mRNA profile of Nellore calves after primary infection with Haemonchus placei

Haemonchus parasites are responsible for many losses in animal production. However, few studies are available, especially of zebu cattle. In this study, we investigated mRNA differences of immune response genes in naive Nellore calves infected with Haemonchus placei, relating these differences to patterns of cellular infiltrate. Calves were infected with 15,000 H. placei 13 larvae and after 7 days lymph node and abomasum tissues were collected. IL-2, IL-4, IL-8, IL-12, IL-13, IFN-gamma, MCP-1, lysozyme, pepsinogen and TNF-alpha genes were evaluated by qPCR. Mast cells, eosinophils and globular leukocytes were counted by abomasum histology. In the infected group, IL-4, IL-13 and TNF-alpha were up-regulated in the abomasal lymph node. In the abomasum, IL-13 increased and TNF-alpha was down-regulated (p < 0.05). No differences were detected for mast cells and eosinophil counts in abomasal tissue (p > 0.05). We conclude that for this infection time, there was Th2 polarization but that cellular infiltrate in abomasal tissue takes longer to develop. (C) 2010 Elsevier B.V. All rights reserved.

Increased electronegative LDL and decreased antibodies against electronegative LDL levels correlate with inflammatory markers and adhesion molecules in hemodialysed patients

Background: Chronic Kidney Disease (CKD) patients present high levels of electronegative LDL (LDL) that can modulate the expression of molecules involved in inflammation and it is closely linked to atherosclerosis. We investigated the association between LDL(-) and inflammatory markers in patients undergoing hemodialysis (HD). Methods: Forty-seven HD patients from a private clinic in Rio de Janeiro, Brazil were studied and compared with 20 age matched healthy individuals. Serum LDL(-) and anti-LDL(-) autoantibody levels were measured by ELISA; TNF-alpha, IL-6, VCAM-1 and ICAM-1 were determined by a multiplex assay kit. Results: HD patients presented higher IL-6 and TNF-alpha concentrations (4.1 +/- 1.6 and 5.5 +/- 2.1 pg/ml, respectively) than healthy subjects (2.6 +/- 0.2 and 2.4 +/- 1.1 pg/ml, respectively) (p = 0.0001). In addition, they presented higher VCAM-1 and ICAM-1 levels and, LDL(-) concentrations were also increased (0.18 +/- 0.12 U/I) when compared to healthy individuals (0.10 +/- 0.08 U/I) (p<0.02). In contrast, the anti-LDL(-) autoantibody levels were lower in HD patients (0.02 +/- 0.01 mg/l) than in healthy subjects (0.05 +/- 0.03 mg/l) (p<0.001). There was a positive correlation between LDL(-) and IL-6 (r = 0.25, p = 0.004) and ICAM-1 (r = 0.36; p = 0.003). There was also a negative correlation between anti-LDL(-) autoantibodies and TNF-alpha (r = -0.37; p = 0.003) and VCAM-1 (r = -0.50; p = 0.0001). Conclusions: The association between LDL(-) and inflammation and the lower levels of anti-LDL(-) autoantibodies are important risk factors related to atherosclerosis in CKD. (C) 2011 Published by Elsevier B.V.

Dietary glutamine supplementation affects macrophage function, hematopoiesis and nutritional status in early weaned mice

Background Et aims: To investigate the effect that early weaning associated with the ingestion of either a glutamine-free or supplemented diet has on the functioning of peritoneal. macrophages, hematopoiesis and nutritional status of mice. Methods: Swiss Webster mice were early weaned on their 14th day of life and distributed to two groups, being fed either a glutamine-free diet (-GLN) or a glutamine-supplemented diet (+GLN). Animals belonging to a control group (CON) were weaned on their 21st day of life. Results: The -GLN and +GLN groups had a lower lean body mass, carcass protein and ash content, plasma glutamine concentration and lymphocyte counts both in the peripheral blood and bone marrow when compared to the CON group (P < 0.05). Dietary supplementation with glutamine reversed both the lower concentrations of protein and DNA in the muscle and liver, as well. as the reduced capacity of spreading and synthesizing nitric oxide, hydrogen peroxide, TNF-alpha, IL-1 beta and IL-6 in cultures of peritoneal. macrophages obtained from the -GLN group (P < 0.05). Conclusion: These data indicate that the ingestion of glutamine modulates the function of peritoneal macrophages in early weaned mice. However, a glutamine-supplemented diet cannot substitute maternal milk in respect to immunological and metabolic parameters. (C) 2008 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Splenectomy Increases Mortality in Murine Trypanosoma cruzi Infection

The spleen is a secondary lymphoid organ that harbours a variety of cells such as T and B lymphocytes and antigen-presenting cells important to immune response development. In this study, we evaluated the impact of spleen removal in the immune response to experimental Trypanosoma cruzi infection. C57BL/6 mice were infected with Y strain of the parasite and infection was followed daily. Mice that underwent splenectomy had fewer parasites in peripheral blood at the peak of infection; however, mortality was increased. Histological analysis of heart and liver tissues revealed an increased number of parasites and inflammatory infiltrates at these sites. Spleen removal was associated with reduction in IFN-gamma and TNF-alpha production during infection as well as with a decrease in specific antibody secretion. Haematological disorders were also detected. Splenectomized mice exhibited severe anaemia and decreased bone marrow cell numbers. Our results indicate that spleen integrity is critical in T. cruzi infection for the immune response against the parasite, as well as for the control of bone marrow haematological function.

Effects of Protein-Energy Malnutrition on NF-KappaB Signalling in Murine Peritoneal Macrophages

Protein-energy malnutrition (PEM) is an important public health problem affecting millions of people worldwide. PEM decreases resistance to infection, impairing a number of physiological processes. In unstimulated cells, NF-kappa B is kept from binding to its consensus sequence by the inhibitor I kappa B alpha, which retains NF-kappa B in the cytoplasm. Upon various signals, such as lipopolysaccharide (LPS), I kappa B alpha is rapidly degraded and NF-kappa B is induced to translocate into the nucleus, where it activates expression of various genes that participate in the inflammatory response, including those involved in the synthesis of TNF-alpha. TRAF-6 is a cytoplasmic adapter protein that links the stimulatory signal from Toll like receptor-4 to NF-kappa B. The aim of this study was to evaluate the effect of malnutrition on induction of TNF-a by LPS in murine peritoneal macrophages. We evaluated peritoneal cellularity, the expression of MyD88, TRAF-6, IKK, I kappa B alpha and NF-kappa B, NF-kappa B activation and TNF-alpha mRNA and protein synthesis inmacrophages. Two-month-old male BALB/Cmice were submitted to PEM with a low-protein diet that contained 2% protein, compared to 12% protein in the control diet. When the experimental group had lost about 20% of the original body weight, it was used in the subsequent experiments. Malnourished animals presented anemia, leucopenia and severe reduction in peritoneal cavity cellularity. TNF-a mRNA and protein levels of macrophages stimulated with LPS were significantly lower in malnourished animals. PEM also decreased TRAF-6 expression and NF-kappa B activation after LPS stimulation. These results led us to conclude that PEM changes NF-kappa B signalling pathway in macrophages to LPS stimulus.

Plasmodium vivax recombinant vaccine candidate AMA-1 plays an important role in adaptive immune response eliciting differentiation of dendritic cells

The Apical Membrane Antigen-1 (AMA-1) is a well-characterized and functionally important merozoite protein and is currently considered a major candidate antigen for a malaria vaccine. Previously, we showed that AMA-1 has an influence on cellular immune responses of malaria-naive subjects, resulting in an alternative activation of monocyte-derived dendritic cells and induction of a pro-inflammatory response by stimulated PBMCs. Although there is evidence, from human and animal malaria model systems that cell-mediated immunity may contribute to both protection and pathogenesis, the knowledge on cellular immune responses in vivax malaria and the factors that may regulate this immunity are poorly understood. In the current work, we describe the maturation of monocyte-derived dendritic cells of P. vivax naturally infected individuals and the effect of P. vivax vaccine candidate Pv-AMA-1 on the immune responses of the same donors. We show that malaria-infected subjects present modulation of DC maturation, demonstrated by a significant decrease in expression of antigen-presenting molecules (CD1a, HLA-ABC and HLA-DR), accessory molecules (CD40, CD80 and CD86) and Fc gamma RI (CD64) receptor (P <= 0.05). Furthermore, Pv-AMA-1 elicits an upregulation of CD1a and HLA-DR molecules on the surface of monocyte-derived dendritic cells (P=0.0356 and P=0.0196, respectively), and it is presented by AMA-1-stimulated DCs. A significant pro-inflammatory response elicited by Pv-AMA-1-pulsed PBMCs is also demonstrated, as determined by significant production of TNF-alpha, IL-12p40 and IFN-gamma (P <= 0.05). Our results suggest that Pv-AMA-1 may partially revert DC down-modulation observed in infected subjects, and exert an important role in the initiation of pro-inflammatory immunity that might contribute substantially to protection. (c) 2009 Elsevier Ltd. All rights reserved.

Serum amyloid A induces CCL20 secretion in mononuclear cells through MAPK (p38 and ERK1/2) signaling pathways

Although the serum levels of SAA had been reported to be upregulated during inflammatory/infectious process, the role of this acute-phase protein has not been completely elucidated. In previous studies, we demonstrated that SAA stimulated the production of TNF-alpha, IL-1 beta, IL-8, NO, and ROS by neutrophils and/or mononuclear cells. Herein we demonstrate that SAA induces the expression and release of CCL20 from Cultured human blood mononuclear cells. We also focus on the signaling pathways triggered by SAA. in THP-1 cells SAA promotes phosphorylation of p38 and ERK1/2. Furthermore, the addition of SB203580 (p38 inhibitor) and PD98059 (ERK 1/2 inhibitor) inhibits the expression and release of CCL20 in mononuclear cells treated with SAA. Our results point to SAA as an important link of innate to adaptive immunity, once it might act on the recruitment of mononuclear cells. (C) 2008 Elsevier B.V. All rights reserved.

Protein-Energy Malnutrition Modifies the Production of Interleukin-10 in Response to Lipopolysaccharide (LPS) in a Murine Model

Malnutrition modifies resistance to infection by impairing a number of physiological processes including hematopoesis and the immune response. In this study, we examined the production of Interleukin-4 (IL-4) and IL-10 in response to lipopolysaccharide (LPS) and also evaluated the cellularity of the blood, bone marrow, and spleen in a mouse model of protein-energy malnutrition. Two-month-old male Swiss mice were subjected to protein-energy malnutrition (PEM) with a low-protein diet (4%) as compared to the control diet (20%). When the experimental group lost approximately 20% of their original body weight, the animals from both groups received 1.25 mu g of LPS intravenously. The Cells ill the blood, bone marrow, and spleen were counted, and circulating levels of IL-4 and IL-10 were evaluated in animals stimulated with LPS. Cells from the spleen, bone marrow, and peritoneal cavity of non-inoculated animals were collected for Culture to evaluate the production of IL-4 and IL-10 after stimulating these cells with 1.25 mu g of LPS in vitro. Malnourished animals presented leucopenia and a severe reduction in bone marrow, spleen, and peritoneal cavity cellularity before and after Stimulus with LPS. The circulating levels of IL-10 were increased in malnourished animals inoculated with LPS when compared to control animals, although the levels of IL-4 did not differ. In cells cultured with LPS, we observed high levels of IL-10 in the bone marrow cells of malnourished animals. These findings suggest that malnourished mice present a deficient immune response to LPS. These alterations may be partly responsible for the immunodeficiency observed in these malnourished mice.

Dietary glutamine supplementation increases the activity of peritoneal macrophages and hemopoiesis in early-weaned mice inoculated with Mycobacterium bovis bacillus Calmette-Guerin

Infants who are breast-fed have been shown to have a lower incidence of certain infectious diseases compared with formula-fed infants. Glutamine is one of the most abundant amino acids found in maternal milk and it is essential for the function of immune system cells such as macrophages. The purpose of this study was to investigate the effect of glutamine supplementation on the function of peritoneal macrophages and on hemopoiesis in early-weaned mice inoculated with Mycobacterium bovis bacillus Calmette-Guerin (BCG). Mice were wearied at 14 d of age and distributed to 2 groups and fed either a glutamine-free diet (n = 16) or a glutamine-supplemented diet (+Gln (n = 16). Both diets were isonitrogenous (with addition of a mixture of nonessential amino acids) and isocaloric. At d 21, 2 subgroups of mice (n = 16) were intraperitoneally injected with BCG and all mice were killed at d 28. Plasma, muscle and liver glutamine concentrations and muscle glutamine synthetase activity were not affected by diet or inoculation with BCG. The +GIn diet led to increased leukocyte and lymphocyte counts in the peripheral blood (P < 0.05) and granulocyte and lymphocyte counts in the bone marrow and spleen (P < 0.05). The +GIn diet increased spreading and adhesion capacities, hydrogen peroxide, nitric oxide, and tumor necrosis factor-alpha (TNF alpha) syntheses and the phagocytic and fungicidal activity of peritoneal macrophages (P < 0.05). The interaction between the +GIn diet and BCG inoculation increased the area under the curve of interleukin (IL)-1 beta and TNF alpha syntheses (P < 0.05). In conclusion, the intake of glutamine increases the function of peritoneal macrophages and hemopoiesis in early-weaned and BCG-inoculated mice. These data have important implications for the design of breast milk substitutes for human infants.

Effects of chlorogenic acid on neutrophil locomotion functions in response to inflammatory stimulus

Aim of the study: Species of Lychnophora are used in Brazilian folk medicine as analgesic and anti-inflammatory agents. Chlorogenic acid (CGA) and their analogues are important components of polar extracts of these species, as well in several European and Asian medicinal plants. Some of these phenolic compounds display anti-inflammatory effects. In this paper we report the isolation of CGA from Lychnophora salicifolia and its effects on functions involved in neutrophils locomotion. Materials and methods: LC-MS(n) data confirmed the presence of CGA in the plant. Actions of CGA were investigated on neutrophils obtained from peritoneal cavity of Wistar rats (4h after 1% oyster glycogen solution injection; 10 ml), and incubated with vehicle or with 50, 100 or 1000 mu M CGA in presence of lipopolysaccharide from Escherichia coil (LPS, 5 mu g/ml). Nitric oxide (NO; Griess reaction); prostaglandin E(2) (PGE(2)), interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha [TNF-alpha; enzyme-linked immunosorbent assay (EIA)]; protein (flow cytometry) and gene (RT-PCR) expression of L-selectin, beta(2)integrin and platelet-endothelial cell adhesion molecule-1 (PECAM-1) were quantified. In vitro neutrophil adhesion to primary culture of microvascular endothelial cell (PMEC) and neutrophil migration in response to formyl-methionil-leucil-phenilalanine (fMLP, 10(-8)M, Boyden chamber) was determined. Results: CGA treatment did not modify the secretion of inflammatory mediators, but inhibited L-selectin cleavage and reduced beta(2) integrin, independently from its mRNA synthesis, and reduced membrane PECAM-1 expression: inhibited neutrophil adhesion and neutrophil migration induced by fMLP. Conclusions: Based on these findings, we highlight the direct inhibitory actions of CGA on adhesive and locomotion properties of neutrophils, which may contribute to its anti-inflammatory effects and help to explain the use of Lychnophora salicifolia as an anti-inflammatory agent. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Endogenous opioids: role in prostaglandin-dependent and -independent fever

This study evaluated the participation of mu-opioid-receptor activation in body temperature (T-b) during normal and febrile conditions (including activation of heat conservation mechanisms) and in different pathways of LPS-induced fever. The intracerebroventricular treatment of male Wistar rats with the selective opioid mu-receptor-antagonist cyclic D-Phe-Cys-Try-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP; 0.1-1.0 mu g) reduced fever induced by LPS (5.0 mu g/kg) but did not change Tb at ambient temperatures of either 20 C or 28 C. The subcutaneous, intracerebroventricular, and intrahypothalamic injection of morphine (1.0 -10.0 mg/kg, 3.0 -30.0 mu g, and 1 -100 ng, respectively) produced a dose-dependent increase in Tb. Intracerebroventricular morphine also produced a peripheral vasoconstriction. Both effects were abolished by CTAP. CTAP (1.0 mu g icv) reduced the fever induced by intracerebroventricular administration of TNF-alpha (250 ng), IL-6 (300 ng), CRF (2.5 mu g), endothelin-1 (1.0 pmol), and macrophage inflammatory protein (500 pg) and the first phase of the fever induced by PGF(2 alpha) (500.0 ng) but not the fever induced by IL-1 beta (3.12 ng) or PGE(2) (125.0 ng) or the second phase of the fever induced by PGF(2 alpha). Morphine-induced fever was not modified by the cyclooxygenase (COX) inhibitor indomethacin (2.0 mg/kg). In addition, morphine injection did not induce the expression of COX-2 in the hypothalamus, and CTAP did not modify PGE2 levels in cerebrospinal fluid or COX-2 expression in the hypothalamus after LPS injection. In conclusion, our results suggest that LPS and endogenous pyrogens (except IL-1 beta and prostaglandins) recruit the opioid system to cause a mu-receptor-mediated fever.

Tityus serrulatus venom and toxins Ts1, Ts2 and Ts6 induce macrophage activation and production of immune mediators

Scorpion envenomation induces a systemic immune response, and neurotoxins of venom act on specific ion channels, modulating neurotransmitter release or activity. However, little is known about the immunomodulatory effects of crude venom from scorpion Tityus serrulatus (TsV) or its toxins (Ts1, Ts2 and Ts6) in combination with lipopolysaccharide (LPS). To investigate the immunomodulatory effects of TsV and its toxins (Ts1, Ts2 and Ts6), J774.1 cells were stimulated with different concentrations (25, 50 and 100 mu g/mL) of venom or toxins pre-stimulated or not with LPS (0.5 mu g/mL). Macrophage cytotoxicity was assessed, and nitric oxide (NO) and cytokine production were analyzed utilizing the culture supernatants. TsV and its toxins did not produce cytotoxic effects. Depending on the concentrations used, TsV, Ts1 and Ts6 stimulated the production of NO, interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha in J774.1 cells, which were enhanced under LPS co-stimulation. However, LPS + Ts2 inhibited NO, IL-6 and TNF-alpha production, and Ts2 alone stimulated the production of IL-10, suggesting an anti-inflammatory activity for this toxin. Our findings are important for the basic understanding of the mechanisms involved in macrophage activation following envenomation: additionally, these findings may contribute to the discovery of new therapeutic compounds to treat immune-mediated diseases. (C) 2011 Elsevier Ltd. All rights reserved.

Isolation and functional characterization of proinflammatory acidic phospholipase A(2) from Bothrops leucurus snake venom

In the present study, an acidic PLA(2), designated BI-PLA(2), was isolated from Bothrops leucurus snake venom through two chromatographic steps: ion-exchange on CM-Sepharose and hydrophobic chromatography on Phenyl-Sepharose. Bl-PLA(2) was homogeneous on SDS-PAGE and when submitted to 2D electrophoresis the molecular mass was 15,000 Da and pl was 5.4. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA(2)s from snake venoms. Its specific activity was 159.9 U/mg and the indirect hemolytic activity was also higher than that of the crude venom. Bl-PLA(2) induced low myotoxic and edema activities as compared to those of the crude venom. Moreover, the enzyme was able to induce increments in IL-12p40, TNF-alpha, IL-1 beta and IL-6 levels and no variation of IL-8 and IL-10 in human PBMC stimulated in vitro, suggesting that Bl-PLA2 induces proinflammatory cytokine production by human mononuclear cells. Bothrops leucurus venom is still not extensively explored and knowledge of its components will contribute for a better understanding of its action mechanism. (C) 2011 Elsevier Inc. All rights reserved.

Quercetin inhibits UV irradiation-induced inflammatory cytokine production in primary human keratinocytes by suppressing NF-kappa B pathway

Background: Topical flavonoids, such as quercetin, have been shown to reduce ultraviolet (UV) irradiation-mediated skin damage. However, the mechanisms and signaling pathways involved in this protective effect are not clear. UV irradiation leads to activation of two major signaling pathways, namely nuclear factor kappa B (NF-kappa B) and activator protein-1 (AP-1) pathways. Activation of NF-kappa B pathway by UV irradiation stimulates inflammatory cytokine expression, whereas activation of AP-1 pathway by UV irradiation promotes matrix metalloproteinase (MMP) production. Both pathways contribute to UV irradiation-induced skin damage, such as photoaging and skin tumor formation. Objective: To elucidate the underlying mechanism, we examined the effect of quercetin on UV irradiation induced activation of NF-kappa B and AP-1 pathways. Methods: Primary human keratinocytes, the major skin cell type subjected to physiological solar UV irradiation, were used to study the effects of quercetin on UV irradiation-induced signal transduction pathways. Results: Quercetin decreased UV irradiation-induced NF-kappa B DNA-binding by 80%. Consequently, quercetin suppressed UV irradiation-induced expression of inflammatory cytokines IL-1 beta (similar to 60%), IL-6 (similar to 80%), IL-8 (similar to 76%) and TNF-alpha (similar to 69%). In contrast, quercetin had no effect on UV irradiation activation of three MAP kinases, ERK, JNK, or p38. Accordingly, induction of AP-1 target genes such as MMP-1 and MMP-3 by UV irradiation was not suppressed by quercetin. Conclusion: Our data indicate that the ability of quercetin to block UV irradiation-induced skin inflammation is mediated, at least in part, by its inhibitory effect on NF-kappa B activation and inflammatory cytokine production. (C) 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Budlein A from Viguiera robusta inhibits leukocyte-endothelial cell interactions, adhesion molecule expression and inflammatory mediators release

Budlein A has been reported to exert some analgesic and anti-inflammatory properties. In this study, we have evaluated its effect on LPS-induced leukocyte recruitment in vivo and the mechanisms involved in its anti-inflammatory activity. In vivo, intravital videomicroscopy was used to determine the effects of budlein A on LPS-induced leukocyte-endothelial cell interactions in the murine cremasteric microcirculation. In vitro, the effects of budlein A on LPS-induced cytokine, chemokine and nitrites release, T-cell proliferative response as well as cell adhesion molecule expression (CAM) were evaluated. In vivo, intraperitoneal administration of budlein A (2.6 mM/kg) caused a significant reduction of LPS-induced leukocyte rolling flux, adhesion and emigration by 84, 92 and 96% respectively. In vitro, T-cell proliferative response was also affected by budlein A. When murine J774 macrophages were incubated with the sesquiterpene lactone, LPS-induced IL-1 beta, tumor necrosis factor-alpha (TNF-alpha) and keratinocyte-derived chemokine (KC) release were concentration-dependently inhibited. In human umbilical vein endothelial cells (HUVECs), budlein A also reduced the production of TNF-alpha, monocyte chemoattractant protein-1 (MCP-1), IL-8, nitrites and CAM expression elicited by LPS. Budlein A is a potent inhibitor of LPS-induced leukocyte accumulation in vivo. This effect appears to be mediated through inhibition of cytokine and chemokine release and down-regulation of CAM expression. Thus, it has potential therapeutic interest for the control of leukocyte recruitment that occurs in different inflammatory disorders. (C) 2009 Elsevier GrnbH. All rights reserved.