Nylane Maria Nunes de Alencar

Antitumor properties of a sulfated polysaccharide from the red seaweed Champia feldmannii (Diaz‐Pifferer)

In recent years, much attention has been focused on polysaccharides isolated from natural sources. The aim of this study was to investigate the in vitro and in vivo antitumor properties of a sulfated polysaccharide isolated from the seaweed C. feldmannii (Cf‐PLS). Hematological, biochemical and histopathological analyses were performed in order to evaluate the toxicological aspects related to Cf‐PLS treatment. Its effects on the immunological system were also investigated. The Cf‐PLS did not show any significant in vitro cytotoxicity at the experimental exposure levels that were used, but showed in vivo antitumor effect. The inhibition rates of sarcoma 180 tumor development were 48.62 and 48.16% at the doses of 10 and 25 mg kg−1, respectively. In addition, Cf‐PLS was also able to increase the response elicited by 5‐fluorouracil (5‐FU) from 48.66 to 68.32%. The histopathological analysis of liver and kidney showed that both organs were moderately affected by Cf‐PLS‐treatment. Neither enzymatic activity of alanine aminotransferase nor urea or creatinine levels were significantly altered. In hematological analysis, leucopeny was observed after 5‐FU treatment, but this effect was prevented when the treatment was associated with the Cf‐PLS. It was also demonstrated that Cf‐PLS acts as an immunomodulatory agent, raising the production of specific antibodies, and increasing the production of OVA‐specific antibodies. It also induced a discreet hyperplasia of lymphoid folicules of the white pulp in the spleen of treated mice. In conclusion, Cf‐PLS has some interesting anticancer activity that could be associated with its immunostimulating properties. Copyright

Hydrogen sulfide augments neutrophil migration through enhancement of adhesion molecule expression and prevention of CXCR2 internalization: role of ATP-sensitive potassium channels.

In this study, we have addressed the role of H2S in modulating neutrophil migration in either innate (LPS-challenged naive mice) or adaptive (methylated BSA (mBSA)-challenged immunized mice) immune responses. Treatment of mice with H2S synthesis inhibitors, dl-propargylglycine (PAG) or β-cyanoalanine, reduced neutrophil migration induced by LPS or methylated BSA (mBSA) into the peritoneal cavity and by mBSA into the femur/tibial joint of immunized mice. This effect was associated with decreased leukocyte rolling, adhesion, and P-selectin and ICAM-1 expression on endothelium. Predictably, treatment of animals with the H2S donors, NaHS or Lawesson’s reagent, enhanced these parameters. Moreover, the NaHS enhancement of neutrophil migration was not observed in ICAM-1-deficient mice. Neither PAG nor NaHS treatment changed LPS-induced CD18 expression on neutrophils, nor did the LPS- and mBSA-induced release of neutrophil chemoattractant mediators TNF-α, keratinocyte-derived chemokine, and LTB4. Furthermore, in vitro MIP-2-induced neutrophil chemotaxis was inhibited by PAG and enhanced by NaHS treatments. Accordingly, MIP-2-induced CXCR2 internalization was enhanced by PAG and inhibited by NaHS treatments. Moreover, NaHS prevented MIP-2-induced CXCR2 desensitization. The PAG and NaHS effects correlated, respectively, with the enhancement and inhibition of MIP-2-induced G protein-coupled receptor kinase 2 expression. The effects of NaHS on neutrophil migration both in vivo and in vitro, together with CXCR2 internalization and G protein-coupled receptor kinase 2 expression were prevented by the ATP-sensitive potassium (KATP+) channel blocker, glybenclamide. Conversely, diazoxide, a KATP+ channel opener, increased neutrophil migration in vivo. Together, our data suggest that during the inflammatory response, H2S augments neutrophil adhesion and locomotion, by a mechanism dependent on KATP+ channels.

Leguminous lectins as tools for studying the role of sugar residues in leukocyte recruitment

The natural physiological ligands for selectins are oligosaccharides found in glycoprotein or glycolipid molecules in cell membranes. In order to study the role of sugar residues in the in vivo lectin anti-inflammatory effect, we tested three leguminous lectins with different carbohydrate binding affinities in the peritonitis and paw oedema models induced by carrageenin in rats. L. sericeus lectin was more anti-inflammatory than D. virgata lectin, the effects being reversed by their specific binding sugars (N-acetylglucosamine and alpha-methylmannoside, respectively). However, V. macrocarpa, a galactose-specific lectin, was not anti-inflammatory. The proposed anti-inflammatory activity of lectins could be due to a blockage of neutrophil-selectin carbohydrate ligands. Thus, according to the present data, we suggest an important role for N-acetylglucosamine residue as the major ligand for selectins on rat neutrophil membranes.

Latex constituents from Calotropis procera (R. Br.) display toxicity upon egg hatching and larvae of Aedes aegypti (Linn.)

Calotropis procera R. Br. (Asclepiadaceae) is a well-known medicinal plant with leaves, roots, and bark being exploited by popular medicine to fight many human and animal diseases. This work deals with the fractionation of the crude latex produced by the green parts of the plant and aims to evaluate its toxic effects upon egg hatching and larval development of Aedes aegypti. The whole latex was shown to cause 100% mortality of 3rd instars within 5 min. It was fractionated into water-soluble dialyzable (DF) and non-dialyzable (NDF) rubber-free materials. Both fractions were partially effective to prevent egg hatching and most of individuals growing under experimental conditions died before reaching 2nd instars or stayed in 1st instars. Besides, the fractions were very toxic to 3rd instars causing 100% mortality within 24 h. When both fractions were submitted to heat-treatment the toxic effects were diminished considerably suggesting low thermostability of the toxic compounds. Polyacrylamide gel electrophoresis of both fractions and their newly fractionated peaks obtained through ion exchange chromatography or desalting attested the presence of proteins in both materials. When submitted to protease digestion prior to larvicidal assays NDF lost most of its toxicity but DF was still strongly active. It may be possible that the highly toxic effects of the whole latex from C. procera upon egg hatching and larvae development should be at least in part due to its protein content found in NDE However the toxicity seems also to involve non protein molecules present in DF.

The galactose-binding lectin from Vatairea macrocarpa seeds induces in vivo neutrophil migration by indirect mechanism

To explore the pathways by which lectins induce an inflammatory response, the lectin from Vatairea macrocarpa (VML) seeds was used to induce neutrophil migration in rats. The lectin was shown to cause cell migration, with the effect partially blocked when galactose was added to inhibit lectin activity. Neutrophil migration was also reduced when peritoneal cavity of the animals was depleted of their resident cells beforehand, suggesting that neutrophil migration was mediated by an indirect mechanism. Pre-treatment of rats with thioglycollate increased recruitment of neutrophils while depletion of mast cells by the addition of compound 48/80 had little effect on neutrophil infiltration, suggesting the involvement of macrophages in the inflammatory process induced by the lectin. Inhibition of the cyclooxigenase, leukotriene and PAF activities by indomethacin, MK886 and BN50730, respectively, did not modify the pro-inflammatory effect previously observed. However, dexamethasone and thalidomide significantly reduced the population of neutrophils in the peritoneal cavity after lectin injection. The present study suggests that the effects produced by a galactose-binding lectin do not involve lipoxygenase, cyclooxygenase or PAF mediators that are well known to be involved in the inflammatory process. The blocking actions of dexamethasone and thalidimide suggest that as yet unidentified pro-inflammatory mediators are involved.

Porcine Spermadhesin PSP-I/PSP-II Stimulates Macrophages to Release a Neutrophil Chemotactic Substance: Modulation by Mast Cells

Abstract The complex of porcine seminal plasma heterodimers I and II (PSP-I/PSP-II), which are heterodimers of glycosylated spermadhesins, is the major component of porcine seminal fluid. The proinflammatory and immunostimulatory activities of this spermadhesin complex suggest its participation in modulation of the uterine immune activity that may ensure reproductive success. Spermadhesin PSP-I/PSP-II induced the migration of neutrophils into the peritoneal cavity of rats via activation of resident cells. In the present study, we have investigated the involvement of macrophages and mast cells in the neutrophil chemotactic activity of PSP-I/PSP-II and the underlying mechanism. Macrophages and mast cells were isolated, cultured, and stimulated with purified PSP-I/PSP-II. Pharmacological modulation was performed using the glucocorticoid dexamethasone, indomethacin (cyclooxygenase inhibitor), MK886 (leukotriene inhibitor), and the supernatant of spermadhesin-stimulated mast cells. Macrophages stimulated with PSP-I/PSP-II released into the culture supernatant a neutrophil chemotactic substance. This activity was partly inhibited by both dexamethasone (85%) and the supernatant of spermadhesin-stimulated mast cells (74%) but not by indomethacin and MK886. An anti-tumor necrosis factor (TNF) α antibody neutralized (by 68%) the neutrophil chemotactic activity of PSP-I/PSP-II-stimulated macrophages. An anti-interleukin (IL)-4 antibody blocked the inhibitory activity of spermadhesin-stimulated mast cells on release of a neutrophil chemotactic substance by PSP-I/PSP-II-stimulated macrophages. As a whole, these data indicate that the neutrophil migration-inducing ability of spermadhesin PSP-I/PSP-II involves the release of the inflammatory cytokine TNFα by stimulated macrophages and that this activity is modulated by the lymphokine IL-4 liberated by mast cells. The balance between these two cytokines may control onset of the local inflammatory reaction, avoiding excessive neutrophil recruitment that would lead to tissue damage.

Anti‐inflammatory and antimicrobial effect of lectin from Lonchocarpus sericeus seeds in an experimental rat model of infectious peritonitis

We have investigated the anti‐inflammatory and antimicrobial effect of the lectin from Lonchocarpus sericeus seeds (LSL) in a model of infectious peritonitis in adult Wistar rats. Animals were treated with saline or LSL (10mg kg−1, i.v) immediately and 6h after the induction of peritonitis via cecal ligation and single puncture. Twelve hours after surgery, animals were killed and the infectious process was monitored by total and differential count of cells from blood and peritoneal washing liquid, adenosine deaminase activity, antibiogram and the number of viable bacteria of the peritoneal cavity. LSL treatment decreased the inflammatory response evoked by the induction of peritonitis, as seen by the inhibition of neutrophil migration into peritoneal cavities, leucocytosis and reduction of adenosine deaminase activity in the peritoneal fluid. All these effects were reversed by the lectin association to N‐acetyl‐glucosamine. LSL in‐vitro did not show any antimicrobial action, but promoted a marked decrease of the viable bacterial population in peritoneal cavities. In conclusion, LSL inhibited the inflammatory response and the bacterial colonization of infectious peritonitis in rats.

Spermadhesin PSP-I/PSP-II Heterodimer and Its Isolated Subunits Induced Neutrophil Migration into the Peritoneal Cavity of Rats

Abstract Spermadhesins are a group of (glyco)proteins from seminal fluid involved in various aspects of porcine fertilization. PSP-I/PSP-II, a heterodimer of glycosylated spermadhesins, is the major component of porcine seminal fluid. Its biological function remains, however, enigmatic. Using an in vitro chemotaxis assay, we showed that PSP-I/PSP-II and its isolated subunits induced migration of purified neutrophils. A possible proinflammatory activity of PSP-I/PSP-II induced upon injection of the spermadhesin heterodimer and its isolated subunits into the peritoneal cavity of rats was investigated. Lavage of peritoneal cavities, thioglycolate treatment, and mast cell depletion were done before spermadhesin administration, and neutrophil migration was evaluated 4 h after injections. Pharmacological modulation was also investigated. Resident cell depletion by lavage reduced the neutrophil migration induced by PSP-I/PSP-II and the PSP-II subunit but had no effect on that induced by isolated PSP-I. Both an increase of macrophage population by thioglycolate treatment and mast cell depletion potentiated the neutrophil migration induced by PSP-I/PSP-II and by PSP-II. The glucocorticoid dexamethasone but not indomethacin (cyclooxygenase inhibitor), MK886 (leukotriene inhibitor), and BN50739 (platelet activation factor [PAF] antagonist) inhibited neutrophil migration induced by PSP-I/PSP-II. Coincubation with mannose-6-phosphate (a PSP-II-specific ligand) inhibited neutrophil recruitment induced by PSP-II but did not alter the PSP-I activity. As a whole, the data suggested that enhancement of the neutrophil migration-inducing activity of PSP-I/PSP-II and PSP-II involved an indirect mechanism, i.e., via activation of resident cells, probably macrophages. On the other hand, PSP-I appeared to act directly on neutrophils. We hypothesize that the neutrophil migration-inducing effect displayed by PSP-II might be due to interaction of its lectin domain with cellular receptors and that neutrophil recruitment induced by PSP-I may involve protein-protein interactions.

In vivo anti-inflammatory effect of a sulfated polysaccharide isolated from the marine brown algae Lobophora variegata.

Context: Lobophora variegata J.V. Lamouroux (Dictyotaceae) is a brown marine alga widely encountered in the Brazilian sea coast that presents high content of fucans. Anti-inflammatory effects of fucans are reported mostly in models in vitro, but little is known about its effects in vivo. Objective: To investigate vascular and cellular effects of a sulfated polysaccharide from the brown marine algae L. variegata (SP-Lv) in acute inflammatory models. Materials and methods: SP-Lv was isolated by DEAE-cellulose and analyzed by agarose gel electrophoresis and evaluated for its inhibitory effect on paw edema, vascular permeability, leukocyte migration and peritoneal nitrite content induced by zymosan in Wistar rats. Anticoagulant activities and possible systemic toxicity were also evaluated. Results: SP-Lv inhibited the paw edema (120 min: 1.42 ± 0.11 vs. 0.95 ± 0.05 mL), plasma exudation (21.53 ± 0.62 vs. 11.96 ± 0.68 μg/g), nitrite content (4.42 ± 0.33 vs. 2.86 ± 0.003 μM) and leukocyte migration (5.15 ± 1.21 vs. 1.99 ± 0.16 cells/103 mL) induced by zymosan. SP-Lv and l-NAME reduced the paw edema (60–120 min) elicited by l-arginine. However, at 180 min SP-Lv effect was more accentuated and sustained until 240 min, while that of l-NAME was abolished. Similarly to indomethacin, SP-Lv inhibited the entire edema time-course induced by phospholipase A2, except for the time of 60 min. Discussion and conclusion: The anti-edematogenic effect of SP-Lv seems to occur via inhibition of nitric oxide synthase and cyclooxygenase activities. These results suggest a potential applicability of polysaccharides from alga origin in acute inflammatory conditions.

Proteins from latex of Calotropis procera prevent septic shock due to lethal infection by Salmonella enterica serovar Typhimurium

AIM OF THE STUDY The latex of Calotropis procera has been used in traditional medicine to treat different inflammatory diseases. The anti-inflammatory activity of latex proteins (LP) has been well documented using different inflammatory models. In this work the anti-inflammatory protein fraction was evaluated in a true inflammatory process by inducing a lethal experimental infection in the murine model caused by Salmonella enterica Subsp. enterica serovar Typhimurium. MATERIALS AND METHODS Experimental Swiss mice were given 0.2 ml of LP (30 or 60 mg/kg) by the intraperitoneal route 24 h before or after lethal challenge (0.2 ml) containing 10(6) CFU/ml of Salmonella Typhimurium using the same route of administration. RESULTS All the control animals succumbed to infection within 6 days. When given before bacterial inoculums LP prevented the death of mice, which remained in observation until day 28. Even, LP-treated animals exhibited only discrete signs of infection which disappeared latter. LP fraction was also protective when given orally or by subcutaneous route. Histopathological examination revealed that necrosis and inflammatory infiltrates were similar in both the experimental and control groups on days 1 and 5 after infection. LP activity did not clear Salmonella Typhimurium, which was still present in the spleen at approximately 10(4) cells/g of organ 28 days after challenge. However, no bacteria were detected in the liver at this stage. LP did not inhibit bacterial growth in culture medium at all. In the early stages of infection bacteria population was similar in organs and in the peritoneal fluid but drastically reduced in blood. Titration of TNF-alpha in serum revealed no differences between experimental and control groups on days 1 and 5 days after infection while IL-12 was only discretely diminished in serum of experimental animals on day 5. Moreover, cultured macrophages treated with LP and stimulated by LPS released significantly less IL-1beta. CONCLUSIONS LP-treated mice did not succumb to septic shock when submitted to a lethal infection. LP did not exhibit in vitro bactericidal activity. It is thought that protection of LP-treated mice against Salmonella Typhimurium possibly involves down-regulation of pro-inflammatory cytokines (other than TNF-alpha). LP inhibited IL-1beta release in cultured macrophages and discretely reduced IL-12 in serum of animals given LP. Results reported here support the folk use of latex to treat skin infections by topic application.