Vinicius F. Carvalho

The Hemolymph of the Ascidian Styela plicata (Chordata-Tunicata) Contains Heparin inside Basophil-like Cells and a Unique Sulfated Galactoglucan in the Plasma

The hemolymph of ascidians (Chordata-Tunicata) contains different types of hemocytes embedded in a liquid plasma. In the present study, heparin and a sulfated heteropolysaccharide were purified from the hemolymph of the ascidian Styela plicata. The heteropolysaccharide occurs free in the plasma, is composed of glucose (∼60%) and galactose (∼40%), and is highly sulfated. Heparin, on the other hand, occurs in the hemocytes, and high performance liquid chromatography of the products formed by degradation with specific lyases revealed that it is composed mainly by the disaccharides ΔUA(2SO4)-1→4-β-d-GlcN(SO4) (39.7%) and ΔUA(2SO4)-1→4-β-d-GlcN(SO4)(6SO4) (38.2%). Small amounts of the 3-O-sulfated disaccharides ΔUA(2SO4)-1→4-β-d-GlcN(SO4)(3SO4) (9.8%) and ΔUA(2SO4)-1→4-β-d-GlcN(SO4)(3SO4)(6SO4) (3.8%) were also detected. These 3-O-sulfated disaccharides were demonstrated to be essential for the binding of the hemocyte heparin to antithrombin III. Electron microscopy techniques were used to characterize the ultrastructure of the hemocytes and to localize heparin and histamine in these cells. At least five cell types were recognized and classified as univacuolated and multivacuolated cells, amebocytes, hemoblasts, and granulocytes. Immunocytochemistry showed that heparin and histamine co-localize in intracellular granules of only one type of hemocyte, the granulocyte. These results show for the first time that in ascidians, a sulfated galactoglucan circulates free in the plasma, and heparin occurs as an intracellular product of a circulating basophil-like cell.

Adoptive transfer of mast cells abolishes the inflammatory refractoriness to allergen in diabetic rats.

Mast cells are pivotal secretory cells primarily implicated in allergen-evoked inflammatory responses and are downregulated following experimental chemical diabetes. Here we tested the hypothesis that a decrease in the number and reactivity of mast cells would account for the hyporesponsiveness of diabetic rats to allergen-induced inflammation. We found that the anaphylactic release of histamine from sensitized ileum, trachea and skin tissues was clearly reduced in rats turned diabetic via intravenous administration of alloxan. Likewise, actively and passively sensitized diabetic rats mounted a weaker allergen-evoked pleural mast cell degranulation and protein extravasation, as compared to sensitized nondiabetic animals, which paralleled a marked reduction in the mast cell population in the pleural cavity. The number of mast cells enumerated in the mesentery from diabetic rats was also clearly reduced. The allergen-evoked plasma leakage in diabetic rats was restored by the transfer of mast cells from sensitized nondiabetic rats. Moreover, the adoptive transfer of sensitized mast cells from diabetics to naive animals led to a lower allergen-induced exudation as compared to the response noted after the transfer of sensitized naive mast cells. Purified mast cells from diabetic rats were hyporesponsive to antigen and compound 48/80 stimulation in vitro as attested by histamine release. Thus, our results show that the phenomenon of refractoriness of diabetic animals to allergen challenge appears to be accounted for by a reduction in the number and reactivity of mast cells.

Thymic atrophy in acute experimental Chagas disease is associated with an imbalance of stress hormones.

Disorders in the hypothalamic–pituitary–adrenal axis are associated with the pathogenesis of Trypanosoma cruzi infection. During the acute phase of this disease, increased levels of circulating glucocorticoids (GCs) correlate with thymic atrophy. Recently, we demonstrated that this phenomenon is paralleled by a decrease of prolactin (PRL) secretion, another stress hormone that seems to counteract many immunosuppressive effects of GCs. Both GCs and PRL are intrathymically produced and exhibit mutual antagonism through the activation of their respective receptors, GR, and PRLR. Considering that GCs induce apoptosis and inhibit double‐positive (DP) thymocyte proliferation and that PRL administration prevents these effects, it seems plausible that a local imbalance of GR–PRLR crosstalk underlies the thymic involution occurring in acute T. cruzi infection. In this respect, preserving PRLR signaling seems to be crucial for protecting DP from GC‐induced apoptosis.

Induction of bone‐marrow eosinophilia in mice submitted to surgery is dependent on stress‐induced secretion of glucocorticoids

We examined bone‐marrow in mice receiving subcutaneous implants of heat‐coagulated egg white, which are known to present chronic eosinophilic inflammation at the implant site. Egg white implants (EWIs) induced marked bone‐marrow eosinophilia, and increased bone‐marrow cell responses to granulocyte‐macrophage colony‐stimulating factor and interleukin‐5 in culture. These effects were observed as early as 24 h and lasted for, at least, 30 days in implant recipients. We found, however, that increased eosinophil production was also observed in control mice which underwent surgery but received no EWI (sham‐implanted mice), up to 15 days post‐surgery. As this suggests an important contribution of nonspecific stress mechanisms to eosinopoiesis, we further evaluated the role of stress hormones produced by the adrenal glands in the bone‐marrow eosinophilia of sham‐implanted mice. Bone‐marrow eosinophilia in mice undergoing surgery was dissociated from increases in other haemopoietic lineages. Surgery by itself increased circulating corticosterone levels by 24 h, and the increase was prevented by inhibition of adrenal glucocorticoid production by metyrapone. The effect of surgery on bone‐marrow eosinophilia was prevented by pretreatment with both the glucocorticoid receptor antagonist, mifepristone, and metyrapone, and by surgical adrenalectomy. By contrast, cathecolamine receptor antagonists (propranolol, prazosin and yohimbine) were ineffective, indicating that cathecolamine release from the adrenal glands was not responsible for the effects on bone‐marrow. These results highlight a critical role for stress‐induced glucocorticoid hormones in selectively upregulating bone‐marrow eosinopoiesis in mice submitted to surgery.

Thymus involution in alloxan diabetes: analysis of mast cells

We previously reported that alloxan-induced diabetes results in reduction in the number and reactivity of mast cells at different body sites. In this study, the influence of diabetes on thymic mast cells was investigated. Thymuses from diabetic rats showed marked alterations including shrinkage, thymocyte depletion, and increase in the extracellular matrix network, as compared to those profiles seen in normal animals. Nevertheless, we noted that the number and reactivity of mast cells remained unchanged. These findings indicate that although diabetes leads to critical alterations in the thymus, the local mast cell population is refractory to its effect. This suggests that thymic mast cells are under a different regulation as compared to those located in other tissues.

Systemic anaphylaxis is prevented in alloxan-diabetic rats by a mechanism dependent on glucocorticoids

This study was undertaken to examine whether glucocorticoids could be implicated in the hyporesponsiveness of diabetic rats to systemic anaphylaxis. Rats were actively sensitized with a mixture of Al(OH)(3) plus ovalbumin and challenged i.v. with ovalbumin 14 days later. Diabetes was induced by alloxan-injected i.v. either before or after sensitization. Elevation of total and specific serum immunoglobulin E (IgE) was abolished in rats turned diabetic and then sensitised, but not in those first sensitised and then turned diabetic. In both conditions, increased serum corticosterone levels occurred in parallel with protection of diabetic animals against fatal shock, intestinal haemorrhage and elevation in plasma histamine levels evoked by antigen challenge. The resistance of diabetic rats to fatal shock was no longer significantly different from that of non-diabetic rats following treatment with the glucocorticoid receptor antagonist RU 486 (mifepristone). These findings indicate that endogenous glucocorticoid plays a pivotal role in the phenomenon of hyporeactivity to systemic anaphylaxis in alloxan-diabetic rats.

Mast cell function and death in Trypanosoma cruzi infection

Although the roles of mast cells (MCs) are essential in many inflammatory and fibrotic diseases, their role in Trypanosoma cruzi-induced cardiomyopathy is unexplored. In this study, we treated infected CBA mice with cromolyn, an MC stabilizer, and observed much greater parasitemia and interferon-γ levels, higher mortality, myocarditis, and cardiac damage. Although these data show that MCs are important in controlling acute infection, we observed MC apoptosis in the cardiac tissue and peritoneal cavity of untreated mice. In the heart, pericardial mucosal MC die, perhaps because of reduced amounts of local stem cell factor. Using RT-PCR in purified cardiac MCs, we observed that infection induced transcription of P2X(7) receptor and Fas, two molecules reportedly involved in cell death and inflammatory regulation. In gld/gld mice (FasL(-/-)), apoptosis of cardiac, but not peritoneal, MCs was decreased. Conversely, infection of P2X(7)(-/-) mice led to reduced peritoneal, but not cardiac, MC death. These data illustrate the immunomodulatory role played by MCs in T. cruzi infection and the complexity of molecular interactions that control inflammatory pathways in different tissues and compartments.

Inhibition of advanced glycation end products by aminoguanidine restores mast cell numbers and reactivity in alloxan-diabetic rats

Mast cell number and reactivity have been shown to be down-regulated under diabetic conditions. This study was undertaken in order to investigate the role of the advanced glycation end products in the reduction of mast cell number and reactivity in diabetic rats. The effect of aminoguanidine on mast cell apoptosis was also evaluated. Diabetes was induced by intravenous injection of alloxan into fasted rats and aminoguanidine was administered after 3 days of diabetes induction, once daily for 18 consecutive days. Mast cell apoptosis and levels of Bax, a pro-apoptotic member of Bcl-2 family, were evaluated by TUNEL and western blot, respectively. Diabetes led to increased levels of fructosamine and AGEs in the plasma, an effect prevented by aminoguanidine. Treatment with aminoguanidine restored mast cell numbers in the pleural cavity and in mesenteric tissue of diabetic rats. Aminoguanidine also significantly reversed the diabetes-induced reduction in histamine release, as measured by fluorescence, following activation with substance P or antigen in vitro. Increased apoptosis and levels of Bax in mast cells from diabetic rats were inhibited by aminoguanidine. In conclusion, our findings showed that aminoguanidine restored the number and reactivity of mast cells in diabetic rats, accompanied by suppression of apoptosis, evidencing that advanced glycation end product formation has a critical role in mast cell behavior of diabetic rats.

Suppression of Allergic Inflammatory Response in the Skin of Alloxan-Diabetic Rats: Relationship with Reduced Local Mast Cell Numbers

Background: Diabetic patients are refractory to allergic inflammatory diseases. In this study, the influence of alloxan-induced diabetes on allergic skin inflammation was investigated. Methods: Diabetes was induced by intravenous injection of alloxan into male Wistar rats, and the analyses were performed 21 days later. Animals were actively sensitized with a mixture of aluminium hydroxide plus ovalbumin and challenged intradermally with ovalbumin on day 14. Results: Diabetic sensitized rats exhibited a less pronounced antigen-induced protein extravasation in the dorsal skin when compared with normal animals. Also, fragments of the dorsal subcutaneous tissue from diabetic sensitized rats showed a reduction in histamine release after stimulation with antigen in vitrowhen compared with fragments obtained from nondiabetic sensitized rats. Optical microscopy analysis revealed that the dorsal skin of diabetic rats showed a marked reduction in dermis thickness, as compared with that seen in normal animals. A significant decrease in the number of skin mast cells was also noted, a phenomenon that paralleled with the reduction in the expression of extracellular matrix components laminin, fibronectin and collagen. Administration of insulin into diabetic rats restored basal mast cell numbers as well as the levels of laminin, fibronectin and collagen. Conclusions: Our findings show that alloxan diabetes induces downregulation of the skin allergic inflammatory response in rats, and this was correlated with reduction in local mast cell numbers and expression of extracellular matrix components. Lastly, these alterations were reversed with insulin treatment.

Mangiferin Prevents Guinea Pig Tracheal Contraction via Activation of the Nitric Oxide-Cyclic GMP Pathway

Previous studies have described the antispasmodic effect of mangiferin, a natural glucoside xanthone (2-C-β-Dgluco-pyranosyl-1,3,6,7-tetrahydroxyxanthone) that is present in mango trees and other plants, but its mechanism of action remains unknown. The aim of this study was to examine the potential contribution of the nitric oxide-cyclic GMP pathway to the antispasmodic effect of mangiferin on isolated tracheal rings preparations. The functional effect of mangiferin on allergic and non-allergic contraction of guinea pig tracheal rings was assessed in conventional organ baths. Cultured tracheal rings were exposed to mangiferin or vehicle, and nitric oxide synthase (NOS) 3 and cyclic GMP (cGMP) levels were quantified using western blotting and enzyme immunoassays, respectively. Mangiferin (0.1–10 µM) inhibited tracheal contractions induced by distinct stimuli, such as allergen, histamine, 5-hydroxytryptamine or carbachol, in a concentration-dependent manner. Mangiferin also caused marked relaxation of tracheal rings that were precontracted by carbachol, suggesting that it has both anti-contraction and relaxant properties that are prevented by removing the epithelium. The effect of mangiferin was inhibited by the nitric oxide synthase inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME) (100 µM), and the soluble guanylate cyclase inhibitor, 1H-[1], [2], [4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 µM), but not the adenylate cyclase inhibitor, 9-(tetrahydro-2-furyl)adenine (SQ22536) (100 µM). The antispasmodic effect of mangiferin was also sensitive to K+ channel blockers, such as tetraethylammonium (TEA), glibenclamide and apamin. Furthermore, mangiferin inhibited Ca2+-induced contractions in K+ (60 mM)-depolarised tracheal rings preparations. In addition, mangiferin increased NOS3 protein levels and cGMP intracellular levels in cultured tracheal rings. Finally, mangiferin-induced increase in cGMP levels was abrogated by co-incubation with either ODQ or L-NAME. These data suggest that the antispasmodic effect of mangiferin is mediated by epithelium-nitric oxide- and cGMP-dependent mechanisms.