Glial cell activity is maintained during prolonged inflammatory challenge in rats

We evaluated the expression of glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), ionized calcium binding adaptor protein-1 (Iba-1), and ferritin in rats after single or repeated lipopolysaccharide (LPS) treatment, which is known to induce endotoxin tolerance and glial activation. Male Wistar rats (200-250 g) received ip injections of LPS (100 µg/kg) or saline for 6 days: 6 saline (N = 5), 5 saline + 1 LPS (N = 6) and 6 LPS (N = 6). After the sixth injection, the rats were perfused and the brains were collected for immunohistochemistry. After a single LPS dose, the number of GFAP-positive cells increased in the hypothalamic arcuate nucleus (ARC; 1 LPS: 35.6 ± 1.4 vs control: 23.1 ± 2.5) and hippocampus (1 LPS: 165.0 ± 3.0 vs control: 137.5 ± 2.5), and interestingly, 6 LPS injections further increased GFAP expression in these regions (ARC = 52.5 ± 4.3; hippocampus = 182.2 ± 4.1). We found a higher GS expression only in the hippocampus of the 6 LPS injections group (56.6 ± 0.8 vs 46.7 ± 1.9). Ferritin-positive cells increased similarly in the hippocampus of rats treated with a single (49.2 ± 1.7 vs 28.1 ± 1.9) or repeated (47.6 ± 1.1 vs 28.1 ± 1.9) LPS dose. Single LPS enhanced Iba-1 in the paraventricular nucleus (PVN: 92.8 ± 4.1 vs 65.2 ± 2.2) and hippocampus (99.4 ± 4.4 vs 73.8 ± 2.1), but had no effect in the retrochiasmatic nucleus (RCA) and ARC. Interestingly, 6 LPS increased the Iba-1 expression in these hypothalamic and hippocampal regions (RCA: 57.8 ± 4.6 vs 36.6 ± 2.2; ARC: 62.4 ± 6.0 vs 37.0 ± 2.2; PVN: 100.7 ± 4.4 vs 65.2 ± 2.2; hippocampus: 123.0 ± 3.8 vs 73.8 ± 2.1). The results suggest that repeated LPS treatment stimulates the expression of glial activation markers, protecting neuronal activity during prolonged inflammatory challenges.

A Novel Stress-Induced Sugarcane Gene Confers Tolerance to Drought, Salt and Oxidative Stress in Transgenic Tobacco Plants

Background: Drought is a major abiotic stress that affects crop productivity worldwide. Sugarcane can withstand periods of water scarcity during the final stage of culm maturation, during which sucrose accumulation occurs. Meanwhile, prolonged periods of drought can cause severe plant losses. Methodology/Principal Findings: In a previous study, we evaluated the transcriptome of drought-stressed plants to better understand sugarcane responses to drought. Among the up-regulated genes was Scdr1 (sugarcane drought-responsive 1). The aim of the research reported here was to characterize this gene. Scdr1 encodes a putative protein containing 248 amino acids with a large number of proline (19%) and cysteine (13%) residues. Phylogenetic analysis showed that ScDR1is in a clade with homologs from other monocotyledonous plants, separate from those of dicotyledonous plants. The expression of Scdr1 in different varieties of sugarcane plants has not shown a clear association with drought tolerance. Conclusions/Significance: The overexpression of Scdr1 in transgenic tobacco plants increased their tolerance to drought, salinity and oxidative stress, as demonstrated by increased photosynthesis, water content, biomass, germination rate, chlorophyll content and reduced accumulation of ROS. Physiological parameters, such as transpiration rate (E), net photosynthesis (A), stomatal conductance (gs) and internal leaf CO2 concentration, were less affected by abiotic stresses in transgenic Scdr1 plants compared with wild-type plants. Overall, our results indicated that Scdr1 conferred tolerance to multiple abiotic stresses, highlighting the potential of this gene for biotechnological applications.

Kinin B1 Receptor in Adipocytes Regulates Glucose Tolerance and Predisposition to Obesity

Background: Kinins participate in the pathophysiology of obesity and type 2 diabetes by mechanisms which are not fully understood. Kinin B-1 receptor knockout mice (B-1(-/-)) are leaner and exhibit improved insulin sensitivity. Methodology/Principal Findings: Here we show that kinin B-1 receptors in adipocytes play a role in controlling whole body insulin action and glucose homeostasis. Adipocytes isolated from mouse white adipose tissue (WAT) constitutively express kinin B-1 receptors. In these cells, treatment with the B-1 receptor agonist des-Arg(9)-bradykinin improved insulin signaling, GLUT4 translocation, and glucose uptake. Adipocytes from B-1(-/-) mice showed reduced GLUT4 expression and impaired glucose uptake at both basal and insulin-stimulated states. To investigate the consequences of these phenomena to whole body metabolism, we generated mice where the expression of the kinin B-1 receptor was limited to cells of the adipose tissue (aP2-B-1/B-1(-/-)). Similarly to B-1(-/-) mice, aP2-B-1/B-1(-/-) mice were leaner than wild type controls. However, exclusive expression of the kinin B1 receptor in adipose tissue completely rescued the improved systemic insulin sensitivity phenotype of B-1(-/-) mice. Adipose tissue gene expression analysis also revealed that genes involved in insulin signaling were significantly affected by the presence of the kinin B-1 receptor in adipose tissue. In agreement, GLUT4 expression and glucose uptake were increased in fat tissue of aP2-B-1/B-1(-/-) when compared to B-1(-/-) mice. When subjected to high fat diet, aP2-B-1/B-1(-/-) mice gained more weight than B-1(-/-) littermates, becoming as obese as the wild types. Conclusions/Significance: Thus, kinin B-1 receptor participates in the modulation of insulin action in adipocytes, contributing to systemic insulin sensitivity and predisposition to obesity.

Genetic variation of germination cold tolerance in Japanese rice germplasm

Low temperatures at the initial stages of rice development prevent fast germination and seedling establishment and may cause significant productivity losses. In order to develop rice cultivars exhibiting cold tolerance, it is necessary to investigate genetic resources, providing basic knowledge to allow the introduction of genes involved in low temperature germination ability from accessions into elite cultivars. Japanese rice accessions were evaluated at the germination under two conditions: 13 degrees C for 28 days (cold stress) and 28 degrees C for seven days (optimal temperature). The traits studied were coleoptile and radicle length under optimal temperature, coleoptile and radicle length under cold and percentage of the reduction in coleptile and radicle length due to low temperature. Among the accessions studied, genetic variation for traits related to germination under low temperatures was observed and accessions exhibiting adequate performance for all investigated traits were identified. The use of multivariate analysis allowed the identification of the genotypes displaying cold tolerance by smaller reductions in coleoptile and radicle lenght in the presence of cold and high vigour, by higher coleoptile and radicle growth under cold.

Studying the genetic basis of drought tolerance in sorghum by managed stress trials and adjustments for phenological and plant height differences

Managed environments in the form of well watered and water stressed trials were performed to study the genetic basis of grain yield and stay green in sorghum with the objective of validating previously detected QTL. As variations in phenology and plant height may influence QTL detection for the target traits, QTL for flowering time and plant height were introduced as cofactors in QTL analyses for yield and stay green. All but one of the flowering time QTL were detected near yield and stay green QTL. Similar co-localization was observed for two plant height QTL. QTL analysis for yield, using flowering time/plant height cofactors, led to yield QTL on chromosomes 2, 3, 6, 8 and 10. For stay green, QTL on chromosomes 3, 4, 8 and 10 were not related to differences in flowering time/plant height. The physical positions for markers in QTL regions projected on the sorghum genome suggest that the previously detected plant height QTL, Sb-HT9-1, and Dw2, in addition to the maturity gene, Ma5, had a major confounding impact on the expression of yield and stay green QTL. Co-localization between an apparently novel stay green QTL and a yield QTL on chromosome 3 suggests there is potential for indirect selection based on stay green to improve drought tolerance in sorghum. Our QTL study was carried out with a moderately sized population and spanned a limited geographic range, but still the results strongly emphasize the necessity of corrections for phenology in QTL mapping for drought tolerance traits in sorghum.

Omega-3 improves glucose tolerance but increases lipid peroxidation and DNA damage in hepatocytes of fructose-fed rats

The high consumption of fructose is linked to the increase in various characteristics of the metabolic syndrome. Fish oil is beneficial for the treatment of these comorbidities, such as insulin resistance, dyslipidemia, and hepatic steatosis. The objective of this study was to evaluate the consequences of the administration of fish oil concomitant to fructose ingestion during the experiment (45 days) and during the final 15 days in high-fructose-fed rats. Male Wistar rats were divided into 5 groups: control; those receiving 10% fish oil (FO); those receiving 60% fructose (Fr); those receiving 60% fructose and 10% fish oil for 45 days (FrFO); and those receiving fructose plus soybean oil for 30 days and fish oil for the final 15 days of the study (FrFO15). There was an increase in triacylglycerol, serum total cholesterol, and hepatic volume in the Fr group. The FO and FrFO groups experienced an increase in lipid peroxidation and a decrease in serum reduced glutathione. The FrFO group suffered greater hepatic injury, with increased alanine aminotransferase levels and DNA damage. Marked n-3 incorporation occurred in the groups receiving fish oil, favoring a better response to the oral glucose tolerance test. Fructose induced comorbidities of the metabolic syndrome, and the use of fish oil promoted a better glucose tolerance, although it was accompanied by more hepatocyte damage.

Cellular Infiltrates and NF kappa B Subunit c-Rel Signaling in Kidney Allografts of Patients With Clinical Operational Tolerance

Background. Nuclear factor kappa B (NF kappa B) plays a potential role in tolerance by orchestrating onset and resolution of inflammation and regulatory T cell differentiation through subunit c-Rel. We characterized cellular infiltrates and expression of NF kappa B1, c-Rel and its upstream regulators phosphatidylinositol 3-kinase/RAC-alpha serine/threonine kinase, in allograft biopsies from patients with spontaneous clinical operational tolerance (COT). Methods. Paraffin-fixed kidney allograft biopsies from 40 patients with COT (n=4), interstitial rejection (IR; n=12), borderline changes (BC; n=12), and long-term allograft function without rejection (NR; n=12) were used in the study. Cellular infiltrates and immunohistochemical expression of key proteins of the NF kappa B pathway were evaluated in the cortical tubulointerstitium and in cellular infiltrates using digital image analysis software. Results were given as mean +/- SEM. Results. Biopsies from patients with COT exhibited a comparable amount of cellular infiltrate to IR, BC, and NR (COT, 191 +/- 81; IR, 291 +/- 62; BC, 178 +/- 45; and NR, 210 +/- 42 cells/mm(2)) but a significantly higher proportion of forkhead box P3-positive cells (COT, 11%+/- 1.7%; IR, 3.5%+/- 0.70%; BC, 3.4%+/- 0.57%; and NR, 3.7%+/- 0.78% of infiltrating cells; P=0.02). c-Rel expression in cellular infiltrates was significantly elevated in IR, BC, and NR when analyzing the number of positive cells per mm(2) (P=0.02) and positive cells per infiltrating cells (P=0.04). In contrast, tubular PI3K and c-Rel expression were significantly higher in IR and BC but not in NR compared with COT (P=0.03 and P=0.006, respectively). With RAC-alpha serine-threonine kinase, similar tendencies were observed (P=0.2). Conclusions. Allografts from COT patients show significant cellular infiltrates but a distinct expression of proteins involved in the NF kappa B pathway and a higher proportion of forkhead box P3-positive cells.

Preserving the B-Cell Compartment Favors Operational Tolerance in Human Renal Transplantation

Transplanted individuals in operational tolerance (OT) maintain long-term stable graft function after completely stopping immunosuppression. Understanding the mechanisms involved in OT can provide valuable information about pathways to human transplantation tolerance. Here we report that operationally tolerant individuals display quantitative and functional preservation of the B-c ell compartment in renal transplantation. OT exhibited normal numbers of circulating total B cells, naive, memory and regulatory B cells (Bregs) as well as preserved B-cell receptor repertoire, similar to healthy individuals. In addition, OT also displayed conserved capacity to activate the cluster of differentiation 40 (CD40)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in Bregs, in contrast, with chronic rejection. Rather than expansion or higher activation, we show that the preservation of the B-cell compartment favors OT. Online address: http://www.molmed.org doi: 10.2119/molmed.2011.00281

ROLE OF FOCAL ADHESION KINASE IN LUNG REMODELING OF ENDOTOXEMIC RATS

Despite significant advances in the care of critically ill patients, acute lung injury continues to be a complex problem with high mortality. The present study was designed to characterize early lipopolysaccharide (LPS)-induced pulmonary injury and small interfering RNA targeting focal adhesion kinase (FAK) as a possible therapeutic tool in the septic lung remodeling process. Male Wistar rats were assigned into endotoxemic group and control group. Total collagen deposition was performed 8, 16, and 24 h after LPS injection. Focal adhesion kinase expression, interstitial and vascular collagen deposition, and pulmonary mechanics were analyzed at 24 h. Intravenous injection of small interfering RNA targeting FAK was used to silence expression of the kinase in pulmonary tissue. Focal adhesion kinase, total collagen deposition, and pulmonary mechanics showed increased in LPS group. Types I, III, and V collagen showed increase in pulmonary parenchyma, but only type V increased in vessels 24 h after LPS injection. Focal adhesion kinase silencing prevented lung remodeling in pulmonary parenchyma at 24 h. In conclusion, LPS induced a precocious and important lung remodeling. There was fibrotic response in the lung characterized by increased amount in total and specific-type collagen. These data may explain the frequent clinical presentation during sepsis of reduced lung compliance, oxygen diffusion, and pulmonary hypertension. The fact that FAK silencing was protective against lung collagen deposition underscores the therapeutic potential of FAK targeting by small interfering RNA.

Prediction of tolerance in children with IgE mediated cow's milk allergy by microarray profiling and chemometric approach

The sera of a retrospective cohort (n = 41) composed of children with well characterized cow's milk allergy collected from multiple visits were analyzed using a protein microarray system measuring four classes of immunoglobulins. The frequency of the visits, age and gender distribution reflected real situation faced by the clinicians at a pediatric reference center for food allergy in 530 Paulo, Brazil. The profiling array results have shown that total IgG and IgA share similar specificity whilst IgM and in particular IgE are distantly related. The correlation of specificity of IgE and IgA is variable amongst the patients and this relationship cannot be used to predict atopy or the onset of tolerance to milk. The array profiling technique has corroborated the clinical selection criteria for this cohort albeit it clearly suggested that 4 out of the 41 patients might have allergies other than milk origin. There was also a good correlation between the array data and ImmunoCAP results, casein in particular. By using qualitative and quantitative multivariate analysis routines it was possible to produce validated statistical models to predict with reasonable accuracy the onset of tolerance to milk proteins. If expanded to larger study groups, the array profiling in combination with the multivariate techniques show potential to improve the prognostic of milk allergic patients. (C) 2012 Elsevier B.V. All rights reserved.

Ventilatory threshold is related to walking tolerance in patients with intermittent claudication

Background: This study assessed the relationship between lower limb hemodynamics and metabolic parameters with walking tolerance in patients with intermittent claudication (IC). Patients and methods: Resting ankle-brachial index (ABI), baseline blood flow (BF), BF response to reactive hyperemia (BFRH), oxygen uptake (VO2), initial claudication distance (ICD) and total walking distance (TWD) were measured in 28 IC patients. Pearson and Spearman correlations were calculated. Results: ABI, baseline BF and BF response to RH did not correlate with ICD or TWD. VO2 at first ventilatory threshold and VO(2)peak were significantly and positively correlated with ICD (r = 0.41 and 0.54, respectively) and TWD (r = 0.65 and 0.71, respectively). Conclusions: VO(2)peak and VO2 at first ventilatory threshold, but not ABI, baseline BF and BFHR were associated with walking tolerance in IC patients. These results suggest that VO2 at first ventilatory threshold may be useful to evaluate walking tolerance and improvements in IC patients.

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.

Increasing Intensity of TENS Prevents Analgesic Tolerance in Rats

Transcutaneous electrical nerve stimulation (TENS) reduces hyperalgesia and pain. Both low-frequency (LF) and high-frequency (HF) TENS, delivered at the same intensity (90% motor threshold [MT]) daily, result in analgesic tolerance with repeated use by the fifth day of treatment. The current study tested 1) whether increasing intensity by 10% per day prevents the development of tolerance to repeated TENS; and 2) whether lower intensity TENS (50% MT) produces an equivalent reduction in hyperalgesia when compared to 90% MT TENS. Sprague-Dawley rats with unilateral knee joint inflammation (3% carrageenan) were separated according to the intensity of TENS used: sham, 50% LF, 50% HF, 90% LF, 90% HF, and increased intensity by 10% per day (IF and HF). The reduced mechanical withdrawal threshold following the induction of inflammation was reversed by application of TENS applied at 90% MT intensity and increasing intensity for the first 4 days. On the fifth day, the groups that received 90% MT intensity showed tolerance. Nevertheless, the group that received an increased intensity on each day still showed a reversal of the mechanical withdrawal threshold with TENS. These results show that the development of tolerance can be delayed by increasing intensity of TENS. Perspective: Our results showed that increasing intensity in both frequencies of TENS was able to prevent analgesic tolerance. Results from this study suggest that increasing intensities could be a clinical method to prevent analgesic tolerance and contribute to the effective use of TENS in reducing inflammatory pain and future clinical trials. (c) 2012 by the American Pain Society

Ectopic expression of a fruit phytoene synthase from Citrus paradisi Macf. promotes abiotic stress tolerance in transgenic tobacco

Abscisic acid (ABA) is an important regulator of plant responses to environmental stresses and an absolute requirement for stress tolerance. Recently, a third phytoene synthase (PSY3) gene paralog was identified in monocots and demonstrated to play a specialized role in stress-induced ABA formation, thus suggesting that the first committed step in carotenogenesis is a key limiting step in ABA biosynthesis. To examine whether the ectopic expression of PSY, other than PSY3, would similarly affect ABA level and stress tolerance, we have produced transgenic tobacco containing a fruit-specific PSY (CpPSY) of grapefruit (Citrus paradisi Macf.). The transgenic plants contained a single- or double-locus insertion and expressed CpPSY at varying transcript levels. In comparison with the wild-type plants, the CpPSY expressing transgenic plants showed a significant increase on root length and shoot biomass under PEG-, NaCl- and mannitol-induced osmotic stress. The enhanced stress tolerance of transgenic plants was correlated with the increased endogenous ABA level and expression of stress-responsive genes, which in turn was correlated with the CpPSY copy number and expression level in different transgenic lines. Collectively, these results provide further evidence that PSY is a key enzyme regulating ABA biosynthesis and that the altered expression of other PSYs in transgenic plants may provide a similar function to that of the monocot's PSY3 in ABA biosynthesis and stress tolerance. The results also pave the way for further use of CpPSY, as well as other PSYs, as potential candidate genes for engineering tolerance to drought and salt stress in crop plants.

Monocyte-derived dendritic cells from breast cancer patients are biased to induce CD4(+)CD25(+)Foxp3(+) regulatory T cells

DCs orchestrate immune responses contributing to the pattern of response developed. In cancer, DCs may play a dysfunctional role in the induction of CD4(+)CD25(+) Foxp3(+) Tregs, contributing to immune evasion. We show here that Mo-DCs from breast cancer patients show an altered phenotype and induce preferentially Tregs, a phenomenon that occurred regardless of DC maturation stimulus (sCD40L, cytokine cocktail, TNF-alpha, and LPS). The Mo-DCs of patients induced low proliferation of allogeneic CD3(+)CD25(neg)Foxp3(neg) cells, which after becoming CD25(+), suppressed mitogen-stimulated T cells. Contrastingly, Mo-DCs from healthy donors induced a stronger proliferative response, a low frequency of CD4(+)CD25(+)Foxp3(+) with no suppressive activity. Furthermore, healthy Mo-DCs induced higher levels of IFN-gamma, whereas the Mo-DCs of patients induced higher levels of bioactive TGF-beta 1 and IL-10 in cocultures with allogeneic T cells. Interestingly, TGF-beta 1 blocking with mAb in cocultures was not enough to completely revert the Mo-DCs of patients' bias toward Treg induction. Altogether, these findings should be considered in immunotherapeutic approaches for cancer based on Mo-DCs. J. Leukoc. Biol. 92: 673-682; 2012.