In vitro inhibitory effect of trichostatin A on canine grade 3 mast cell tumor

Mast cell tumor (MCT) is one of the most prevalent neoplasms that affect skin and soft tissue in dogs. Because mast cell tumors present a great variety of clinical appearance and behavior, their treatment becomes a challenge. Trichostatin A (TSA), an antifungal antibiotic, has shown inhibitory effects on the proliferation and induction of apoptosis in various types of cancer cells. In order to evaluate the potential of trichostatin A as a therapeutic drug, cells of grade 3 MCT were cultured and treated with concentrations of 1 nM to 400 nM of TSA. MTT assay and trypan blue exclusion assays were performed to estimate cell growth and cell viability, and cell cycle analysis was evaluated. TSA treatment showed a reduction in numbers of viable cells and an increase of cell death by apoptosis. The cell cycle analysis showed an increase of hypodiploid cells and a reduction of G0/G1 and G2/M -phases. According to these results, trichostatin A may be an interesting potential chemotherapeutic agent for the treatment of canine MCT.

A novel regeneration system for a wild passion fruit species (Passiflora cincinnata Mast.) based on somatic embryogenesis from mature zygotic embryos

The objective of the present work was to induce somatic embryogenesis from zygotic embryos of Passiflora cincinnata Masters. Zygotic embryos formed calli on media with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.5 mu M benzyladenine (BA) after 30 days of in vitro culture. A concentration of 18.1 mu M 2,4-D resulted in the largest number of somatic embryos. Embryogenic calli were yellowish and friable, forming whitish proembryogenic masses. Morphologically, embryogenic cells were small and had large nuclei and dense cytoplasm, whereas non-embryogenic cells were elongated, with small nuclei and less dense cytoplasm. Calli cultured under white light on basal Murashige and Skoog`s medium with activated charcoal produced embryos in all developmental stages. There were differences among the treatments, with some leading to the production of calli with embryos and some only to callus formation. Some abnormalities were associated with somatic embryos, including fused axes, fused cotyledons and polycotyledonary embryos. Production of secondary somatic embryos occurred in the first cycle of primary embryo development. Secondary embryos differentiated from the surface of the protodermal layer of primary embryos with intense cell proliferation, successive mitotic divisions in the initial phase of embryoid development, and a vascular system formed with no connection to the parental tissue. This secondary embryogenic system of P. cincinnata is characterized by intense proliferation and maintenance of embryogenic competence after successive subcultures. This reproducible protocol opens new prospects for massive propagation and is an alternative to the current organogenesis-based transformation protocol.

Small bowel injury associated to allergy is triggered by platelet-activating factor, mast cells, neutrophils and protected by nitric oxide

Allergy to components of the diet is followed by gut inflammation which in children, sometimes progress to mucosal lesions and anaphylaxis. In newborns suffering of cow`s milk allergy, bloody stools, rectal. bleeding and ulcerations are found. The rat systemic anaphylaxis is a suitable model to study the intestinal lesions associated to allergy. In the present study we used this model to investigate some mechanisms involved. We found that 15 min after antigen challenge of sensitized rats, hemorrhagic lesions develop in the small intestine. The lesions were more severe in jejunum and ileum compared to duodenum. Pretreatment of the rats with a platelet-activating factor-receptor antagonist (WEB-2170) reduced the lesions whereas inhibition of endogenous nitric oxide by L-NAME, greatly increased the hemorrhagic lesions and mortality. Both, lesions and mortality were reversed by L-arginine. The hemorrhagic lesions were also significantly reduced by the mast cell stabilizers, disodium cromoglycate and ketotifen as well as by neutrophils depletion (with anti-PMN antibodies) or inhibition of selectin binding (by treatment with fucoidan). Thus, the intestinal hemorrhagic lesions in this model are dependent on ptatelet-activating factor, mast cell granule-derived mediators and neutrophils. Endogenous nitric oxide and supplementation with L-arginine has a protective role, reducing the lesions and preventing mortality. These results contributed to elucidate mechanisms involved in intestinal lesions which could be of relevance to human small bowel injury associated to allergy. (c) 2007 Elsevier B.V. All rights reserved.

Connective tissue mast cells are the target of formaldehyde to induce tracheal hyperresponsiveness in rats: Putative role of leukotriene B(4) and nitric oxide

Formaldehyde (FA) exposure induces upper airways irritation and respiratory abnormalities, but its mechanisms are not understood. Since mast cells are widely distributed in the airways, we hypothesized that FA might modify the airways reactivity by mechanism involving their activation. Tracheal rings of rats were incubated with Dulbecco`s modified medium culture containing FA (0.1 ppm) in 96-well plastic microplates in a humid atmosphere. After 30 min, 6 h, and 24-72 h, the rings were suspended in an organ bath and dose-response curve to methacholine (MCh) were determined. incubation with FA caused a transient tracheal hyperresponsiveness to MCh that was independent from tracheal epithelium integrity. Connective tissue mast cell depletion caused by compound 48/80 or mast cell activation by the allergic reaction, before exposure of tracheal rings to FA prevented the increased responsiveness to MCh. LTB(4) concentrations were increased in the culture medium of tracheas incubated with FA for 48 h, whereas the LTB(4)-receptor antagonist MK886 (1 mu M) added before FA exposure rendered the tracheal rings normoreactive to MCh. In addition, FA exposure did not cause hyperresponsiveness in tracheal segments incubated with L-arginine (1 mu M). We suggest that airway connective tissue mast cells constitute the target and may provide the increased LTB(4) generation as well as an elevated consumption of NO leading to tracheal hyperresponsiveness to MCh. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Interactions of mast cell degranulating peptides with model membranes: A comparative biophysical study

In the last decade, there has been renewed interest in biologically active peptides in fields like allergy, autoimmume diseases and antibiotic therapy. Mast cell degranulating peptides mimic G-protein receptors, showing different activity levels even among homologous peptides. Another important feature is their ability to interact directly with membrane phospholipids, in a fast and concentration-dependent way. The mechanism of action of peptide HR1 on model membranes was investigated comparatively to other mast cell degranulating peptides (Mastoparan, Eumenitin and Anoplin) to evidence the features that modulate their selectivity. Using vesicle leakage, single-channel recordings and zeta-potential measurements, we demonstrated that HR1 preferentially binds to anionic bilayers, accumulates, folds, and at very low concentrations, is able to insert and create membrane spanning ion-selective pores. We discuss the ion selectivity character of the pores based on the neutralization or screening of the peptides charges by the bilayer head group charges or dipoles. (C) 2009 Elsevier Inc. All rights reserved.