TLR2 and TLR4 expression after kidney ischemia and reperfusion injury in mice treated with FTY720

Ischemia and reperfusion injury (IR) is an antigen independent inflammatory process that causes tissue damage. After IR, kidneys up-regulate leukocyte adhesion molecules and toll-like receptors (TLRs). Moreover, injured kidneys can also secrete factors (i.e. heat shock protein) which bind to TLRs and trigger intracellular events culminating with the increase in the gene expression of inflammatory cytokines. FTY720 is an immunomodulatory compound and protects at least in part kidneys submitted to IR. The mechanisms associated with FTY720`s beneficial effects on kidneys after IR remain elusive. We investigated whether FTY720 administration in mice submitted to kidney IR is associated with modulation of TLR2 and TLR4 expression. C57BL/6 mice submitted to 30 min of renal pedicles clamp were evaluated for serum parameters (creatinine, urea and nitric oxide), kidney histology, spleen and kidney infiltrating cells expression of TLR2 and TLR4, resident kidney cells expression of TLR2 and TLR4 and IL-6 protein expression in kidney. FTY720-treated mice presented decrease in serum creatinine, urea and nitric oxide, diminished expression of TLR2 and TLR4 both in spleen and kidney infiltrating cells, and reduced kidney IL-6 protein expression in comparison with IR non-treated mice. However, acute tubular necrosis was present both in IR non-treated and IR + FTY720-treated groups. Also, FTY720 did not prevent TLR2 and TLR4 expression in kidney resident cells. In conclusion, FTY720 can promote kidney function recovery after IR by reducing the inflammatory process. Further studies are needed in order to establish whether TLR2 and TLR4 down regulation should be therapeutically addressed as protective targets of renal function and structure after IR. (C) 2011 Elsevier B.V. All rights reserved.

Serum amyloid A induces reactive oxygen species (ROS) production and proliferation of fibroblast

Serum amyloid A (SAA) levels are elevated highly in acute phase response and elevated slightly and persistently in chronic diseases such as rheumatoid arthritis and diabetes. Given that fibroblasts exert profound effects on progression of inflammatory chronic diseases, the aim of this study was to investigate the response of fibroblasts to SAA. A dose-dependent increase in O(2)(-) levels was observed by treatment of fibroblasts with SAA (r = 0.99 and P <= 0.001). In addition, the expression of p47-phox was up-regulated by SAA (P < 0.001) and diphenyliodonium (DPI), a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, reduced the release of O(2)(-) by 50%. Also, SAA raised fibroblast proliferation (P < 0.001) and this effect was completely abolished by the addition of anti-oxidants (P < 0.001). These findings support the notion that, in chronic inflammatory sites, SAA activated fibroblast proliferation and ROS production.

Moderate exercise improves leucocyte function and decreases inflammation in diabetes

P>The genesis and progression of diabetes occur due in part to an uncontrolled inflammation profile with insulin resistance, increased serum levels of free fatty acids (FFA), proinflammatory cytokines and leucocyte dysfunction. In this study, an investigation was made of the effect of a 3-week moderate exercise regimen on a treadmill (60% of VO(2max), 30 min/day, 6 days a week) on inflammatory markers and leucocyte functions in diabetic rats. The exercise decreased serum levels of tumour necrosis factor (TNF)-alpha (6%), cytokine-induced neutrophil chemotactic factor 2 alpha/beta (CINC-2 alpha/beta) (9%), interleukin (IL)-1 beta (34%), IL-6 (86%), C-reactive protein (CRP) (41%) and FFA (40%) in diabetic rats when compared with sedentary diabetic animals. Exercise also attenuated the increased responsiveness of leucocytes from diabetics when compared to controls, diminishing the reactive oxygen species (ROS) release by neutrophils (21%) and macrophages (28%). Exercise did not change neutrophil migration and the proportion of neutrophils and macrophages in necrosis (loss of plasma membrane integrity) and apoptosis (DNA fragmentation). Serum activities of creatine kinase (CK) and lactate dehydrogenase (LDH) were not modified in the conditions studied. Therefore, physical training did not alter the integrity of muscle cells. We conclude that moderate physical exercise has marked anti-inflammatory effects on diabetic rats. This may be an efficient strategy to protect diabetics against microorganism infection, insulin resistance and vascular complications.

Effects of single or repeated amphetamine treatment and withdrawal on lung allergic inflammation in rats

The effects of single or repeated amphetamine (AMPH) treatment and those of AMPH withdrawals on immune-mediated lung inflammatory response were studied in rats. Two experiments were done. In the first, rats egg-albumin (OVA) sensitized were singularly or repeatedly (21 days, once daily) treated with AMPH (1.0 mg/kg) or with a similar number and volume of 0.9% NaCl. The OVA aerosol challenge was performed 12 h after the single or last repeated AMPH treatment and also 72 and 120 h after AMPH withdrawal. In the second experiment, the effects of reserpine (1.0 mg/kg/day for 5 consecutive days) on single AMPH actions on lung allergic response of rats were analyzed. Single and repeated AMPH treatment induced opposite actions on Bronchoalveolar lavage fluid (BAL) cellularity of allergic rats: single treatment decreased and repeated treatment increased the total number of cells as well as those of macrophages, neutrophils and eosinophils. Our data also showed that single but not repeated AMPH treatment decreased the number of neutrophils, monocytes and lymphocytes in the peripheral blood, and increased the total number of bone marrow cells in rats sensitized and challenged with OVA. Furthermore, it was shown that reserpine treatment precluded the effects of single AMPH treatment on cellular migration to the lung of OVA-sensitized and challenged rats. It was concluded that AMPH effects on lung inflammatory response and cell recruitment to the lung in allergic rats rely at least partially on corticosterone serum levels. The possible involvement of vesicular monoamine transporter type 2 (VMAT2) with these observed effects was discussed. (c) 2008 Elsevier B.V. All rights reserved.

PAS-1, an Ascaris suum Protein, Modulates Allergic Airway Inflammation via CD8(+) gamma delta TCR(+) and CD4(+) CD25(+) FoxP3(+) T Cells

We have previously demonstrated that PAS-1, a 200 kDa protein from Ascaris suum, has a potent immunomodulatory effect on humoral and cell-mediated responses induced by APAS-3 (an allergenic protein from A. suum) or unrelated antigens. In this study, we investigated the mechanisms by which PAS-1 is able to induce this effect on an allergic airway inflammation induced by OVA in mice. C57BL/6 mice were adoptively transferred on day 0 with seven different PAS-1-primed cell populations: PAS-1-primed CD19(+) or B220(+) or CD3(+) or CD4(+) or CD8(+) or CD4(+) CD25) or CD4(+) CD25(+) lymphocytes. These mice were immunized twice with OVA and alum by intraperitoneal route (days 0 and 7) and challenged twice by intranasal route (days 14 and 21). Two days after the last challenge, the airway inflammation was evaluated by antibody levels, cellular migration, eosinophil peroxidase levels, cytokine and eotaxin production, and pulmonary mechanical parameters. Among the adoptively transferred primed lymphocytes, only CD4(+) CD25(+), CD8(+) or the combination of both T cells impaired the production of total IgE and OVA-specific IgE and IgG1 antibodies, eosinophilic airway inflammation, Th2-type cytokines (IL-4, IL-5 and IL-13), eotaxin release and airway hyperreactivity. Moreover, airway recruited cells from CD4(+) CD25(+) and CD8(+) T-cell recipient secreted more IL-10/TGF-beta and IFN-gamma, respectively. Moreover, we found that PAS-1 expands significantly the number of CD4(+) CD25(+) FoxP3(+) and CD8(+) gamma delta TCR(+) cells. In conclusion, these findings demonstrate that the immunomodulatory effect of PAS-1 is mediated by these T-cell subsets.

Lung inflammation is induced by renal ischemia and reperfusion injury as part of the systemic inflammatory syndrome

Ischemia and reperfusion injury (IRI) are mainly caused by leukocyte activation, endothelial dysfunction and production of reactive oxygen species. Moreover, IRI can lead to a systemic response affecting distant organs, such as the lungs. The objective was to study the pulmonary inflammatory systemic response after renal IRI. Male C57Bl/6 mice were subjected to 45 min of bilateral renal ischemia, followed by 4, 6, 12, 24 and 48 h of reperfusion. Blood was collected to measure serum creatinine and cytokine concentrations. Bronchoalveolar lavage fluid (BALF) was collected to determine the number of cells and PGE(2) concentration. Expressions of iNOS and COX-2 in lung were determined by Western blot. Gene analyses were quantified by real time PCR. Serum creatinine increased in the IRI group compared to sham mainly at 24 h after IRI (2.57 +/- A 0.16 vs. 0.43 +/- A 0.07, p < 0.01). The total number of cells in BAL fluid was higher in the IRI group in comparison with sham, 12 h (100 x 10(4) +/- A 15.63 vs. 18.1x10(4) +/- A 10.5, p < 0.05) 24 h (124 x 10(4) +/- A 8.94 vs. 23.2x10(4) +/- A 3.5, p < 0.05) and 48 h (79 x 10(4) +/- A 15.72 vs. 22.2 x 10(4) +/- A 4.2, p < 0.05), mainly by mononuclear cells and neutrophils. Pulmonary COX-2 and iNOS were up-regulated in the IRI group. TNF-alpha, IL-1 beta, MCP-1, KC and IL-6 mRNA expression were up-regulated in kidney and lungs 24 h after renal IRI. ICAM-1 mRNA was up-regulated in lungs 24 h after renal IRI. Serum TNF-alpha, IL-1 beta and MCP-1 and BALF PGE(2) concentrations were increased 24 h after renal IRI. Renal IRI induces an increase of cellular infiltration, up-regulation of COX-2, iNOS and ICAM-1, enhanced chemokine expression and a Th1 cytokine profile in lung demonstrating that the inflammatory response is indeed systemic, possibly leading to an amplification of renal injury.

Early modulation of inflammation by mesenchymal stem cell after acute kidney injury

Therapy with stem cells has showed to be promising for acute kidney injury (AKI), although how it works is still controversial. Modulation of the inflammatory response is one possible mechanism. Most of published data relies on early time and whether the protection is still maintained after that is not known. Here, we analyzed whether immune modulation continues after 24 h of reperfusion. MSC were obtained from male Wistar rats. After 3-5 passages, cells were screened for CD73, CD90, CD44, CD45, CD29 and CD 31. In addition, MSC were submitted to differentiation in adipocyte and in osteocyte. AKI was induced by bilaterally clamping of renal pedicles for 60 min. Six hours after injury, MSC (2 x 105 cells) were administered intravenously. MSC-treated animals presented the lowest serum creatinine compared to non-treated animals (24 h: 1.3 +/- 0.21 vs. 3.23 +/- 0.89 mg/dl, p<0.05). The improvement in renal function was followed by a lower expression of IL-1b, IL-6 and TNF-alpha and higher expression of IL-4 and IL-10. However, 48 h after reperfusion, this cytokine profile has changed. The decrease in Th1 cytokines was less evident and IL-6 was markedly up regulated. PCNA analysis showed that regeneration occurs faster in kidney tissues of MSC-treated animals than in controls at 24 h. And also ratio of Bcl-2/Bad was higher at treated animals after 24 and 48 h. Our data demonstrated that the immunomodulatory effects of MSC occur at very early time point, changing the inflammation profile toward a Th2 profile. (C) 2009 Elsevier B.V. All rights reserved.

PAS-1, a protein from Ascaris suum, modulates allergic inflammation via IL-10 and IFN-gamma, but not IL-12

Helminths and their products have a profound immunomodulatory effect upon the inductive and effector phases of inflammatory responses, including allergy. We have demonstrated that PAS-1, a protein isolated from Ascaris strum worms, has an inhibitory effect on lung allergic inflammation due to its ability to down-regulate eosinophilic inflammation, Th2 cytokine release and IgE antibody production. Here, we investigated the role of IL-12, IFN-gamma and IL-10 in the PAS-1-induced inhibitory mechanism using a murine model of asthma. Wild type C57BL/6, IL-12(-/-), IFN-gamma(-/-) and IL-10(-/-) mice were immunized with PAS-1 and/or OVA and challenged with the same antigens intranasally. The suppressive effect of PAS-I was demonstrated on the cellular influx into airways, with reduction of eosinophil number and eosinophil peroxidase activity in OVA + PAS-1-immunized wild type mice. This effect well correlated with a significant reduction in the levels of IL-4, IL-5, IL-13 and eotaxin in BAL fluid. Levels of IgE and IgG1 antibodies were also impaired in serum from these mice. The inhibitory activity of PAS-I was also observed in IL-12(-/-) mice, but not in IFN-gamma(-/-) and IL-10(-/-) animals. These data show that IFN-gamma and IL-10, but not IL-12, play an important role in the PAS-1 modulatory effect. (C) 2008 Elsevier Ltd. All rights reserved.

Low level laser therapy (LLLT) decreases pulmonary microvascular leakage, neutrophil influx and IL-1 beta levels in airway and lung from rat subjected to LPS-induced inflammation

Background and Objective. Low level laser therapy (LLLT) is a known anti-inflammatory therapy. Herein we studied the effect of LLLT on lung permeability and the IL-1 beta level in LPS-induced pulmonary inflammation. Study Design/Methodology. Rats were divided into 12 groups (n = 7 for each group). Lung permeability was measured by quantifying extravasated albumin concentration in lung homogenate, inflammatory cells influx was determined by myeloperoxidase activity, IL-1P in BAL was determined by ELISA and IL-1P mRNA expression in trachea was evaluated by RT-PCR. The rats were irradiated on the skin over the upper bronchus at the site of tracheotomy after LPS. Results. LLLT attenuated lung permeability. In addition, there was reduced neutrophil influx, myeloperoxidase activity and both IL-1 beta in BAL and IL-1 beta mRNA expression in trachea obtained from animals subjected to LPS-induced inflammation. Conclusion. LLLT reduced the lung permeability by a mechanism in which the IL-1 beta seems to have an important role.

Effects of supplementation with free glutamine and the dipeptide alanyl-glutamine on parameters of muscle damage and inflammation in rats submitted to prolonged exercise

In this study, we investigated the effect of the supplementation with the dipeptide L-alanyl-L-glutamine (DIP) and a solution containing L-glutamine and L-alanine on plasma levels markers of muscle damage and levels of pro-inflammatory cytokines and glutamine metabolism in rats submitted to prolonged exercise. Rats were submitted to sessions of swim training for 6 weeks. Twenty-one days prior to euthanasia, the animals were supplemented with DIP (n = 8) (1.5 g.kg(-1)), a solution of free L-glutamine (1 g.kg(-1)) and free L-alanine (0.61 g.kg(-1)) (G&A, n = 8) or water (control (CON), n = 8). Animals were killed at rest before (R), after prolonged exercise (PE-2 h of exercise). Plasma concentrations of glutamine, glutamate, tumour necrosis factor-alpha (TNF-alpha), prostaglandin E2 (PGE2) and activity of creatine kinase (CK), lactate dehydrogenase (LDH) and muscle concentrations Of glutamine and glutamate were measured. The concentrations of plasma TNF-alpha, PGE2 and the activity of CK were lower in the G&A-R and DIP-R groups, compared to the CON-R. Glutamine in plasma (p < 0.04) and soleus muscle (p < 0.001) was higher in the DIP-R and G&A-R groups relative to the CON-R group. G&A-PE and DIP-PE groups exhibited lower concentrations of plasma PGE2 (p < 0.05) and TNF-alpha (p < 0.05), and higher concert I rations of glutamine and glutamate in soleus (p < 0.001) and gastrocnemius muscles (p < 0.05) relative to the CON-PE group. We concluded that supplementation with free L-glutamine and the dipeptide LL-alanyl-LL-glutamine represents an effective source of glutamine, which may attenuate inflammation biomarkers after periods of training and plasma levels of CK and the inflammatory response induced by prolonged exercise. Copyright (C) 2009 John Wiley & Sons, Ltd.

The Genetics of Alzheimer`s Disease in Brazil: 10 Years of Analysis in a Unique Population

Alzheimer`s Disease (AD) is the most common type of dementia among the elderly, with devastating consequences for the patient, their relatives, and caregivers. More than 300 genetic polymorphisms have been involved with AD, demonstrating that this condition is polygenic and with a complex pattern of inheritance. This paper aims to report and compare the results of AD genetics studies in case-control and familial analysis performed in Brazil since our first publication, 10 years ago. They include the following genes/markers: Apolipoprotein E (APOE), 5-hidroxytryptamine transporter length polymorphic region (5-HTTLPR), brain-derived neurotrophin factor (BDNF), monoamine oxidase A (MAO-A), and two simple-sequence tandem repeat polymorphisms (DXS1047 and D10S1423). Previously unpublished data of the interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta) genes are reported here briefly. Results from others Brazilian studies with AD patients are also reported at this short review. Four local families studied with various markers at the chromosome 21, 19, 14, and 1 are briefly reported for the first time. The importance of studying DNA samples from Brazil is highlighted because of the uniqueness of its population, which presents both intense ethnical miscegenation, mainly at the east coast, but also clusters with high inbreeding rates in rural areas at the countryside. We discuss the current stage of extending these studies using high-throughput methods of large-scale genotyping, such as single nucleotide polymorphism microarrays, associated with bioinformatics tools that allow the analysis of such extensive number of genetics variables, with different levels of penetrance. There is still a long way between the huge amount of data gathered so far and the actual application toward the full understanding of AD, but the final goal is to develop precise tools for diagnosis and prognosis, creating new strategies for better treatments based on genetic profile.

Structural changes in the epithelium of the small intestine and immune cell infiltration of enteric ganglia following acute mucosal damage and local inflammation

An acute enteritis is commonly followed by intestinal neuromuscular dysfunction, including prolonged hyperexcitability of enteric neurons. Such motility disorders are associated with maintained increases in immune cells adjacent to enteric ganglia and in the mucosa. However, whether the commonly used animal model, trinitrobenzene sulphonate (TNBS)-induced enteritis, causes histological and immune cell changes similar to human enteric neuropathies is not clear. We have made a detailed study of the mucosal damage and repair and immune cell invasion following intralumenal administration of TNBS. Intestines from untreated, sham-operated and TNBS-treated animals were examined at 3 h to 56 days. At 3 h, the mucosal surface was completely ablated, by 6 h an epithelial covering was substantially restored and by 1 day there was full re-epithelialisation. The lumenal epithelium developed from a squamous cell covering to a fully differentiated columnar epithelium with mature villi at about 7 days. Prominent phagocytic activity of enterocytes occurred at 1-7 days. A surge of eosinophils and T lymphocytes associated with the enteric nerve ganglia occurred at 3 h to 3 days. However, elevated immune cell numbers occurred in the lamina propria of the mucosa until 56 days, when eosinophils were still three times normal. We conclude that the disruption of the mucosal surface that causes TNBS-induced ileitis is brief, a little more than 6 h, and causes a transient immune cell surge adjacent to enteric ganglia. This is much briefer than the enteric neuropathy that ensues. Ongoing mucosal inflammatory reaction may contribute to the persistence of enteric neuropathy.

Beneficial Effects of Physical Training on the Cardio-Inflammatory Disorder Induced by Lung Ischemia/Reperfusion in Rats

Our laboratory demonstrated that training program attenuated the inflammatory responses in lung ischemia/reperfusion (IR). Considering the importance of the inflammatory responses on the cardiovascular system, we evaluate the effect of physical training on the vascular responsiveness and its underlying mechanism after lung IR. Male Wistar rats were submitted to run training and lung IR. Concentration-response curves for relaxing and contracting agents were obtained. Protein expressions of SOD-1 and p47(phox), plasma nitritre/nitrate (NO (x) (-) ) and interleukin 6 (IL-6) were evaluated. A decreased in the potency for acetylcholine and phenylephrine associated with an upregulation of the p47(phox) expression were found after Lung IR as well as an increase in IL-6 and NO (x) (-) levels. Run training attenuated the vascular dysfunction that was accompanied by reduction of the p47(phox) protein expression and IL-6 levels. Our findings show the beneficial effect of training on the vascular function that was associated with reduction in inflammatory response in lung IR.

Effect of medium/omega-6 long chain triglyceride-based emulsion on leucocyte death and inflammatory gene expression

Lipid emulsion (LE) containing medium/omega-6 long chain triglyceride-based emulsion (MCT/omega-6 LCT LE) has been recommended in the place of omega-6 LCT-based emulsion to prevent impairment of immune function. The impact of MCT/omega-6 LCT LE on lymphocyte and neutrophil death and expression of genes related to inflammation was investigated. Seven volunteers were recruited and infusion of MCT/omega-6 LCT LE was performed for 6 h. Four volunteers received saline and no change was found. Blood samples were collected before, immediately afterwards and 18 h after LE infusion. Lymphocytes and neutrophils were studied immediately after isolation and after 24 and 48 h in culture. The following determinations were carried out: plasma-free fatty acids, triacylglycerol and cholesterol concentrations, plasma fatty acid composition, neutral lipid accumulation in lymphocytes and neutrophils, signs of lymphocyte and neutrophil death and lymphocyte expression of genes related to inflammation. MCT/omega-6 LCT LE induced lymphocyte and neutrophil death. The mechanism for MCT/omega-6 LCT LE-dependent induction of leucocyte death may involve changes in neutral lipid content and modulation of expression of genes related to cell death, proteolysis, cell signalling, inflammatory response, oxidative stress and transcription.

Innate immune response in the sea urchin Echinometra lucunter (Echinodermata)

Echinometra lucunter, (Pinda) is a sea urchin encountered in the Brazilian coast and exposed to high and low temperatures related to low and high tides. Despite their great distribution and importance, few studies have been done on the biological function of their coelomocytes. Thus, Echinometra lucunter perivisceral coelomocytes were characterized under optical and transmission electron microscopy. Phagocytic amoebocytes in the perivisceral coelom were labelled by injecting ferritin, and ferritin labelled phagocytic amoebocytes were found in the peristomial connective tissue after injecting India ink into the tissue, indicating the amoebocytes ability to respond to an inflammatory stimulus. Results showed that the phagocytic amoebocytes were the main inflammatory cells found in the innate immune response of E lucunter. While other works have recorded these phenomena in sea urchins found in moderate and constant temperature, this study reports on these same phenomena in a tropical sea urchin under great variation of temperature, thus providing new data to inflammatory studies in invertebrate pathology. (C) 2007 Elsevier Inc. All rights reserved.