Involvement of PGE(2) and RANTES in Staphylococcus aureus-induced fever in rats

Martins JM, Longhi-Balbinot DT, Soares DM, Figueiredo MJ, Malvar D do C, de Melo MC, Rae GA, Souza GE. Involvement of PGE(2) and RANTES in Staphylococcus aureus-induced fever in rats. J Appl Physiol 113: 1456-1465, 2012. First published August 30, 2012; doi:10.1152/japplphysiol.00936.2011.-This study investigated the involvement of prostaglandins and regulated on activation, normal T cell expressed and secreted (RANTES), in fever induced by live Staphylococcus aureus (no. 25923, American Type Culture Collection) injection in rats. S. aureus was injected intraperitoneally at 10(9), 10(10), and 2 x 10(10) colony-forming units (CFU)/cavity, and body temperature (T-b) was measured by radiotelemetry. The lowest dose of S. aureus induced a modest transient increase in T-b, whereas the two higher doses promoted similar long-lasting and sustained T-b increases. Thus, the 10(10) CFU/cavity dose was chosen for the remaining experiments. The T-b increase induced by S. aureus was accompanied by significant decreases in tail skin temperature and increases in PGE(2) levels in the cerebrospinal fluid (CSF) and hypothalamus but not in the venous plasma. Celecoxib (selective cyclooxygenase-2 inhibitor, 2.5 mg/kg po) inhibited the fever and the increases in PGE(2) concentration in the CSF and hypothalamus induced by S. aureus. Dipyrone (120 mg/kg ip) reduced the fever from 2.5 to 4 h and the PGE(2) increase in the CSF but not in the hypothalamus. S. aureus increased RANTES in the peritoneal exudate but not in the CSF or hypothalamus. Met-RANTES (100 mu g/kg iv), a chemokine (C-C motif) receptor (CCR)1/CCR5 antagonist, reduced the first 6 h of fever induced by S. aureus. This study suggests that peripheral (local) RANTES and central PGE(2) production are key events in the febrile response to live S. aureus injection. As dipyrone does not reduce PGE(2) synthesis in the hypothalamus, it is plausible that S. aureus induces fever, in part, via a dipyrone-sensitive PGE(2)-independent pathway.

Lipopolysaccharide-Induced Sickness Behaviour Evaluated in Different Models of Anxiety and Innate Fear in Rats

The fact that there is a complex and bidirectional communication between the immune and nervous systems has been well demonstrated. Lipopolysaccharide (LPS), a component of gram-negative bacteria, is widely used to systematically stimulate the immune system and generate profound physiological and behavioural changes, also known as sickness behaviour (e.g. anhedonia, lethargy, loss of appetite, anxiety, sleepiness). Different ethological tools have been used to analyse the behavioural modifications induced by LPS; however, many researchers analysed only individual tests, a single LPS dose or a unique ethological parameter, thus leading to disagreements regarding the data. In the present study, we investigated the effects of different doses of LPS (10, 50, 200 and 500 mu g/kg, i.p.) in young male Wistar rats (weighing 180200 g; 89 weeks old) on the ethological and spatiotemporal parameters of the elevated plus maze, light-dark box, elevated T maze, open-field tests and emission of ultrasound vocalizations. There was a dose-dependent increase in anxiety-like behaviours caused by LPS, forming an inverted U curve peaked at LPS 200 mu g/kg dose. However, these anxiety-like behaviours were detected only by complementary ethological analysis (stretching, grooming, immobility responses and alarm calls), and these reactions seem to be a very sensitive tool in assessing the first signs of sickness behaviour. In summary, the present work clearly showed that there are resting and alertness reactions induced by opposite neuroimmune mechanisms (neuroimmune bias) that could lead to anxiety behaviours, suggesting that misunderstanding data could occur when only few ethological variables or single doses of LPS are analysed. Finally, it is hypothesized that this bias is an evolutionary tool that increases animals security while the body recovers from a systemic infection.

A crucial role for IL-6 in the CNS of rats during fever induced by the injection of live E. coli

Interleukin (IL)-1 beta, tumor necrosis factor (TNF)-alpha, and IL-6 have been established as important mediators of fever induced by lipopolysaccharide (LPS) from Gram-negative bacteria. Whether these pro-inflammatory cytokines are also important in mediating fever induced by live bacteria remains less certain. We therefore investigated the following: (1) the synthesis of TNF-alpha, IL-1 beta, and IL-6 during E. coli-induced fever and (2) the effect of blocking the action of cytokines within the brain on E. coli-induced fever. Body or tail skin temperature (bT or Tsk, respectively) was measured by biotelemetry or telethermometry, every 30 min, during 6 or 24 h. Depending on the number of colony-forming units (CFU) injected i.p., administration of E. coli induced a long-lasting increase in bT of male Wistar rats. The duration of fever did not correlate with the number of CFU found in peritoneal cavity or blood. Because 2.5 x 10(8) CFU induced a sustained fever without inducing a state of sepsis/severe infection, this dose was used in subsequent experiments. The E. coli-induced increase in bT was preceded by a decrease in Tsk, reflecting a thermoregulatory response. TNF-alpha, IL-1 beta, and IL-6 were detected at 3 h in serum of animals injected i.p. with E. coli. In the peritoneal exudates, TNF-alpha, IL-1 beta, and IL-6 were detected at 0.5 and 3 h after E. coli administration. Moreover, both IL-1 beta and IL-6, but not TNF-alpha, were found in the cerebrospinal fluid (CSF) and hypothalamus of animals injected with E. coli. Although pre-treatment (i.c.v., 2 mu l, 15 min before) with anti-IL-6 antibody (anti-IL-6, 5 mu g) reduced E. coli-induced fever, pre-treatment with either IL-1 receptor antagonist (IL-1ra, 200 mu g) or soluble TNF receptor I (sTNFRI, 500 ng) had no effect on the fever response. In conclusion, replicating E. coli promotes an integrated thermoregulatory response in which the central action of IL-6, but not IL-1 and TNF, appears to be important.

Febrile response induced by cecal ligation and puncture (CLP) in rats: involvement of prostaglandin E-2 and cytokines

The purpose of the present study was to better understand the events involved in the febrile response induced by cecal ligation and puncture (CLP), a complex infectious process. To this end, we conducted in vivo experiments in rats examining (1) fever development, (2) bacterial number in the infection focus and in blood, (3) peripheral and hypothalamic synthesis of cytokines, (4) hypothalamic and cerebrospinal fluid (CSF) synthesis of prostaglandin E-2 (PGE(2)), (5) the effect of anti-IL-6 antibody on fever, and (6) the effect of celecoxib on fever and hypothalamic synthesis of PGE(2) after CLP induction. We found that CLP promotes fever and animal death depending on the number of punctures. The peak of CLP-induced fever overlapped with the maximal increase in the number of bacteria in the infectious focus and blood, which occurred at 6 and 12 h. The peak of the febrile response also coincided with increased amounts of interleukin (IL)-1 beta, IL-6 and IL-10 in the peritoneal exudate and serum; IL-6 in the hypothalamus and PGE(2) in the CSF and predominantly in the hypothalamus. Moreover, intracerebroventricularly injected anti-IL-6 antibody reduced the febrile response while celecoxib reduced the fever and PGE(2) amount in the hypothalamus induced by CLP. Tumor necrosis factor (TNF)-alpha peaked at 3 h at all sites studied. Conversely, IL-10 concentration decreased in the hypothalamus. These findings show that the peak of CLP-induced fever is accompanied by an increase of bacteria in peritoneal fluid (local infection) and blood; local synthesis of pyrogenic (IL-1 beta, IL-6) and antipyretic (IL-10) cytokines and central production of IL-6 and PGE(2), suggesting that these last are the central mediators of this response.

Cannabidiol, a non-psychotropic plant-derived cannabinoid, decreases inflammation in a murine model of acute lung injury: Role for the adenosine A(2A) receptor

Acute lung injury is an inflammatory condition for which treatment is mainly supportive because effective therapies have not been developed. Cannabidiol, a non-psychotropic cannabinoid component of marijuana (Cannabis sativa), has potent immunosuppressive and anti-inflammatory properties. Therefore, we investigated the possible anti-inflammatory effect of cannabidiol in a murine model of acute lung injury. Analysis of total inflammatory cells and differential in bronchoalveolar lavage fluid was used to characterize leukocyte migration into the lungs; myeloperoxidase activity of lung tissue and albumin concentration in the bronchoalveolar lavage fluid were analyzed by colorimetric assays; cytokine/chemokine production in the bronchoalveolar lavage fluid was also analyzed by Cytometric Bead Arrays and Enzyme-Linked Immunosorbent Assay (ELISA). A single dose of cannabidiol (20 mg/kg) administered prior to the induction of LPS (lipopolysaccharide)-induced acute lung injury decreases leukocyte (specifically neutrophil) migration into the lungs, albumin concentration in the bronchoalveolar lavage fluid, myeloperoxidase activity in the lung tissue, and production of pro-inflammatory cytokines (TNF and IL-6) and chemokines (MCP-1 and MIP-2) 1, 2, and 4 days after the induction of LPS-induced acute lung injury. Additionally, adenosine A(2A) receptor is involved in the anti-inflammatory effects of cannabidiol on LPS-induced acute lung injury because ZM241385 (4-(2[7-Amino-2-(2-furyl)[1,2,4] triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl) phenol) (a highly selective antagonist of adenosine A(2A) receptor) abrogated all of the anti-inflammatory effects of cannabidiol previously described. Thus, we show that cannabidiol has anti-inflammatory effects in a murine model of acute lung injury and that this effect is most likely associated with an increase in the extracellular adenosine offer and signaling through adenosine A(2A) receptor. (C) 2012 Elsevier B. V. All rights reserved.

Hypoactivity of the central dopaminergic system and autistic-like behavior induced by a single early prenatal exposure to lipopolysaccharide

The aim of the present study was to evaluate the behavioral patterns associated with autism and the prevalence of these behaviors in males and females, to verify whether our model of lipopolysaccharide (LPS) administration represents an experimental model of autism. For this, we prenatally exposed Wistar rats to LPS (100 mu g/kg, intraperitoneally, on gestational day 9.5), which mimics infection by gram-negative bacteria. Furthermore, because the exact mechanisms by which autism develops are still unknown, we investigated the neurological mechanisms that might underlie the behavioral alterations that were observed. Because we previously had demonstrated that prenatal LPS decreases striatal dopamine (DA) and metabolite levels, the striatal dopaminergic system (tyrosine hydroxylase [TH] and DA receptors D1a and D2) and glial cells (astrocytes and microglia) were analyzed by using immunohistochemistry, immunoblotting, and real-time PCR. Our results show that prenatal LPS exposure impaired communication (ultrasonic vocalizations) in male pups and learning and memory (T-maze spontaneous alternation) in male adults, as well as inducing repetitive/restricted behavior, but did not change social interactions in either infancy (play behavior) or adulthood in females. Moreover, although the expression of DA receptors was unchanged, the experimental animals exhibited reduced striatal TH levels, indicating that reduced DA synthesis impaired the striatal dopaminergic system. The expression of glial cell markers was not increased, which suggests that prenatal LPS did not induce permanent neuroinflammation in the striatum. Together with our previous finding of social impairments in males, the present findings demonstrate that prenatal LPS induced autism-like effects and also a hypoactivation of the dopaminergic system. (c) 2012 Wiley Periodicals, Inc.

Molecular confirmation of ovine herpesvirus 2-induced malignant catarrhal fever lesions in cattle from Rio Grande do Norte, Brazil

Molecular findings that confirmed the participation of ovine herpesvirus 2 (OVH-2) in the lesions that were consistent with those observed in malignant catarrhal fever of cattle are described. Three mixed-breed cattle from Rio Grande do Norte state demonstrated clinical manifestations that included mucopurulent nasal discharge, corneal opacity and motor incoordination. Routine necropsy examination demonstrated ulcerations and hemorrhage of the oral cavity, corneal opacity, and lymph node enlargement. Significant histopathological findings included widespread necrotizing vasculitis, non-suppurative meningoencephalitis, lymphocytic interstitial nephritis and hepatitis, and thrombosis. PCR assay performed on DNA extracted from kidney and mesenteric lymph node of one animal amplified a product of 423 base pairs corresponding to a target sequence within the ovine herpesvirus 2 (OVH-2) tegument protein gene. Direct sequencing of the PCR products, from extracted DNA of the kidney and mesenteric lymph node of one cow, amplified the partial nucleotide sequences (423 base pairs) of OVH-2 tegument protein gene. Blast analysis confirmed that these sequences have 98-100% identity with similar OVH-2 sequences deposited in GenBank. Phylogenetic analyses, based on the deduced amino acid sequences, demonstrated that the strain of OVH-2 circulating in ruminants from the Brazilian states of Rio Grande do Norte and Minas Gerais are similar to that identified in other geographical locations. These findings confirmed the active participation of OVH-2 in the classical manifestations of sheep associated malignant catarrhal fever.

Antidepressants prevent hierarchy destabilization induced by lipopolysaccharide administration in mice: a neurobiological approach to depression

In spite of the high prevalence and negative impact of depression, little is known about its pathophysiology. Basic research on depression needs new animal models in order to increase knowledge of the disease and search for new therapies. The work presented here aims to provide a neurobiologically validated model for investigating the relationships among sickness behavior, antidepressants treatment, and social dominance behavior. For this purpose, dominant individuals from dyads of male Swiss mice were treated with the bacterial endotoxin lipopolysaccharide (LPS) to induce social hierarchy destabilization. Two groups were treated with the antidepressants imipramine and fluoxetine prior to LPS administration. In these groups, antidepressant treatment prevented the occurrence of social destabilization. These results indicate that this model could be useful in providing new insights into the understanding of the brain systems involved in depression.

Cannabinoid CB1 receptor restrains accentuated activity of hypothalamic corticotropin-releasing factor and brainstem tyrosine hydroxylase neurons in endotoxemia-induced hypophagia in rats

It is well known that endocannabinoids play an important role in the regulation of food intake and body weight. Endocannabinoids and cannabinoid receptors are found in the hypothalamus and brainstem, which are central areas involved in the control of food intake and energy expenditure. Activation of these areas is related to hypophagia observed during inflammatory stimulus. This study investigated the effects of cannabinoid (CB1) receptor blockade on lipopolysaccharide (LPS)-induced hypophagia. Male Wistar rats were pretreated with rimonabant (10 mg/kg, by gavage) or vehicle; 30 min later they received an injection of either LPS (100 mu g/kg, intraperitoneal) or saline. Food intake, body weight, corticosterone response, CRF and CART mRNA expression, Fos-CRF and Fos-alpha-MSH immunoreactivity in the hypothalamus and Fos-tyrosine hydroxylase (TH) immunoreactivity in the brainstem were evaluated. LPS administration decreased food intake and body weight gain and increased plasma corticosterone levels and CRF mRNA expression in the PVN. We also observed an increase in Fos-CRF and Fos-TH double-labeled neurons after LPS injection in vehicle-pretreated rats, with no changes in CART mRNA or Fos-alpha-MSH immunoreactive neurons in the ARC. In saline-treated animals, rimonabant pretreatment decreased food intake and body weight gain but did not modify hormone response or Fos expression in the hypothalamus and brainstem compared with vehicle-pretreated rats. Rimonabant pretreatment potentiated LPS-induced hypophagia, body weight loss and Fos-CRF and Fos-TH expressing neurons. Rimonabant did not modify corticosterone, CRF mRNA or Fos-alpha-MSH responses in rats treated with LPS. These data suggest that the endocannabinoid system, mediated by CB1 receptors, modulates hypothalamic and brainstem circuitry underlying the hypophagic effect during endotoxemia to prevent an exaggerated food intake decrease. This article is part of a Special Issue entitled 'Central Control of Food Intake'. (C) 2011 Elsevier Ltd. All rights reserved.

INTERACTION BETWEEN THE CARBON MONOXIDE AND NITRIC OXIDE PATHWAYS IN THE LOCUS COERULEUS DURING FEVER

FAPESP [2010/50882-1]

DETERMINATION OF DICLOFENAC SODIUM IN RABBIT PLASMA BY HPLC/UV: EVALUATION AND CHARACTERIZATION OF ITS CRYSTALLINE FORMS, ANHYDROUS AND HYDRATE, ON THE ANTIPYRETIC EFFECT

Diclofenac sodium (DS) is a non-steroidal anti-inflammatory drug that is widely prescribed for the treatment of rheumatoid arthritis and post-surgery analgesia. The active pharmaceutical ingredient is the anhydrous form; however, it can also exist in hydrate form. In this context, knowing the properties of the solid state is important and relevant in the pharmaceutical area because they have a significant impact on the solubility, bioavailability, and chemical stability of the drugs. In the present study, data from XRPD, FTIR spectroscopy, and thermal analysis were used for the identification and characterization of DS forms (anhydrous and hydrate). An HPLC method was optimized to evaluate the plasma concentration of DS in rabbits. The optimized method exhibited good linearity over the range 0.1-60 mu g/mL with correlation coefficients of >0.9991. The mean recovery was 100%. Precision and accuracy were determined within acceptable limits. Finally, to compare the pharmacological properties of anhydrous and hydrate DS forms, we investigated their effects in the febrile response induced by lipopolysaccharide from E. coli in rabbits. The results show that the antipyretic effect of anhydrous and hydrate DS forms are similar.

TLR2, TLR4 and the MYD88 Signaling Pathway Are Crucial for Neutrophil Migration in Acute Kidney Injury Induced by Sepsis

The aim of this study was to investigate the role of TLR2, TLR4 and MyD88 in sepsis-induced AKI. C57BL/6 TLR2(-/-), TLR4(-/-) and MyD88(-/-) male mice were subjected to sepsis by cecal ligation and puncture (CLP). Twenty four hours later, kidney tissue and blood samples were collected for analysis. The TLR2(-/-), TLR4(-/-) and MyD88(-/-) mice that were subjected to CLP had preserved renal morphology, and fewer areas of hypoxia and apoptosis compared with the wild-type C57BL/6 mice (WT). MyD88(-/-) mice were completely protected compared with the WT mice. We also observed reduced expression of proinflammatory cytokines in the kidneys of the knockout mice compared with those of the WT mice and subsequent inhibition of increased vascular permeability in the kidneys of the knockout mice. The WT mice had increased GR1(+low) cells migration compared with the knockout mice and decreased in GR1(+high) cells migration into the peritoneal cavity. The TLR2(-/-), TLR4(-/-), and MyD88(-/-) mice had lower neutrophil infiltration in the kidneys. Depletion of neutrophils in the WT mice led to protection of renal function and less inflammation in the kidneys of these mice. Innate immunity participates in polymicrobial sepsis-induced AKI, mainly through the MyD88 pathway, by leading to an increased migration of neutrophils to the kidney, increased production of proinflammatory cytokines, vascular permeability, hypoxia and apoptosis of tubular cells.

Both adiponectin and interleukin-10 inhibit LPS-induced activation of the NF-kappa B pathway in 3T3-L1 adipocytes

Adiponectin and interleukin 10 (IL-10) are adipokines that are predominantly secreted by differentiated adipocytes and are involved in energy homeostasis, insulin sensitivity, and the anti-inflammatory response. These two adipokines are reduced in obese subjects, which favors increased activation of nuclear factor kappa B (NF-kappa B) and leads to elevation of pro-inflammatory adipokines. However, the effects of adiponectin and IL-10 on NF-kappa B DNA binding activity (NF-kappa Bp50 and NF-kappa Bp65) and proteins involved with the toll-like receptor (TLR-2 and TLR-4) pathway, such as MYD88 and TRAF6 expression, in lipopolysaccharide-treated 3T3-L1 adipocytes are unknown. Stimulation of lipopolysaccharide-treated 3T3-L1 adipocytes for 24 h elevated IL-6 levels; activated the NF-kappa B pathway cascade; increased protein expression of IL-6R, TLR-4, MYD88, and TRAF6; and increased the nuclear activity of NF-kappa B (p50 and p65) DNA binding. Adiponectin and IL-10 inhibited the elevation of IL-6 levels and activated NF-kappa B (p50 and p65) DNA binding. Taken together, the present results provide evidence that adiponectin and IL-10 have an important role in the anti-inflammatory response in adipocytes. In addition, inhibition of NF-kappa B signaling pathways may be an excellent strategy for the treatment of inflammation in obese individuals. (C) 2011 Elsevier Ltd. All rights reserved.

Pathogenesis of reproductive failure induced by Trypanosoma vivax in experimentally infected pregnant ewes

The present study was aimed at investigating the effect of experimental infection by Trypanosoma vivax in different stages of pregnancy, determining the pathogenesis of reproductive failure, and confirming transplacental transmission. We used 12 pregnant ewes distributed into four experimental groups: G1, was formed by three ewes infected with T. vivax in the first third of pregnancy (30 days); G2 comprised three infected ewes in the final third of pregnancy (100 days); G3 and G4 were composed of three non-infected ewes with the same gestational period, respectively. Each ewe of G1 and G2 was inoculated with 1.25 × 105 tripomastigotes. Clinical examination, determination of parasitemia, serum biochemistry (albumin, total protein, glucose, cholesterol, and urea), packed cell volume (PCV), serum progesterone, and pathological examination were performed. Placenta, amniotic fluid, blood and tissues from the fetuses and stillbirths were submitted to PCR. Two ewes of G1 (Ewe 1 and 3) presented severe infection and died in the 34th and 35th days post-infection (dpi), respectively; but both fetuses were recovered during necropsy. In G2, Ewe 5 aborted two fetuses on the 130th day (30 dpi) of pregnancy; and Ewe 6 aborted one fetus in the 140th day (40 dpi) of gestation. Ewes 2 and 4 delivered two weak lambs that died five days after birth. Factors possibly involved with the reproductive failure included high parasitemia, fever, low PCV, body score, serum glucose, total protein, cholesterol, and progesterone. Hepatitis, pericarditis, and encephalitis were observed in the aborted fetuses. The presence of T. vivax DNA in the placenta, amniotic fluid, blood, and tissues from the fetuses confirms the transplacental transmission of the parasite. Histological lesion in the fetuses and placenta also suggest the involvement of the parasite in the etiopathogenesis of reproductive failure in ewes.

Protective effects of aerobic exercise on acute lung injury induced by LPS in mice

Abstract Introduction The regular practice of physical exercise has been associated with beneficial effects on various pulmonary conditions. We investigated the mechanisms involved in the protective effect of exercise in a model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Methods Mice were divided into four groups: Control (CTR), Exercise (Exe), LPS, and Exercise + LPS (Exe + LPS). Exercised mice were trained using low intensity daily exercise for five weeks. LPS and Exe + LPS mice received 200 µg of LPS intratracheally 48 hours after the last physical test. We measured exhaled nitric oxide (eNO); respiratory mechanics; neutrophil density in lung tissue; protein leakage; bronchoalveolar lavage fluid (BALF) cell counts; cytokine levels in BALF, plasma and lung tissue; antioxidant activity in lung tissue; and tissue expression of glucocorticoid receptors (Gre). Results LPS instillation resulted in increased eNO, neutrophils in BALF and tissue, pulmonary resistance and elastance, protein leakage, TNF-alpha in lung tissue, plasma levels of IL-6 and IL-10, and IL-1beta, IL-6 and KC levels in BALF compared to CTR (P ≤0.02). Aerobic exercise resulted in decreases in eNO levels, neutrophil density and TNF-alpha expression in lung tissue, pulmonary resistance and elastance, and increased the levels of IL-6, IL-10, superoxide dismutase (SOD-2) and Gre in lung tissue and IL-1beta in BALF compared to the LPS group (P ≤0.04). Conclusions Aerobic exercise plays important roles in protecting the lungs from the inflammatory effects of LPS-induced ALI. The effects of exercise are mainly mediated by the expression of anti-inflammatory cytokines and antioxidants, suggesting that exercise can modulate the inflammatory-anti-inflammatory and the oxidative-antioxidative balance in the early phase of ALI.