors of NO synthase remarkably abrogated the 15d-PGJ(2)-mediated suppression of neutrophil migration to the inflammatory site. Moreover, inducible NOS(-/-) mice were not susceptible to 15d-PGJ(2)-mediated suppression of neutrophil migration to the inflammatory sites when compared with their wild type. In addition, 15d-PGJ(2)-mediated suppression of neutrophil migration appeared to be independent of the production of cytokines and chemokines, since their production were not significantly affected in the carrageenan-injected peritoneal cavities. Finally, up-regulation of carrageenan-triggered ICAM-I expression in the mesenteric microcirculation vessels was abrogated by pretreatment of wild-type mice with 15d-PGJ(2), whereas 15d-PGJ(2) inhibited F-actin rearrangement process in neutrophils. Taken together these findings demonstrated that 15d-PGJ(2) suppresses inflammation-initiated neutrophil migration in a mechanism dependent on NO production in mesenteric tissues.

Gastroprotective effect of heme-oxygenase 1/biliverdin/CO pathway in ethanol-induced gastric damage in mice

Our objective was to evaluate the role of heme-oxygenase 1 (HO-1)/biliverdin/CO pathway in gastric defense against ethanol-induced gastric damage in mice. Mice were pre-treated with saline, hemin (HO-1 inducer), biliverdin (HO-1 product), dimanganese decacarbonyl (DMDC, CO donor) or zinc protoporphyrin IX (ZnPP IX, HO-1 antagonist). Another group received soluble guanylate cyclase (sGC) inhibitor (ODQ) 30 min before hemin, biliverdin or DMDC. After 30 min, gastric damage was induced by ethanol. After one hour, rats were sacrificed. Gastric lesions were measured using a computer planimetry program, and gastric corpus pieces were assayed for malonylaldehyde (MDA), glutathione (GSH) or bilirubin. HO-1 expression was determined after saline or ethanol administration by polymerase chain reaction (PCR) or immunohistochemistry. Ethanol (25% or 50%) induced gastric damage, increased MDA levels and reduced GSH in the gastric tissue. Ethanol 50% increased HO-1 mRNA transcripts, HO-1 immunoreactivity, and bilirubin concentration in gastric mucosa. Pre-treatment with hemin reduced gastric damage and MDA formation and increased GSH concentration in the gastric mucosa. ZnPP IX amplified the ethanol-induced gastric lesion, increased MDA formation and decreased GSH concentration in gastric mucosa. Biliverdin and DMDC reduced gastric damage and MDA formation and increased GSH concentration in the gastric tissue. ODQ completely abolished the DMDC protective gastric effect However, effects of hemin or biliverdin did not change with ODQ treatment. Our results suggest that HO-1/biliverdin/CO pathway plays a protective role against ethanol-induced gastric damage through mechanisms that can be dependent (CO) or independent (biliverdin) of sGC activation. (C) 2010 Elsevier B.V. All rights reserved.

Role of the spinal cord NO/cGMP pathway in the control of arterial pressure and heart rate

The modulatory effect of nitric oxide/cyclic guanosine monophosphate (NO/cGMP) pathway on sympathetic preganglionic neurons still deserves further investigation. The present study was designed to examine the role of the spinal cord NO/cGMP pathway in controlling mean arterial pressure and heart rate. We observed that intrathecal administration of the NO synthase inhibitor N omega-Nitro-L-arginine methyl ester hydrochloride (L-NAME) causes an increase in mean arterial pressure but does not affect heart rate. Intrathecal administration of the soluble guanylyl cyclase inhibitor 1H-[1,2,4] Oxadiazolo[4,3-a] quinoxalin-1-one (ODQ) does not change mean arterial pressure and heart rate. The precursor for NO synthesis, L-arginine, reduces both mean arterial pressure and heart rate while administration of ODQ before L-arginine impaired decreases in mean arterial pressure and heart rate. Administration of the N-methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino-5-phosphonopentanoic acid (AP5) after L-NAME does not affect increases in mean arterial pressure promoted by NO synthase inhibition. Although the hypotensive and bradycardic responses induced by intrathecal administration of L-arginine depend on cGMP, our results indicate that NO acts to tonically inhibit SPNs, independent of either cGMP or NMDA receptors.

SAGE analysis demonstrates increased expression of TOSO contributing to Fas-mediated resistance in CLL

To identify novel genes involved in the molecular pathogenesis of chronic lymphocytic leukemia (CLL) we performed a serial analysis of gene expression (SAGE) in CLL cells, and compared this with healthy B cells (nCD19(+)). We found a high level of similarity among CLL subtypes, but a comparison of CLL versus nCD19(+) libraries revealed 55 genes that were over-represented and 49 genes that were down-regulated in CLL. A gene ontology analysis revealed that TOSO, which plays a functional role upstream of Fas extrinsic apoptosis pathway, was over-expressed in CLL cells. This finding was confirmed by real-time reverse transcription-polymerase chain reaction in 78 CLL and 12 nCD19(+) cases (P <.001). We validated expression using flow cytometry and tissue microarray and demonstrated a 5.6-fold increase of TOSO protein in circulating CLL cells (P =.013) and lymph nodes (P =.006). Our SAGE results have demonstrated that TOSO is a novel overexpressed antiapoptotic gene in CLL.

Number of expressed cancer/testis antigens identifies focal adhesion pathway genes as possible targets for multiple myeloma therapy

Considering that the importance of cancer/testis (CT) antigens in multiple myeloma (MM) biology is still under investigation, the present study aimed to: (1) identify genes differentially expressed in MM using microarray analysis of plasma cell samples, separated according to the number of expressed CTs; (2) examine possible pathways related to MM pathogenesis; (3) validate the expression of candidate genes by quantitative real-time PCR (RQ-PCR). Three samples predominantly positive (>6 expressed), including the U266 cell line, and three samples predominantly negative (0 or 1 expressed CT for the 13 analyzed CT antigens), were submitted for microarray analysis. Validation by RQ-PCR from 24 MM samples showed that the ITGAS gene was downregulated in predominantly positive (>6 expressed CTs, p = 0.0030) and in tumor versus normal plasma cells (p = 0.0182). The RhoD gene was overexpressed in tumor plasma cells when compared to normal plasma cells (p = 0.0339). Results of the microarray analysis corroborate the hypothesis that MM could be separated into predominantly positive and predominantly negative expression. The differential expression of ITGA5 and RhoD suggests disruption of the focal adhesion pathway in MM and offers a new target field to be explored in this disease.

Syndromic and non-syndromic aneurysms of the human ascending aorta share activation of the Smad2 pathway

Common features such as elastic fibre destruction, mucoid accumulation, and smooth muscle cell apoptosis are co-localized in aneurysms of the ascending aorta of various aetiologies. Recent experimental studies reported an activation of TGF-beta in aneurysms related to Marfan (and Loeys-Dietz) syndrome. Here we investigate TGF-beta signalling in normal and pathological human ascending aortic wall in syndromic and non-syndromic aneurysmal disease. Aneurysmal ascending aortic specimens, classified according to aetiology: syndromic MFS (n = 15, including two mutations in TGFBR2), associated with BAV (n = 15) or degenerative forms (n = 19), were examined. We show that the amounts of TGF-beta 1 protein retained within and released by aneurysmal tissue were greater than for control aortic tissue, whatever the aetiology, contrasting with an unchanged TGF-beta 1 mRNA level. The increase in stored TGF-beta 1 was associated with enhanced LTBP-I protein and mRNA levels. These dysiregulations of the extracellular ligand are associated with higher phosphorylated Smad2 and Smad2 mRNA levels in the ascending aortic wall from all types of aneurysm. This activation correlated with the degree of elastic fibre fragmentation. Surprisingly, there was no consistent association between the nuclear location of pSmad2 and extracellular TGF-beta 1 and LTBP-I staining and between their respective mRNA expressions. In parallel, decorin. was focally increased in aneurysmal media, whereas biglycan was globally decreased in aneurysmal aortas. In conclusion, this study highlights independent dysregulations of TGF-beta retention and Smad2 signalling in syndromic and non-syndromic aneurysms of the ascending aorta. Copyright (C) 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Evaluation of lignin contents in tropical forages using different analytical methods and their correlations with degradation of insoluble fiber

We compared the lignin contents of tropical forages by different analytical methods and evaluated their correlations with parameters related to the degradation of neutral detergent fiber (NDF). The lignin content was evaluated by five methods: cellulose solubilization in sulfuric acid [Lignin (sa)], oxidation with potassium permanganate [Lignin (pm)], the Klason lignin method (KL), solubilization in acetyl bromide from acid detergent fiber (ABLadf) and solubilization in acetyl bromide from the cell wall (ABLcw). Samples from ten grasses and ten legumes were used. The lignin content values obtained by gravimetric methods were also corrected for protein contamination, and the corrected values were referred to as Lignin (sa)p, Lignin (pm)p and KLp. The indigestible fraction of NDF (iNDF), the discrete lag (LAG) and the fractional rate of degradation (kd) of NDF were estimated using an in vitro assay. Correcting for protein resulted in reductions (P < 0.05) in the lignin contents as measured by the Lignin (sa), Lignin (pm) and, especially, the KL methods. There was an interaction (P < 0.05) of analytical method and forage group for lignin content. In general, LKp method provided the higher (P < 0.05) lignin contents. The estimates of lignin content obtained by the Lignin (sa)p, Lignin (pm)p and LKp methods were associated (P > 0.05) with all of the NDF degradation parameters. However, the strongest correlation coefficients for all methods evaluated were obtained with Lignin (pm)p and KLp. The lignin content estimated by the ABLcw method did not correlate (P > 0.05) with any parameters of NDF degradation. There was a correlation (P < 0.05) between the lignin content estimated by the ABLadf method and iNDF content. Nonetheless, this correlation was weaker than those found with gravimetric methods. From these results, we concluded that the gravimetric methods produce residues that are contaminated by nitrogenous compounds. Adjustment for these contaminants is suggested, particularly for the KL method, to express lignin content with greater accuracy. The relationships between lignin content measurements and NDF degradation parameters can be better determined using KLp and Lignin (pm)p methods. (C) 2011 Elsevier B.V. All rights reserved.

Plasminogen Acquisition and Activation at the Surface of Leptospira Species Lead to Fibronectin Degradation

Pathogenic Leptospira species are the etiological agents of leptospirosis, a widespread disease of human and veterinary concern. In this study, we report that Leptospira species are capable of binding plasminogen (PLG) in vitro. The binding to the leptospiral surface was demonstrated by indirect immunofluorescence confocal microscopy with living bacteria. The PLG binding to the bacteria seems to occur via lysine residues because the ligation is inhibited by addition of the lysine analog 6-aminocaproic acid. Exogenously provided urokinase-type PLG activator (uPA) converts surface-bound PLG into enzymatically active plasmin, as evaluated by the reaction with the chromogenic plasmin substrate D-Val-Leu-Lys 4-nitroanilide dihydrochloridein. The PLG activation system on the surface of Leptospira is PLG dose dependent and does not cause injury to the organism, as cellular growth in culture was not impaired. The generation of active plasmin within Leptospira was observed with several nonvirulent high-passage strains and with the nonpathogenic saprophytic organism Leptospira biflexa. Statistically significant higher activation of plasmin was detected with a low-passage infectious strain of Leptospira. Plasmin-coated virulent Leptospira interrogans bacteria were capable of degrading purified extracellular matrix fibronectin. The breakdown of fibronectin was not observed with untreated bacteria. Our data provide for the first time in vitro evidence for the generation of active plasmin on the surface of Leptospira, a step that may contribute to leptospiral invasiveness.

Ethanol Wet-bonding Challenges Current Anti-degradation Strategy

The long-term effectiveness of chlorhexidine as a matrix metalloproteinase (MMP) inhibitor may be compromised when water is incompletely removed during dentin bonding. This study challenged this anti-bond degradation strategy by testing the null hypothesis that wet-bonding with water or ethanol has no effect on the effectiveness of chlorhexidine in preventing hybrid layer degradation over an 18-month period. Acid-etched dentin was bonded under pulpal pressure simulation with Scotchbond MP and Single Bond 2, with water wet-bonding or with a hydrophobic adhesive with ethanol wet-bonding, with or without pre-treatment with chlorhexidine diacetate (CHD). Resin-dentin beams were prepared for bond strength and TEM evaluation after 24 hrs and after aging in artificial saliva for 9 and 18 mos. Bonds made to ethanol-saturated dentin did not change over time with preservation of hybrid layer integrity. Bonds made to CHD pre-treated acid-etched dentin with commercial adhesives with water wet-bonding were preserved after 9 mos but not after 18 mos, with severe hybrid layer degradation. The results led to rejection of the null hypothesis and highlight the concept of biomimetic water replacement from the collagen intrafibrillar compartments as the ultimate goal in extending the longevity of resin-dentin bonds.

Bis-GMA co-polymerizations: Influence on conversion, flexural properties, fracture toughness and susceptibility to ethanol degradation of experimental composites

Objectives. The aim of this study was to evaluate the influence of monomer content on fracture toughness (K(Ic)) before and after ethanol solution storage, flexural properties and degree of conversion (DC) of bisphenol A glycidyl methacrylate (Bis-GMA) co-polymers. Methods. Five formulations were tested, containing Bis-GMA (B) combined with TEGDMA (T), UDMA (U) or Bis-EMA (E), as follows (in mol%): 30B:70T; 30B:35T:35U; 30B:70U; 30B:35T:35E; 30B:70E. Bimodal filler was introduced at 80 wt%. Single-edge notched beams for fracture toughness (FT, 25 mm x 5 mm x 2.5 mm, a/w = 0.5, n = 20) and 10 mm x 2 mm x 1 mm beams for flexural strength (FS) and modulus (FM) determination (10 mm x 2 mm x 1 mm, n = 10) were built and then stored in distilled water for 24 h at 37 degrees C. All FS/FM beams and half of the FT specimens were immediately submitted to three-point bending test. The remaining FT specimens were stored in a 75%ethanol/25%water (v/v) solution for 3 months prior to testing. DC was determined with FT-Raman spectroscopy in fragments of both FT and FS/FM specimens at 24 h. Data were submitted to one-way ANOVA/Tukey test (alpha = 5%). Results. The 30B:70T composite presented the highest K(Ic) value (in MPa m(1/2)) at 24 h (1.3 +/- 0.4), statistically similar to 30B:35T:35U and 30B:70U, while 30B:70E presented the lowest value (0.5 +/- 0.1). After ethanol storage, reductions in K(Ic) ranged from 33 to 72%. The 30B:70E material presented the lowest reduction in FT and 30B:70U, the highest. DC was similar among groups (69-73%), except for 30B:70U (52 +/- 4%, p < 0.001). 30B:70U and 30B:35T:35U presented the highest FS (125 +/- 21 and 122 +/- 14 MPa, respectively), statistically different from 30B:70T or 30B:70E (92 +/- 20 and 94 +/- 16 MPa, respectively). Composites containing UDMA or Bis-EMA associated with Bis-GMA presented similar FM, statistically lower than 30B:35T:35U. Significance. Composites formulated with Bis-GMA:TEGDMA:UDMA presented the best compromise between conversion and mechanical properties. (C) 2009 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Insights from Studies with Oral Cleft Genes Suggest Associations between WNT-pathway Genes and Risk of Oral Cancer

Oral squamous cell carcinoma (OSCC) accounts for more than 90% of the malignant neoplasms that arise in the mucosa of the upper aerodigestive tract. Recent studies of cleft lip/palate have shown the association of genes involved in cancer. WNT pathway genes have been associated with several types of cancer and recently with cleft lip/palate. To investigate if genes associated with cleft lip/palate were also associated with oral cancer, we genotyped 188 individuals with OSCC and 225 control individuals for markers in AXIN2, AXIN1, GSK3 beta, WNT3A, WNT5A, WNT8A, WNT11, WNT3, and WNT9B. Statistical analysis was performed with PLINK 1.06 software to test for differences in allele frequencies of each polymorphism between cases and controls. We found association of SNPs in GSK3B (p = 0.0008) and WNT11 (p = 0.03) with OSCC. We also found overtransmission of GSK3B haplotypes in OSCC cases. Expression analyses showed up-regulation of WNT3A, GSK3B, and AXIN1 and down-regulation of WNT11 in OSCC in comparison with control tissues (P < 0.001). Additional studies should focus on the identification of potentially functional variants in these genes as contributors to human clefting and oral cancer.

Differential bonds degradation of two resin-modified glass-ionomer cements in primary and permanent teeth

Objectives: To evaluate the effect of chemical degradation on bond strength of resin-modified glass-ionomer cements bonded to primary and permanent dentin. Methods: Class I cavities of permanent and primary extracted human molars were restored with two resin-modified glass-ionomer cements: Fuji 11 LC and Vitremer, and stored in water for 24 h. Half samples were immersed in 10% NaOCl aqueous solution for 5 h. Teeth were sectioned into beams and tested for microtensile bond strengths. Results were analyzed with multiple ANOVA and Tukey`s tests (p < 0.05). Analysis of debonded surfaces was performed by SEM. Results: 24 h bond strengths for Vitremer and Fuji 11 LC were similar. For Fuji 11, bond strength values were higher for primary than for permanent dentin. Vitremer bond strength was similar for both. Chemical degradation did not affect Fuji I] LC bond strength to dentin. However, decreases in bond strength were found for Vitremer groups after NaOCl immersion. Signs of glass ionomer-dentin interaction were evident by SEM analysis for Fuji 11 LC specimens. Conclusions: Vitremer and Fuji II presented similar bond strength at 24. Vitremer dentin bonds were prone to chemical degradation. Fuji II LC-dentin bonds showed typical features of glass-ionomer dentin interaction at the bonded interfaces, and were resistant to in vitro degradation. (C) 2009 Elsevier Ltd. All rights reserved.

Resistance to degradation of resin-modified glass-ionomer cements dentine bonds

Objective: To evaluate the effect of EDTA pre-treatment of dentine on resistance to degradation of the bond between dentine and resin-modified glass-ionomer cements. Methods: Sixty non-carious human molars underwent cavity preparations. Teeth were restored with Fuji II LC or Vitremer. Half of the cavities were restored following manufacturers` instructions whereas the other half was pre-treated with EDTA (0.1 M, pH 7.4) for 60 s. Teeth were stored in water at 37 degrees C for 24 h, 3 months or submitted to 10% NaOCl immersion for 5 h. Teeth were sectioned into beams (1 +/- 0.1 mm) and tested to failure in tension at 0.5 mm/min. Bond strength data (MPa) were analyzed by ANOVA and SNK multiple-comparisons tests (p < 0.05). Results: When EDTA was used for pre-treatment of dentine, higher bond strengths were observed for both cements. Degradation challenges produced a decrease in bond strength values only in the Vitremer group. This decrease was avoided when EDTA was used for dentine treatment before restoring with Vitremer. Conclusions: EDTA pre-treatment of dentine increases bond strength of resin modified glass-ionomers cements to dentine and improves resistance to degradation of the bond between Vitremer and dentine. (C) 2009 Elsevier Ltd. All rights reserved.

Role of the spinal cord heme oxygenase-carbon monoxide-cGMP pathway in the nociceptive response of rats

The aim of the present study was to investigate the role of the spinal cord heme oxygenase (HO)-carbon monoxide (CO)-soluble guanylate cyclase (sGC)-cGMP pathway in nociceptive response of rats to the formalin experimental nociceptive model. Animals were handled and adapted to the experimental environment for a few days before the formalin test was applied. For the formalin test 50 mu l of a 1% formalin solution was injected subcutaneously in the dorsal surface of the right hind paw. Following injections, animals were observed for I h and flinching behavior was measured as the nociceptive response. Thirty min before the test, rats were pretreated with intrathecal injections with the HO inhibitor, zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG) or heme-lysinate, which is known to induce the HO pathway. Control animals were treated with vehicles. We observed a significant increase in nociceptive response of rats treated with ZnDPBG, and a drastic reduction of flinching nociceptive behavioral response in the heme-lysinate treated animals. Furthermore, the HO pathway seems to act via cGMP, since methylene blue (a sGC inhibitor) prevented the reduction of flinching nociceptive behavioral response caused by heme-lysinate. These findings strongly indicate that the HO pathway plays a spinal antinociceptive role during the formalin test, acting via cGMP. (C) 2007 Elsevier B.V. All rights reserved.