A crucial role for TNF-alpha in mediating neutrophil influx induced by endogenously generated or exogenous chemokines, KC/CXCL1 and LIX/CXCL5

Background and purpose: Chemokines orchestrate neutrophil recruitment to inflammatory foci. In the present study, we evaluated the participation of three chemokines, KC/CXCL1, MIP-2/CXCL2 and LIX/CXCL5, which are ligands for chemokine receptor 2 (CXCR2), in mediating neutrophil recruitment in immune inflammation induced by antigen in immunized mice. Experimental approach: Neutrophil recruitment was assessed in immunized mice challenged with methylated bovine serum albumin, KC/CXCL1, LIX/CXCL5 or tumour necrosis factor (TNF)-alpha. Cytokine and chemokine levels were determined in peritoneal exudates and in supernatants of macrophages and mast cells by elisa. CXCR2 and intercellular adhesion molecule 1 (ICAM-1) expression was determined using immunohistochemistry and confocal microscopy. Key results: Antigen challenge induced dose- and time-dependent neutrophil recruitment and production of KC/CXCL1, LIX/CXCL5 and TNF-alpha, but not MIP-2/CXCL2, in peritoneal exudates. Neutrophil recruitment was inhibited by treatment with reparixin (CXCR1/2 antagonist), anti-KC/CXCL1, anti-LIX/CXCL5 or anti-TNF-alpha antibodies and in tumour necrosis factor receptor 1-deficient mice. Intraperitoneal injection of KC/CXCL1 and LIX/CXCL5 induced dose- and time-dependent neutrophil recruitment and TNF-alpha production, which were inhibited by reparixin or anti-TNF-alpha treatment. Macrophages and mast cells expressed CXCR2 receptors. Increased macrophage numbers enhanced, while cromolyn sodium (mast cell stabilizer) diminished, LIX/CXCL5-induced neutrophil recruitment. Macrophages and mast cells from immunized mice produced TNF-alpha upon LIX/CXCL5 stimulation. Methylated bovine serum albumin induced expression of ICAM-1 on mesenteric vascular endothelium, which was inhibited by anti-TNF-alpha or anti-LIX/CXCL5. Conclusion and implications: Following antigen challenge, CXCR2 ligands are produced and act on macrophages and mast cells triggering the production of TNF-alpha, which synergistically contribute to neutrophil recruitment through induction of the expression of ICAM-1.

Involvement of NO in the inhibitory effect of Calotropis procera latex protein fractions on leukocyte rolling, adhesion and infiltration in rat peritonitis model

Aim of the study: The latex of Calotropis procera has been used in the traditional medicinal system for the treatment of leprosy, ulcers, tumors, piles and diseases of liver, spleen, abdomen and toothache. it comprises of a non-dialyzable protein fraction (LP) that exhibits anti-inflammatory properties and a dialyzable fraction (DF) exhibiting pro-inflammatory properties. The present study was carried out to evaluate the effect of LP sub-fractions on neutrophil functions and nociception in rodent models and to elucidate the mediatory role of nitric oxide (NO). Material and methods: The LP was subjected to ion exchange chromatography and the effect of its three sub-fractions (LP(PI), LP(PII), and LP(PIII)) thus obtained was evaluated on leukocyte functions in the rat peritonitis model and on nociception in the mouse model. Results: LP sub-fractions exhibit distinct protein profile and produce a significant decrease in the carrageenan and DF induced neutrophil influx and exhibit anti-nociceptive property. The LP and its sub-fractions produced a marked reduction in the number of rolling and adherent leukocytes in the mesenteric microvasculature as revealed by intravital microscopy. The anti-inflammatory effect of LP(PI), the most potent anti-inflammatory fraction of LP, was accompanied by an increase in the serum levels of NO. Further, our study shows that NO is also involved in the inhibitory effect of LP(PI) on neutrophil influx. Conclusions: Our study shows that LP fraction of Calotropis procera comprises of three distinct sets of proteins exhibiting anti-inflammatory and anti-nociceptive properties of which LP(PI) was most potent in inhibiting neutrophil functions and its effects are mediated through NO production. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

PPAR-gamma agonist rosiglitazone prevents inflammatory periodontal bone loss by inhibiting osteoclastogenesis

Rosiglitazone (RGZ), an oral anti-hyperglycemic agent used for non-insulin-dependent diabetes mellitus, is a high-affinity synthetic agonist for peroxisome proliferator-activated receptor-gamma (PPAR-gamma). Both in vitro and in vivo experiments have also revealed that RGZ possesses anti-inflammatory properties. Therefore, in the present study, we investigated the anti-inflammatory effects of RGZ in a rat model of periodontal disease induced by ligature placed around the mandible first molars of each animal. Male Wister rats were divided into four groups: 1) animals without ligature placement receiving administration of empty vehicle (control); 2) animals with ligature receiving administration of empty vehicle; 3) animals with ligature receiving administration with oral RGZ (10 mg/kg/day); and 4) animals with ligature receiving administration of subcutaneous RGZ (10 mg/kg/day). Thirty days after induction of periodontal disease, the animals were sacrificed, and mandibles and gingival tissues were removed for further analysis. An in vitro assay was also employed to test the inhibitory effects of RGZ on osteoclastogenesis. Histomorphological and immunohistochemical analyses of periodontal tissue demonstrated that RGZ-treated animals presented decreased bone resorption, along with reduced RANKL expression, compared to those animals with ligature, but treated with empty vehicle. Corresponding to such results obtained from in vivo experiments, RGZ also suppressed in vitro osteoclast differentiation in the presence of RANKL in MOCP-5 osteoclast precursor cells, along with the down-regulation of the expression of RANKL-induced TRAP mRNA. These data indicated that RGZ may suppress the bone resorption by inhibiting RANKL-mediated osteoclastogenesis elicited during the course of experimental periodontitis in rats. (C) 2009 Elsevier B.V. All rights reserved.

Hydrogen Sulfide Prevents Ethanol-Induced Gastric Damage in Mice: Role of ATP-Sensitive Potassium Channels and Capsaicin-Sensitive Primary Afferent Neurons

The aim of this study was to evaluate the protective effect of hydrogen sulfide (H(2)S) on ethanol-induced gastric lesions in mice and the influence of ATP-sensitive potassium (K(ATP)) channels, capsaicin-sensitive sensory afferent neurons, and transient receptor potential vanilloid (TRPV) 1 receptors on such an effect. Saline and L-cysteine alone or with propargylglycine, sodium hydrogen sulfide (NaHS), or Lawesson`s reagent were administrated for testing purposes. For other experiments, mice were pretreated with glibenclamide, neurotoxic doses of capsaicin, or capsazepine. Afterward, mice received L-cysteine, NaHS, or Lawesson`s reagent. After 30 min, 50% ethanol was administrated by gavage. After 1 h, mice were sacrificed, and gastric damage was evaluated by macroscopic and microscopic analyses. L-Cysteine, NaHS, and Lawesson`s reagent treatment prevented ethanol-induced macroscopic and microscopic gastric damage in a dose-dependent manner. Administration of propargylglycine, an inhibitor of endogenous H(2)S synthesis, reversed gastric protection induced by L-cysteine. Glibenclamide reversed L-cysteine, NaHS, or Lawesson`s reagent gastroprotective effects against ethanol-induced macroscopic damage in a dose-dependent manner. Chemical ablation of sensory afferent neurons by capsaicin reversed gastroprotective effects of L-cysteine or H(2)S donors (NaHS or Lawesson`s reagent) in ethanol-induced macroscopic gastric damage. Likewise, in the presence of the TRPV1 antagonist capsazepine, the gastroprotective effects of L-cysteine, NaHS, or Lawesson`s reagent were also abolished. Our results suggest that H(2)S prevents ethanol-induced gastric damage. Although there are many mechanisms through which this effect can occur, our data support the hypothesis that the activation of K(ATP) channels and afferent neurons/TRPV1 receptors is of primary importance.

Fructose-1,6-bisphosphate reduces inflammatory pain-like behaviour in mice: role of adenosine acting on A(1) receptors

Background and purpose: D-Fructose-1,6-bisphosphate (FBP) is an intermediate in the glycolytic pathway, exerting pharmacological actions on inflammation by inhibiting cytokine production or interfering with adenosine production. Here, the possible antinociceptive effect of FBP and its mechanism of action in the carrageenin paw inflammation model in mice were addressed, focusing on the two mechanisms described above. Experimental approach: Mechanical hyperalgesia (decrease in the nociceptive threshold) was evaluated by the electronic pressure-metre test; cytokine levels were measured by elisa and adenosine was determined by high performance liquid chromatography. Key results: Pretreatment of mice with FBP reduced hyperalgesia induced by intraplantar injection of carrageenin (up to 54%), tumour necrosis factor alpha (40%), interleukin-1 beta (46%), CXCL1 (33%), prostaglandin E(2) (41%) or dopamine (55%). However, FBP treatment did not alter carrageenin-induced cytokine (tumour necrosis factor alpha and interleukin-1 beta) or chemokine (CXCL1) production. On the other hand, the antinociceptive effect of FBP was prevented by systemic and intraplantar treatment with an adenosine A(1) receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine), suggesting that the FBP effect is mediated by peripheral adenosine acting on A(1) receptors. Giving FBP to mice increased adenosine levels in plasma, and adenosine treatment of paw inflammation presented a similar antinociceptive mechanism to that of FBP. Conclusions and implications: In addition to anti-inflammatory action, FBP also presents an antinociceptive effect upon inflammatory hyperalgesia. Its mechanism of action seems dependent on adenosine production but not on modulation of hyperalgesic cytokine/chemokine production. In turn, adenosine acts peripherally on its A(1) receptor inhibiting hyperalgesia. FBP may have possible therapeutic applications in reducing inflammatory pain.

Essential role of platelet-activating factor receptor in the pathogenesis of Dengue virus infection

Severe dengue infection in humans causes a disease characterized by thrombocytopenia, increased levels of cytokines, increased vascular permeability, hemorrhage, and shock. Treatment is supportive. Activation of platelet-activating factor (PAF) receptor (PAFR) on endothelial cells and leukocytes induces increase in vascular permeability, hypotension, and production of cytokines. We hypothesized that activation of PAFR could account for the major systemic manifestations of dengue infection. Inoculation of adult mice with an adapted strain of Dengue virus caused a systemic disease, with several features of the infection in humans. In PAFR(-/-) mice, there was decreased thrombocytopenia, hemoconcentration, decreased systemic levels of cytokines, and delay of lethality, when compared with WT infected mice. Treatment with UK-74,505, an orally active PAFR antagonist, prevented the above-mentioned manifestations, as well as hypotension and increased vascular permeability, and decreased lethality, even when started 5 days after virus inoculation. Similar results were obtained with a distinct PAFR antagonist, PCA-4246. Despite decreased disease manifestation, viral loads were similar (PAFR(-/-)) or lower (PAFR antagonist) than in WT mice. Thus, activation of PAFR plays a major role in the pathogenesis of experimental dengue infection, and its blockade prevents more severe disease manifestation after infection with no increase in systemic viral titers, suggesting that there is no interference in the ability of the murine host to deal with the infection. PAFR antagonists are disease-modifying agents in experimental dengue infection.

Anti-inflammatory activity and possible mechanism of extract from Mikania laevigata in carrageenan-induced peritonitis

Objectives The aim was to test the potential use of an extract of Mikania laevigata (popularly known in Brazil as guaco), made from leaves harvested in different months of the year, oil neutrophil migration after all inflammatory Stimulus and investigate the underlying molecular mechanisms. Methods We examined the effect of guaco on vascular permeability and leucocyte function in carrageenan-induced peritonitis in mice. Key findings Our results demonstrated that guaco extract administered subcutaneously (3 mg/kg) decreased the vascular permeability and also leucocyte rolling and adhesion to the inflamed tissues by a mechanism dependent on nitric oxide. Specifically, inhibitors of nitric oxide synthase remarkably abrogated the guaco extract-mediated suppression of neutrophil migration to the inflammatory site. In addition, guaco extract-mediated suppression of neutrophil migration appeared to be dependent on the production of the cytokines interleukin-1 beta and tumour necrosis factor-alpha. One of the major constituents of the guaco extract, coumarin, was able to inhibit the neutrophil migration towards the inflammatory focus. Conclusions In conclusion the anti-inflammatory effect induced by guaco extract may be by inhibition of pro-inflammatory cytokine production at the inflammatory site.

Inhibition of hydrogen sulphide formation reduces cisplatin-induced renal damage

Background. Cisplatin (CP)-induced renal damage is associated with inflammation. Hydrogen sulphide (H(2)S) is involved in models of inflammation. This study evaluates the effect of DL-propargylglycine (PAG), an inhibitor of endogenous H(2)S formation, on the renal damage induced by CP. Methods. The rats were injected with CP (5 mg/kg, i.p.) or PAG(5 mg/kg twice a day, i.p.) for 4 days, starting 1 h before CP injection. Control rats were injected with 0.15 M NaCl or PAG only. Blood and urine samples were collected 5 days after saline or CP injections for renal function evaluation. The kidneys were removed for tumour necrosis factor (TNF)-alpha quantification, histological, immunohistochemical and Western blot analysis. The cystathionine gamma-lyase (CSE) activity and expression were assessed. The direct toxicity of H(2)S in renal tubular cells was evaluated by the incubation of these cells with NaHS, a donor of H(2)S. Results. CP-treated rats presented increases in plasma creatinine levels and in sodium and potassium fractional excretions associated with tubulointerstitial lesions in the outer medulla. Increased expression of TNF-alpha, macrophages, neutrophils and T lymphocytes, associated with increased H(2)S formation rate and CSE expression, were also observed in the outer medulla from CP-injected rats. All these alterations were reduced by treatment with PAG. A direct toxicity of NaHS for renal tubular epithelial cells was not observed. Conclusions. Treatment with PAG reduces the renal damage induced by CP. This effect seems to be related to the H2S formation and the restriction of the inflammation in the kidneys from PAG+CP-treated rats.

Carbon monoxide and nitric oxide modulate hyperosmolality-induced oxytocin secretion by the hypothalamus in vitro

OT (oxytocin) is secreted from the posterior pituitary gland, and its secretion has been shown to be modulated by NO (nitric oxide). In rats, OT secretion is also stimulated by hyperosmolarity of the extracellular fluid. Furthermore, NOS (nitric oxide synthase) is located in hypothalamic areas involved in fluid balance control. In the present study, we evaluated the role of the NOS/NO and HO (haem oxygenase)/CO (carbon monoxide) systems in the osmotic regulation of OT release from rat hypothalamus in vitro. We conducted experiments on hypothalamic fragments to determine the following: (i) whether NO donors and NOS inhibitors modulate OT release and (ii) whether the changes in OT response occur concurrently with changes in NOS or HO activity in the hypothalamus. Hyperosmotic stimulation induced a significant increase in OT release that was associated with a reduction in nitrite production. Osmotic stimulation of OT release was inhibited by NO donors. NOS inhibitors did not affect either basal or osmotically stimulated OT release. Blockade of HO inhibited both basal and osmotically stimulated OT release, and induced a marked increase in NOS activity. These results indicate the involvement of CO in the regulation of NOS activity. The present data demonstrate that hypothalamic OT release induced by osmotic stimuli is modulated, at least in part, by interactions between NO and CO.

Polymorphisms of Lewis and Secretor genes are related to breast cancer and metastasis in axillary lymph nodes

ABH and Lewis antigen expression has been associated with cancer development and prognosis, tumor differentiation, and metastasis. Considering that invasive ductal breast carcinoma (IDC) presents multiple molecular alterations, the aim of the present study was to determine whether the polymorphism of ABO, Lewis, and Secretor genes, as well as ABO phenotyping, could be associated with tumor differentiation and lymph nodes metastasis. Seventy-six women with IDC and 78 healthy female blood donors were submitted to ABO phenotyping/genotyping and Lewis and Secretor genotyping. Phenotyping was performed by hemagglutination and genotyping by the polymerase chain reaction with sequence-specific primers. ABO, Lewis, and Secretor genes were classified by individual single nucleotide polymorphism at sites 59, 1067, 202, and 314 of the Lewis gene, 428 of the Secretor gene, and 261 (O1 allele), 526 (O2 and B allele), and 703 (B allele). No association was found between breast cancer and ABO antigen expression (P = 0.9323) or genotype (P = 0.9356). Lewis-negative genotype was associated with IDC (P = 0.0126) but not with anatomoclinical parameters. Nonsecretor genotype was associated with axillary lymph node metastasis (P = 0.0149). In conclusion, Lewis and Secretor genotyping could be useful to predict respectively breast cancer susceptibility and axillary lymph nodes metastasis.

Distribution of fentanyl in the placental intervillous space and in the different maternal and fetal compartments in term pregnant women

Fentanyl is used in obstetrical practice to promote analgesia and anesthesia during labor and in cesarean delivery, with rapid and short-term effects. To determine fentanyl concentrations in maternal plasma, in the placental intervillous space, and in the umbilical artery and vein in term pregnant women. Ten healthy pregnant women underwent epidural anesthesia with fentanyl plus bupivacaine and lidocaine, and fentanyl concentrations were determined in the various maternal and fetal compartments, including the placental intervillous space, which has not been previously studied in the literature. The ratios of fentanyl concentrations in the various maternal and fetal compartments revealed an 86% rate of placental fentanyl transfer. The highest fentanyl concentrations were detected in the placental intervillous space, being 2.19 times higher than in maternal plasma, 2.8 times higher than in the umbilical vein and 3.6 times higher than in the umbilical artery, with no significant differences between the umbilical vein and artery, demonstrating that there was no drug uptake by fetal tissues nor metabolism of the drug by the fetus despite the high rates of placental transfer. The present study demonstrated that the placental intervillous space acted as a site of fentanyl deposit, a fact that may be explained by two hypotheses: (1) the blood collected from the placental intervillous space is arterial and, according to some investigators, the arterial plasma concentrations of the drugs administered to patients undergoing epidural anesthesia are higher than the venous concentrations, and (2) a possible role of P-glycoprotein (P-gp).

Pharmacokinetics of lidocaine and its metabolite in peridural anesthesia administered to pregnant women with gestational diabetes mellitus

Background Peridural blockade with lidocaine, bupivacaine, and fentanyl is an anesthetic procedure extensively used in obstetrics, justifying the pharmacokinetic study of these drugs during labor. Objective To investigate the influence of the physiopathological changes of gestational diabetes mellitus (GDM) on the pharmacokinetics of lidocaine and its metabolite monoethylglycinexylidide (MEGX) in pregnant women subjected to peridural anesthesia. Patients and methods Ten normal pregnant women (group 1) and six pregnant women with GDM (group 2) were studied, all of them at term. The patients received 200 mg 2% lidocaine hydrochloride without a vasoconstrictor by the peridural locoregional route. Maternal blood samples were collected at predetermined times for the analysis of lidocaine and MEGX by chromatography and pharmacokinetic analysis. Results The median pharmacokinetic parameters of lidocaine for groups 1 and 2 (P <= 0.05), respectively, were as follows: for Cmax 879.11 and 1,145.58 ng/ml, AUC(0-infinity) 256.01 and 455.95 wg min(-1) ml(-1), Cl/f/kg 10.61 and 5.64 ml min(-1) kg(-1), and Vd/f/kg 3.26 and 2.19 L/kg. The median pharmacokinetic parameters of MEGX for groups 1 and 2 (P <= 0.05), respectively, were as follows: for Cmax 82.71 and 141.38 ng/ml, Tmax 44.71 and 193.14 min, t(1/2)alpha 7.64 and 59.77 min, alpha 0.097 and 0.012/min, and AUC(0-infinity) 29.91 and 108.23 mu g min(-1) ml(-1). Conclusion The present data permit us to conclude that the apparent clearance of lidocaine and MEGX was reduced in diabetic patients compared to normal women, suggesting that GDM inhibits the CYP1A2/CYP3A4 isoforms responsible for the metabolism of this drug and its metabolite.

Distribution of Bupivacaine Enantiomers and Lidocaine and Its Metabolite in the Placental Intervillous Space and in the Different Maternal and Fetal Compartments in Term Pregnant Women

The aim of this study is to determine the concentrations of lidocaine and its metabolite, monoethylglycine xylidide (MEGX), and of the enantiomers of bupivacaine in maternal and fetal compartments. Ten healthy pregnant women were submitted to epidural anesthesia. Drug concentrations were determined in the maternal vein, fetal umbilical artery and vein, and the placental intervillous space. The highest concentrations of the bupivacaine enantiomers lidocaine and of lidocaine and of its MEGX metabolite were detected in maternal plasma and in the placental intervillous space. The placental transfer was 33% for the (+)-(R)-bupivacaine enantiomer and 31% for the (-)-(S)-bupivacaine enantiomer. For lidocaine and its MEGX metabolite, respective placental transfers were 60% and 43%. Lidocaine concentration in the fetal umbilical vein was 1.46 times higher than in the fetal umbilical artery. The highest concentrations of lidocaine and its metabolite and of the enantiomers of bupivacaine were detected in the placental intervillous space. The higher lidocaine concentrations in the fetal umbilical vein than in the fetal umbilical artery suggest that there was tissue uptake of the drug or drug metabolization by the fetus.

Knowledge and practices of medical students to prevent tuberculosis transmission in Rio de Janeiro, Brazil

Objectives. To describe knowledge, practices, and associated factors of medical students to prevent transmission of tuberculosis (TB) in five medical schools. Methods. Cross-sectional survey of undergraduate medical students in preclinical and in early and late clinical years. Information was obtained on sociodemographic profile, previous lectures on TB, knowledge about TB transmission, exposure to patients with active pulmonary TB, and use of respiratory protective masks. Results. Among 1 094 respondents, 575 (52.6%) correctly answered that coughing, speaking, and sneezing can transmit TB. Early [adjusted odds ratio = 4.0 (3.0, 5.5)] and late [adjusted odds ratio = 4.2 (3.1, 5.8)] clinical years were associated with correct answers, but having had previous lectures on TB was not. Among those who had previous lectures on TB, the rate of correct answers increased from 42.1% to 61.6%. Among 332 medical students who reported exposure to TB patients, 194 (58.4%) had not used protective masks. More years of clinical experience was associated with the use of masks [adjusted odds ratio = 2.9 (1.4, 6.1)], while knowledge was inversely associated with the use of masks [adjusted odds ratio = 0.4 (0.2, 0.6)]. Conclusions. Many medical students are not aware of the main routes of TB infection, and lectures on TB are not sufficient to change knowledge and practices. Regardless of knowledge about TB transmission, students engage in risky behaviors: more than two-thirds do not use a protective mask when examining an active TB case. We suggest innovative, effective active learning experiences to change this scenario.