Local inflammatory events induced by Bothrops atrox snake venom and the release of distinct classes of inflammatory mediators

Bothrops atrox is responsible for most accidents involving snakes in the Brazilian Amazon and its venom induces serious systemic and local effects. The local effects are not neutralized effectively by commercial antivenoms, resulting in serious sequelae in individuals bitten by this species. This study investigates the local inflammatory events induced in mice by B. atrox venom (Bay), such as vascular permeability, leukocyte influx and the release of important inflammatory mediators such as cytokines, eicosanoids and the chemokine CCL-2, at the injection site. The effect of Bay on cyclooxygenase (COX-1 and COX-2) expression was also investigated. The results showed that intraperitoneal (i.p.) injection of BaV promoted a rapid and significant increase in vascular permeability, which reached a peak 1 h after venom administration. Furthermore, BaV caused leukocyte infiltration into the peritoneal cavity between 1 and 8 h after i.p. injection, with mononuclear leukocytes (MNs) predominating in the first 4 h, and polymorphonuclear leukocytes (PMNs) in the last 4 h. Increased protein expression of COX-2, but not of COX-1, was detected in leukocytes recruited in the first and fourth hours after injection of BaV. The venom caused the release of eicosanoids PGD(2), PGE(2), TXA(2) and LTB4, cytokines TNF-alpha, IL-6, IL-10 and IL-12p70, but not IFN-gamma, and chemokine CCL-2 at different times. The results show that Bay is able to induce an early increase in vascular permeability and a leukocyte influx to the injection site consisting mainly of MNs initially and PMNs during the later stages. These phenomena are associated with the production of cytokines, the chemokine CCL-2 and eicosanoids derived from COX-1 and COX-2. (c) 2012 Elsevier Ltd. All rights reserved.

Actions of translocator protein ligands on neutrophil adhesion and motility induced by G-protein coupled receptor signaling

The 18 kDa translocator protein (TSPO) also known as the peripheral benzodiazepine receptor (PBR), mediates the transportation of cholesterol and anions from the outer to the inner mitochondrial membrane in different cells types. Although recent evidences indicate a potential role for TSPO in the development of inflammatory processes, the mechanisms involved have not been elucidated. The present study investigated the ability of the specific TSPO ligands, the isoquinoline carboxamide PK11195 and benzodiazepine Ro5-4864, on neutrophil recruitment promoted by the N-formylmethionyl-leucyl-phenylalanine peptide (fMLP), an agonist of G-protein coupled receptor (GPCR). Pre-treatment with Ro5-4864 abrograted fMLP-induced leukocyte-endothelial interactions in mesenteric postcapillary venules in vivo. Moreover, in vitro Ro5-4864 treatment prevented fMLP-induced: (i) L-selectin shedding and overexpression of PECAM-1 on the neutrophil cell surface; (ii) neutrophil chemotaxis and (iii) enhancement of intracellular calcium cations (iCa(+2)). Intriguingly, the two latter effects were augmented by cell treatment with PK11195. An allosteric agonist/antagonist relation may be suggested, as the effects of Ro5-4864 on fMLP-stimulated neutrophils were reverted by simultaneous treatment with PK11195. Taken together, these data highlight TSPO as a modulator of pathways of neutrophil adhesion and locomotion induced by GPCR, connecting TSPO actions and the onset of an innate inflammatory response. (C) 2011 Elsevier Inc. All rights reserved.

5-Lipoxygenase Deficiency Impairs Innate and Adaptive Immune Responses during Fungal Infection

5-lipoxygenase-derived products have been implicated in both the inhibition and promotion of chronic infection. Here, we sought to investigate the roles of endogenous 5-lipoxygenase products and exogenous leukotrienes during Histoplasma capsulatum infection in vivo and in vitro. 5-LO deficiency led to increased lung CFU, decreased nitric oxide production and a deficient primary immune response during active fungal infection. Moreover, H. capsulatum-infected 5-LO-/- mice showed an intense influx of neutrophils and an impaired ability to generate and recruit effector T cells to the lung. The fungal susceptibility of 5-LO-/- mice correlated with a lower rate of macrophage ingestion of IgG-H. capsulatum relative to WT macrophages. Conversely, exogenous LTB4 and LTC4 restored macrophage phagocytosis in 5-LO deficient mice. Our results demonstrate that leukotrienes are required to control chronic fungal infection by amplifying both the innate and adaptive immune response during histoplasmosis.

Hypertonic saline solution reduces the inflammatory response in endotoxemic rats

OBJECTIVE: Volume replacement in septic patients improves hemodynamic stability. This effect can reduce the inflammatory response. The objective of this study was to evaluate the effect of 7.5% hypertonic saline solution versus 0.9% normal saline solution for volume replacement during an inflammatory response in endotoxemic rats. METHODS: We measured cytokines (serum and gut), nitrite, and lipid peroxidation (TBARS) as indicators of oxidative stress in the gut. Rats were divided into four groups: control group (C) that did not receive lipopolysaccharide; lipopolysaccharide injection without treatment (LPS); lipopolysaccharide injection with saline treatment (LPS + S); and lipopolysaccharide injection with hypertonic saline treatment (LPS + H). Serum and intestine were collected. Measurements were taken at 1.5, 8, and 24 h after lipopolysaccharide administration. RESULTS: Of the four groups, the LPS + H group had the highest survival rate. Hypertonic saline solution treatment led to lower levels of IL-6, IL-10, nitric oxide, and thiobarbituric acid reactive substances compared to 0.9% normal saline. In addition, hypertonic saline treatment resulted in a lower mortality compared to 0.9% normal saline treatment in endotoxemic rats. Volume replacement reduced levels of inflammatory mediators in the plasma and gut. CONCLUSION: Hypertonic saline treatment reduced mortality and lowered levels of inflammatory mediators in endotoxemic rats. Hypertonic saline also has the advantage of requiring less volume replacement.

Signaling Events During Cyclic Guanosine Monophosphate-Regulated Pigment Aggregation in Freshwater Shrimp Chromatophores

Crustacean color change results partly from granule aggregation induced by red pigment concentrating hormone (RPCH). In shrimp chromatophores, both the cyclic GMP (3', 5'-guanosine monophosphate) and Ca2+ cascades mediate pigment aggregation. However, the signaling elements upstream and downstream from cGMP synthesis by GC-S (cytosolic guanylyl cyclase) remain obscure. We investigate post-RPCH binding events in perfused red ovarian chromatophores to disclose the steps modulating cGMP concentration, which regulates granule translocation. The inhibition of calcium/calmodulin complex (Ca2+/CaM) by N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7) induces spontaneous aggregation but inhibits RPCH-triggered aggregation, suggesting a role in pigment aggregation and dispersion. Nitric oxide synthase inhibition by N omega-nitro-L-arginine methyl ester hydrochloride (L-NAME) strongly diminishes RPCH-induced aggregation; protein kinase G inhibition (by rp-cGMPs-triethylamine) reduces RPCH-triggered aggregation and provokes spontaneous dispersion, disclosing NO/PKG participation in aggregation signaling. Myosin light chain phosphatase inhibition (by cantharidin) accelerates RPCH-triggered aggregation, whereas Rho-associated protein kinase inhibition (by Y-27632, H-11522) reduces RPCH-induced aggregation and accelerates dispersion. MLCP (myosin light chain kinase) and ROCK (Rho-associated protein kinase) may antagonistically regulate myosin light chain (MLC) dephosphorylation/phosphorylation during pigment dispersion/aggregation. We propose the following general hypothesis for the cGMP/Ca2+ cascades that regulate pigment aggregation in crustacean chromatophores: RPCH binding increases Ca2+ (int), activating the Ca2+/CaM complex, releasing NOS-produced nitric oxide, and causing GC-S to synthesize cGMP that activates PKG, which phosphorylates an MLC activation site. Myosin motor activity is initiated by phosphorylation of an MLC regulatory site by ROCK activity and terminated by MLCP-mediated dephosphorylation. Qualitative comparison reveals that this signaling pathway is conserved in vertebrate and invertebrate chromatophores alike.

Partial Replacement of omega-6 Fatty Acids With Medium-Chain Triglycerides, but Not Olive Oil, Improves Colon Cytokine Response and Damage in Experimental Colitis

Background: Soybean oil is rich in omega-6 fatty acids, which are associated with higher incidence and more severe cases of inflammatory bowel diseases. The authors evaluated whether partial replacement of soybean oil by medium-chain triglycerides (MCTs) or olive oil influenced the incidence and severity of experimental ulcerative colitis by using different parenteral lipid emulsions (LEs). Methods: Wistar rats (n = 40) were randomized to receive parenteral infusion of the following LE: 100% soybean oil (SO), 50% MCT mixed with 50% soybean oil (MCT/SO), 80% olive oil mixed with 20% soybean oil (OO/SO), or saline (CC). After 72 hours of infusion, acetic acid experimental colitis was induced. After 24 hours, colon histology and cytokine expression were analyzed. Results: SO was not significantly associated with overall tissue damage. MCT/SO was not associated with necrosis (P < .005), whereas OO/SO had higher frequencies of ulcer and necrosis (P < .005). SO was associated with increased expression of interferon-gamma (P = .005) and OO/SO with increased interleukin (IL)-6 and decreased tumor necrosis factor-alpha expression (P < .05). MCT/SO appeared to decrease IL-1 (P < .05) and increase IL-4 (P < .001) expression. Conclusions: Parenteral SO with high concentration of omega-6 fatty acids was not associated with greater tissue damage in experimental colitis. SO partial replacement with MCT/SO decreased the frequency of histological necrosis and favorably modulated cytokine expression in the colon; however, replacement with OO/SO had unfavorable effects. (JPEN J Parenter Enteral Nutr. 2012; 36: 442-448)

Doxycycline ameliorates 2K-1C hypertension-induced vascular dysfunction in rats by attenuating oxidative stress and improving nitric oxide bioavailability

Vascular dysfunction associated with two-kidney, one-clip (2K-1C) hypertension may result from both altered matrix metalloproteinase (MMP) activity and higher concentrations of reactive oxygen species (ROS). Doxycycline is considering the most potent MMP inhibitor of tetracyclines and attenuates 2K-1C hypertension-induced high blood pressure and chronic vascular remodeling. Doxycycline might also act as a ROS scavenger and this may contribute to the amelioration of some cardiovascular diseases associated with increased concentrations of ROS. We hypothesized that in addition to its MMP inhibitory effect, doxycycline attenuates oxidative stress and improves nitric oxide (NO) bioavailability in 2K-1C hypertension, thus improving hypertension-induced arterial endothelial dysfunction. Sham operated or 2K-1C hypertensive rats were treated with doxycycline 30 mg/kg/day (or vehicle). After 8 weeks of treatment, aortic rings were isolated to assess endothelium dependent vasorelaxation to A23187. Arterial and systemic levels of ROS were respectively measured using dihydroethidine (DHE) and thiobarbituric acid reactive substances (TBARS). Neutrophils-derived ROS were tested in vitro using the fluoroprobe Carboxy-H(2)DCFDA and human neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA). NO levels were assessed in rat aortic endothelial cells by confocal microscopy. Aortic MMP activity was determined by in situ zymography. Doxycycline attenuated 2K-1C hypertension (169 +/- 17.3 versus 209 +/- 10.9 mm Hg in hypertensive controls, p < 0.05) and protected against hypertension-induced reduction in endothelium-dependent vasorelaxation to A23187 (p < 0.05). Doxycycline also decreased hypertension-induced oxidative stress (p <= 0.05), higher MMP activity (p < 0.01) and improved NO levels in aortic endothelial cells (p < 0.01). Therefore, doxycycline ameliorates 2K-1C hypertension-induced endothelial dysfunction in aortas by inhibiting oxidative stress generation and improving NO bioavailability, in addition to its inhibitory effects on MMP activity. (C) 2012 Elsevier Inc. All rights reserved.

Decreased RNA expression of interleukin 17A in skin of leprosy

Interleukin-17A (IL-17A) is a proinflamatory cytokine that plays an important role in fighting pathogens at mucosal interfaces, by summoning neutrophils and upregulating cytoplasmatic antimicrobial peptides. So far, the presence of IL-17A in leprosy has not been demonstrated. The expression of IL-17A and related cytokines (IL-6 and IL-23p19) was addressed through RNA extraction and cDNA quantitative amplification in macerated biopsies of active lesions of 48 leprosy patients and 20 fragments of normal skin of individuals. Blood levels of IL-17A, IL-23p19 and IL-6 were determined by ELISA. We found an abrogated mRNA IL-17A response in all biopsies of leprosy patients, as compared with controls. Circulating IL-17A and IL-23p19 were undetectable in both patients and controls, but IL-6 was higher in lepromatous patients. Although at low levels, IL-17A mRNA in lepromatous patients had an inverse linear correlation with bacillary burden. Low expression of IL-17A in patients may be a constitutive genetic feature of leprosy patients or a circumstantial event induced by the local presence of the pathogen, as an escape mechanism.

Expression of recombinant human mutant granulocyte colony stimulating factor (Nartograstim) in Escherichia coli

The human granulocyte colony stimulating factor (hG-CSF) plays an important role in hematopoietic cell proliferation/differentiation and has been widely used as a therapeutic agent for treating neutropenias. Nartograstim is a commercial G-CSF that presents amino acid changes in specific positions when compared to the wildtype form, which potentially increase its activity and stability. The aim of this work was to develop an expression system in Escherichia coli that leads to the production of large amounts of a recombinant hG-CSF (rhG-CSF) biosimilar to Nartograstim. The nucleotide sequence of hg-csf was codon-optimized for expression in E. coli. As a result, high yields of the recombinant protein were obtained with adequate purity, structural integrity and biological activity. This protein has also been successfully used for the production of specific polyclonal antibodies in mice, which could be used in the control of the expression and purification in an industrial production process of this recombinant protein. These results will allow the planning of large-scale production of this mutant version of hG-CSF (Nartograstim), as a potential new biosimilar in the market.

Increased vascular contractility and oxidative stress in beta(2)-adrenoceptor knockout mice: the role of NADPH oxidase

Background/Aims: beta(2)-adrenoceptor (beta(2)-AR) activation induces smooth muscle relaxation and endothelium-derived nitric oxide (NO) release. However, whether endogenous basal beta(2)-AR activity controls vascular redox status and NO bioavailability is unclear. Thus, we aimed to evaluate vascular reactivity in mice lacking functional beta(2)-AR (beta 2KO), focusing on the role of NO and superoxide anion. Methods and Results: Isolated thoracic aortas from beta 2KO and wild-type mice (WT) were studied. beta 2KO aortas exhibited an enhanced contractile response to phenylephrine compared to WT. Endothelial removal and L-NAME incubation increased phenylephrine-induced contraction, abolishing the differences between beta 2KO and WT mice. Basal NO availability was reduced in aortas from beta 2KO mice. Incubation of beta 2KO aortas with superoxide dismutase or NADPH inhibitor apocynin restored the enhanced contractile response to phenylephrine to WT levels. beta 2KO aortas exhibited oxidative stress detected by enhanced dihydroethidium fluorescence, which was normalized by apocynin. Protein expression of eNOS was reduced, while p47(phox) expression was enhanced in beta 2KO aortas. Conclusions: The present results demonstrate for the first time that enhanced NADPH-derived superoxide anion production is associated with reduced NO bioavailability in aortas of beta 2KO mice. This study extends the knowledge of the relevance of the endogenous activity of beta(2)-AR to the maintenance of the vascular physiology. Copyright (C) 2012 S. Karger AG, Basel

Infrared (810-nm) low-level laser therapy on rat experimental knee inflammation

Arthritis of the knee is the most common type of joint inflammatory disorder and it is associated with pain and inflammation of the joint capsule. Few studies address the effects of the 810-nm laser in such conditions. Here we investigated the effects of low-level laser therapy (LLLT; infrared, 810-nm) in experimentally induced rat knee inflammation. Thirty male Wistar rats (230-250 g) were anesthetized and injected with carrageenan by an intra-articular route. After 6 and 12 h, all animals were killed by CO(2) inhalation and the articular cavity was washed for cellular and biochemical analysis. Articular tissue was carefully removed for real-time PCR analysis in order to evaluate COX-1 and COX-2 expression. LLLT was able to significantly inhibit the total number of leukocytes, as well as the myeloperoxidase activity with 1, 3, and 6 J (Joules) of energy. This result was corroborated by cell counting showing the reduction of polymorphonuclear cells at the inflammatory site. Vascular extravasation was significantly inhibited at the higher dose of energy of 10 J. Both COX-1 and 2 gene expression were significantly enhanced by laser irradiation while PGE(2) production was inhibited. Low-level laser therapy operating at 810 nm markedly reduced inflammatory signs of inflammation but increased COX-1 and 2 gene expression. Further studies are necessary to investigate the possible production of antiinflammatory mediators by COX enzymes induced by laser irradiation in knee inflammation.

Neutrophil Paralysis in Plasmodium vivax Malaria

Background: The activation of innate immune responses by Plasmodium vivax results in activation of effector cells and an excessive production of pro-inflammatory cytokines that may culminate in deleterious effects. Here, we examined the activation and function of neutrophils during acute episodes of malaria. Materials and Methods: Blood samples were collected from P. vivax-infected patients at admission (day 0) and 30-45 days after treatment with chloroquine and primaquine. Expression of activation markers and cytokine levels produced by highly purified monocytes and neutrophils were measured by the Cytometric Bead Assay. Phagocytic activity, superoxide production, chemotaxis and the presence of G protein-coupled receptor (GRK2) were also evaluated in neutrophils from malaria patients. Principal Findings: Both monocytes and neutrophils from P. vivax-infected patients were highly activated. While monocytes were found to be the main source of cytokines in response to TLR ligands, neutrophils showed enhanced phagocytic activity and superoxide production. Interestingly, neutrophils from the malaria patients expressed high levels of GRK2, low levels of CXCR2, and displayed impaired chemotaxis towards IL-8 (CXCL8). Conclusion: Activated neutrophils from malaria patients are a poor source of pro-inflammatory cytokines and display reduced chemotactic activity, suggesting a possible mechanism for an enhanced susceptibility to secondary bacterial infection during malaria.

Expression of Mast Cell Proteases Correlates with Mast Cell Maturation and Angiogenesis during Tumor Progression

Tumor cells are surrounded by infiltrating inflammatory cells, such as lymphocytes, neutrophils, macrophages, and mast cells. A body of evidence indicates that mast cells are associated with various types of tumors. Although role of mast cells can be directly related to their granule content, their function in angiogenesis and tumor progression remains obscure. This study aims to understand the role of mast cells in these processes. Tumors were chemically induced in BALB/c mice and tumor progression was divided into Phases I, II and III. Phase I tumors exhibited a large number of mast cells, which increased in phase II and remained unchanged in phase III. The expression of mouse mast cell protease (mMCP)-4, mMCP-5, mMCP-6, mMCP-7, and carboxypeptidase A were analyzed at the 3 stages. Our results show that with the exception of mMCP-4 expression of these mast cell chymase (mMCP-5), tryptases (mMCP-6 and 7), and carboxypeptidase A (mMC-CPA) increased during tumor progression. Chymase and tryptase activity increased at all stages of tumor progression whereas the number of mast cells remained constant from phase II to III. The number of new blood vessels increased significantly in phase I, while in phases II and III an enlargement of existing blood vessels occurred. In vitro, mMCP-6 and 7 are able to induce vessel formation. The present study suggests that mast cells are involved in induction of angiogenesis in the early stages of tumor development and in modulating blood vessel growth in the later stages of tumor progression.

Human Leucocytes Response to Viable, Extended Freeze-Drying or Heat-Killed Mycobacterium bovis bacillus Calmette-Guerin

We investigated the effects of viable, extended freeze-drying (EFD) or heat-killed (HK) Mycobacterium bovis bacillus CalmetteGuerin (BCG) in respiratory burst activity, gene expression of CYBB and NCF1 encoding components of the human phagocyte nicotinamide adenine dinucleotide (NADPH) oxidase, TLR2 expression, and in IL-10 and TNF-a cytokine production by human peripheral blood mononuclear cells (PBMCs). Viable BCG significantly inhibited TLR2 and CYBB gene expression, as well as superoxide release by human PBMC. All BCG stimuli augmented IL-10 release, but only HK BCG or viable BCG increased TNF-a release by PBMCs. Our studies show that viable BCG can impair the NADPH oxidase system activation and the TLR2 route in human PBMCs. As well, different BCG preparations can distinctly influence cytokine production by human PBMCs.

Modulation of Lung Allergic Response by Renal Ischemia and Reperfusion Injury

The Th1/Th2 balance represents an important factor in the pathogenesis of renal ischemia-reperfusion injury (IRI). In addition, IRI causes a systemic inflammation that can affect other tissues, such as the lungs. To investigate the ability of renal IRI to modulate pulmonary function in a specific model of allergic inflammation, C57Bl/6 mice were immunized with ovalbumin/albumen on days 0 and 7 and challenged with an ovalbumin (OA) aerosol on days 14 and 21. After 24 h of the second antigen challenge, the animals were subjected to 45 minutes of ischemia. After 24 h of reperfusion, the bronchoalveolar lavage (BAL) fluid, blood and lung tissue were collected for analysis. Serum creatinine levels increased in both allergic and non-immunized animals subjected to IRI. However, BAL analysis showed a reduction in the total cells (46%) and neutrophils (58%) compared with control allergic animals not submitted to IRI. In addition, OA challenge induced the phosphorylation of ERK and Akt and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lung homogenates. After renal IRI, the phosphorylation of ERK and expression of COX-2 and iNOS were markedly reduced; however, there was no difference in the phosphorylation of Akt between sham and ischemic OA-challenged animals. Mucus production was also reduced in allergic mice after renal IRI. IL-4, IL-5 and IL-13 were markedly down-regulated in immunized/challenged mice subjected to IRI. These results suggest that renal IRI can modulate lung allergic inflammation, probably by altering the Th1/Th2 balance and, at least in part, by changing cellular signal transduction factors. Copyright (C) 2012 S. Karger AG, Basel