The Value of Immunohistochemical Expression of BAX in Formulating a Prognosis for Canine Cutaneous Mast Cell Tumours

Irnmunohistochcmical expression of BAX was evaluated in 24 canine cutaneous mast cell tumours in order to verify the relationship of this expression to the histopathological grade of the lesions and its prognostic value for clinical outcome. BAX expression increased with higher histopathological grades (P = 0.0148; P < 0.05 between grades I and III). Animals with high levels of BAX expression were 4.25 times more likely to die from the disease and had shorter post-surgical survival times (P = 0.0009). These results suggest that alterations in BAX expression may be related to the aggressiveness of canine cutaneous mast cell tumours, indicating that immunohistochemical detection of BAX may be predictive of clinical outcome. (C) 2011 Elsevier Ltd. All rights reserved.

The Natural Cell-Penetrating Peptide Crotamine Targets Tumor Tissue in Vivo and Triggers a Lethal Calcium-Dependent Pathway in Cultured Cells

Our goal was to demonstrate the in vivo tumor specific accumulation of crotamine, a natural peptide from the venom of the South American rattlesnake Crotalus durissus terrificus, which has been characterized by our group as a cell penetrating peptide with a high specificity for actively proliferating cells and with a concentration-dependent cytotoxic effect. Crotamine cytotoxicity has been shown to be dependent on the disruption of lysosomes and subsequent activation of intracellular proteases. In this work, we show that the cytotoxic effect of crotamine also involves rapid intracellular calcium release and loss of mitochondrial membrane potential as observed in real time by confocal microscopy. The intracellular calcium overload induced by crotamine was almost completely blocked by thapsigargin. Microfluorimetry assays confirmed the importance of internal organelles, such as lysosomes and the endoplasmic reticulum, as contributors for the intracellular calcium increase, as well as the extracellular medium. Finally, we demonstrate here that crotamine injected intraperitoneally can efficiently target remote subcutaneous tumors engrafted in nude mice, as demonstrated by a noninvasive optical imaging procedure that permits in vivo real-time monitoring of crotamine uptake into tumor tissue. Taken together, our data indicate that the cytotoxic peptide crotamine can be used potentially for a dual purpose: to target and detect growing tumor tissues and to selectively trigger tumor cell death.

E-cadherin in canine mast cell tumors: Decreased expression and altered subcellular localization in Grade 3 tumors

Mast cell tumors (MCTs) are the most frequent round cell tumors in dogs and comprise approximately 21% of all canine cutaneous tumors. MCTs are highly invasive and metastatic corresponding to the histological grade. E-cadherin is an adhesion molecule expressed in epithelial cells and although it is an epithelial cellular marker, studies have shown expression of E-cadherin in canine round cell tumors. To better characterize the expression pattern of E-cadherin in several different histological grades of MCTs in dogs, the expression and localization of the adhesion molecule was investigated using immunohistochemistry. For this purpose, 18 cutaneous MCTs were classified into three histological grades, 1, 2 or 3. Clinical history and follow-up data were available for all of the dogs. Cytoplasmic and nuclear expressions of E-cadherin in all three types of tumors were verified by immunostaining using two different antibodies. There was decreased E-cadherin expression in the more aggressive MCTs (Grade 3), suggesting an association between E-cadherin and tumor aggressiveness. Additionally, the loss of E-cadherin expression in either the cytoplasm or nucleus in more aggressive and undifferentiated tumor types confirmed the importance of cellular adhesion in tumor behavior. (C) 2012 Published by Elsevier Ltd.

Expression of Mast Cell Proteases Correlates with Mast Cell Maturation and Angiogenesis during Tumor Progression

Tumor cells are surrounded by infiltrating inflammatory cells, such as lymphocytes, neutrophils, macrophages, and mast cells. A body of evidence indicates that mast cells are associated with various types of tumors. Although role of mast cells can be directly related to their granule content, their function in angiogenesis and tumor progression remains obscure. This study aims to understand the role of mast cells in these processes. Tumors were chemically induced in BALB/c mice and tumor progression was divided into Phases I, II and III. Phase I tumors exhibited a large number of mast cells, which increased in phase II and remained unchanged in phase III. The expression of mouse mast cell protease (mMCP)-4, mMCP-5, mMCP-6, mMCP-7, and carboxypeptidase A were analyzed at the 3 stages. Our results show that with the exception of mMCP-4 expression of these mast cell chymase (mMCP-5), tryptases (mMCP-6 and 7), and carboxypeptidase A (mMC-CPA) increased during tumor progression. Chymase and tryptase activity increased at all stages of tumor progression whereas the number of mast cells remained constant from phase II to III. The number of new blood vessels increased significantly in phase I, while in phases II and III an enlargement of existing blood vessels occurred. In vitro, mMCP-6 and 7 are able to induce vessel formation. The present study suggests that mast cells are involved in induction of angiogenesis in the early stages of tumor development and in modulating blood vessel growth in the later stages of tumor progression.

Phospholipase D2:A Pivotal Player Modulating RBL-2H3 Mast Cell Structure

The current study examined the role of PLD2 in the maintenance of mast cell structure. Phospholipase D (PLD) catalyzes hydrolysis of phosphatidylcholine to produce choline and phosphatidic acid (PA). PLD has two isoforms, PLD1 and PLD2, which vary in expression and localization depending on the cell type. The mast cell line RBL-2H3 was transfected to overexpress catalytically active (PLD2CA) and inactive (PLD2CI) forms of PLD2. The results of this study show that PLD2CI cells have a distinct star-shaped morphology, whereas PLD2CA and RBL-2H3 cells are spindle shaped. In PLD2CI cells, the Golgi complex was also disorganized with dilated cisternae, and more Golgi-associated vesicles were present as compared with the PLD2CA and RBL-2H3 cells. Treatment with exogenous PA led to the restoration of the wild-type Golgi complex phenotype in PLD2CI cells. Conversely, treatment of RBL-2H3 and PLD2CA cells with 1% 1-Butanol led to a disruption of the Golgi complex. The distribution of acidic compartments, including secretory granules and lysosomes, was also modified in PLD2CI cells, where they concentrated in the perinuclear region. These results suggest that the PA produced by PLD2 plays an important role in regulating cell morphology in mast cells. (J Histochem Cytochem 60:386-396, 2012)

Effects of myenteric denervation on extracellular matrix fibers and mast cell distribution in normal stomach and gastric lesions

Abstract Background In this study the effect of myenteric denervation induced by benzalconium chloride (BAC) on distribution of fibrillar components of extracellular matrix (ECM) and inflammatory cells was investigated in gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Rats were divided in four experimental groups: non-denervated (I) and denervated stomach (II) without MNNG treatment; non-denervated (III) and denervated stomachs (IV) treated with MNNG. For histopathological, histochemical and stereological analysis, sections of gastric fragments were stained with Hematoxylin-Eosin, Picrosirius-Hematoxylin, Gomori reticulin, Weigert's Resorcin-Fuchsin, Toluidine Blue and Alcian-Blue/Safranin (AB-SAF). Results BAC denervation causes an increase in the frequency of reticular and elastic fibers in the denervated (group II) compared to the non-denervated stomachs (group I). The treatment of the animals with MNNG induced the development of adenocarcinomas in non-denervated and denervated stomachs (groups III and IV, respectively) with a notable increase in the relative volume of the stroma, the frequency of reticular fibers and the inflammatory infiltrate that was more intense in group IV. An increase in the frequency of elastic fibers was observed in adenocarcinomas of denervated (group IV) compared to the non-denervated stomachs (group III) that showed degradation of these fibers. The development of lesions (groups III and IV) was also associated with an increase in the mast cell population, especially AB and AB-SAF positives, the latter mainly in the denervated group IV. Conclusions The results show a strong association in the morphological alteration of the ECM fibrillar components, the increased density of mast cells and the development of tumors induced by MNNG in the non-denervated rat stomach or denervated by BAC. This suggests that the study of extracellular and intracellular components of tumor microenvironment contributes to understanding of tumor biology by action of myenteric denervation.

Cellular Proliferation in the Prognosis of Intermediate Grade Mast Cell Tumour in Dogs

This was a retrospective study including ninety samples of dogs with a histological diagnosis of intermediate grade cutaneous mast cell tumour (MCT). The objectives of the study were to validate Minichromosome Maintenance Protein 7 (MCM7) as a prognostic marker in MCTs and to compare the ability of mitotic index (MI), Ki67 and MCM7 to predict outcome. The median survival for the entire population was not reached at 2099 days. The mean survival time was 1708 days. Seventy-two cases were censored after a median follow up of 1136 days and eighteen dogs died for causes related to the MCT after a median of 116 days. For each sample MI, Ki67 and MCM7 were determined. The Receiver Operating Characteristic (ROC) curve was obtained for each prognostic marker to evaluate the performance of the test, expressed as area under the curve, and whether the published threshold value was adequate. Kaplan-Meier and corresponding logrank test for MI, Ki67 and MCM7 as binary variables was highly significant (P<0.0001). Multivariable regression analysis of MI, Ki67 and MCM7 corrected for age and surgical margins indicated that the higher risk of dying of MCT was associated with MCM7 > 0.18 (Hazard Ration [HR] 14.7; P<0.001) followed by MI > 5 (HR 13.9; P<0.001) and Ki67 > 0.018 (HR 8.9; P<0.001). Concluding, the present study confirmed that MCM7 is an excellent prognostic marker in cutaneous MCTs being able to divide Patnaik intermediate grade tumours in two categories with different prognosis. Ki67 was equally good confirming its value as a prognostic marker in intermediate grade MCTs. The mitotic index was extremely specific, but lacked of sensitivity. Interestingly, mitotic index, Ki67 and MCM7 were independent from each other suggesting that their combination would improve their individual prognostic value.

Eotaxin/CCL11 levels correlate with myocardial fibrosis and mast cell density in native and transplanted rat hearts

Myocardial fibrosis contributes to hemodynamic and cardiac functional alterations commonly observed posttransplantation. Cardiac mast cells (MC) have been linked to fibrosis in posttransplantation hearts. Eotaxin, which has been shown to be involved in fibrogenesis, has been demonstrated to be increased in production in cardiac macrophages. The aim of our study was to correlate myocardial fibrosis during heart transplant rejection in the rat with eotaxin/chemokine [c-c motif] ligand 11 (CCL11) expression, and with various subtypes of infiltrating cardiac MC, namely connective-type MC (CTMC) and mucosa-type MC (MMC).

An increase in serum tryptase even below 11.4 ng/mL may indicate a mast cell-mediated hypersensitivity reaction: a prospective study in Hymenoptera venom allergic patients

During a systemic hypersensitivity reaction (SR), an increase in serum tryptase compared to the baseline value is an indicator of mast cell activation, most often due to an IgE-mediated mechanism.

Human basophils activated by mast cell-derived IL-3 express retinaldehyde dehydrogenase-II and produce the immunoregulatory mediator retinoic acid

The vitamin A metabolite retinoic acid (RA) plays a fundamental role in cellular functions by activating nuclear receptors. Retinaldehyde dehydrogenase-II (RALDH2) creates localized RA gradients needed for proper embryonic development, but very little is known regarding its regulated expression in adults. Using a human ex vivo model of allergic inflammation by coincubating IgE receptor-activated mast cells (MCs) with blood basophils, we observed prominent induction of a protein that was identified as RALDH2 by mass spectroscopy. RALDH2 was selectively induced in basophils by MC-derived interleukin-3 (IL-3) involving PI3-kinase and NF-kappaB pathways. Importantly, neither constitutive nor inducible RALDH2 expression was detectable in any other human myeloid or lymphoid leukocyte, including dendritic cells. RA generated by RALDH2 in basophils modulates IL-3-induced gene expression in an autocrine manner, providing positive (CD25) as well as negative (granzyme B) regulation. It also acts in a paracrine fashion on T-helper cells promoting the expression of CD38 and alpha4/beta7 integrins. Furthermore, RA derived from IL-3-activated basophils provides a novel mechanism of Th2 polarization. Thus, RA must be viewed as a tightly controlled basophil-derived mediator with a high potential for regulating diverse functions of immune and resident cells in allergic diseases and other Th2-type immune responses.

Immunological microenvironment in prostate cancer: high mast cell densities are associated with favorable tumor characteristics and good prognosis

BACKGROUND: Number of intratumoral mast cells predicts survival in various cancers. The prognostic significance of such mast cells in surgically treated prostate cancer is unknown. METHODS: Mast cell densities were determined in prostate cancer samples of more than 2,300 hormone-naïve patients using a tissue microarray format in correlation with clinical follow-up data. Mast cells were visualized immunohistochemically (c-kit). All patients were homogeneously treated by radical prostatectomy at a single institution. RESULTS: Mast cells were present in 95.9% of the tumor samples. Median mast cell number on the tissue spot was 9 (range: 0-90; median density: 31 mast cells/mm(2)). High mast cell densities were significantly associated with more favorable tumors having lower preoperative prostate-specific antigen (P = 0.0021), Gleason score (P < 0.0001) and tumor stage (P < 0.0001) than tumors with low mast cell densities. Prostate-specific antigen recurrence-free survival significantly (P = 0.0001) decreased with decline of mast cell density showing poorest outcome for patients without intratumoral mast cells. In multivariate analysis mast cell density narrowly missed to add independent prognostic information (P = 0.0815) for prostate-specific antigen recurrence. CONCLUSION: High intratumoral mast cell density is associated with favorable tumor characteristics and good prognosis in prostate cancer. This finding is consistent with a role of mast cells in the immunological host-defense reaction on prostate cancer. Triggering mast cell activity might expand immunotherapeutic strategies in prostate cancer.

Sphingosine kinase 1 is pivotal for Fc epsilon RI-mediated mast cell signaling and functional responses in vitro and in vivo

Mast cell degranulation is pivotal to allergic diseases; investigating novel pathways triggering mast cell degranulation would undoubtedly have important therapeutic potential. FcepsilonRI-mediated degranulation has contradictorily been shown to require SphK1 or SphK2, depending on the reports. We investigated the in vitro and in vivo specific role(s) of SphK1 and SphK2 in FcepsilonRI-mediated responses, using specific small interfering RNA-gene silencing. The small interfering RNA-knockdown of SphK1 in mast cells inhibited several signaling mechanisms and effector functions, triggered by FcepsilonRI stimulation including: Ca(2+) signals, NFkappaB activation, degranulation, cytokine/chemokine, and eicosanoid production, whereas silencing SphK2 had no effect at all. Moreover, silencing SPHK1 in vivo, in different strains of mice, strongly inhibited mast cell-mediated anaphylaxis, including inhibition of vascular permeability, tissue mast cell degranulation, changes in temperature, and serum histamine and cytokine levels, whereas silencing SPHK2 had no effect and the mice developed anaphylaxis. Our data differ from a recent report using SPHK1(-/-) and SPHK2(-/-) mice, which showed that SphK2 was required for FcepsilonRI-mediated mast cell responses. We performed experiments in mast cells derived from SPHK1(-/-) and SPHK2(-/-) mice and show that the calcium response and degranulation, triggered by FcepsilonRI-cross-linking, is not different from that triggered in wild-type cells. Moreover, IgE-mediated anaphylaxis in the knockout mice showed similar levels in temperature changes and serum histamine to that from wild-type mice, indicating that there was no protection from anaphylaxis for either knockout mice. Thus, our data strongly suggest a previously unrecognized compensatory mechanism in the knockout mice, and establishes a role for SphK1 in IgE-mediated mast cell responses.

Early stages of experimental colon carcinogenesis inhibit mast cell-dependent mucosal immune function of the distal colon

Inhibition of local host immune reactions is one mechanism contributing to tumor progression. To determine if alterations in local immune functioning occur during colon carcinogenesis, a model mucosal immune response, type I hypersensitivity against the intestinal parasite Trichinella spiralis, was first characterized in normal mice and then examined during experimental colon carcinogenesis. Segments of sensitized colon mounted in Ussing chambers and challenged with T. spiralis-derived antigen resulted in a rise in short-circuit current ($\rm\Delta I\sb{sc}$) that was antigen-specific and inhibited by furosemide, implicating epithelial Cl$\sp-$ secretion as the ionic mechanism. The immune-regulated Cl$\sp-$ secretion by colonic epithelial cells required the presence of mast cells with surface IgE. Inhibition of potential anaphylactic mediators with various pharmacological agents in vitro implicated prostaglandins and leukotrienes as the principal mediators of the antigen-induced $\rm\Delta I\sb{sc}$, with 5-hydroxytryptamine also playing a role. Distal colon from immune mice fed an aspirin-containing diet (800 mg/kg powdered diet) ad libitum for 6 wk had a decreased response to antigen, confirming the major role of prostaglandins in generating the colonic I$\sb{\rm sc}$. To determine the effects of early stages of colon carcinogenesis on this mucosal immune response, mice were immunized with T. spiralis 1 day after or 8 wk prior to the first of 6 weekly injections of the procarcinogen 1,2-dimethylhydrazine (DMH). Responsiveness to antigenic challenge was suppressed in the distal colon 4-6 wk after the final injection of DMH. One injection of DMH was not sufficient to inhibit antigen responsiveness. The colonic epithelium remained sensitive to direct stimulation by exogenous Cl$\sp-$ secretagogues. Decreased antigen-induced $\rm\Delta I\sb{sc}$ in the distal colon was not due to systemic immune suppression by DMH, as the proximal colon and jejunum maintained responsiveness to antigen. Also, rejection of a secondary T. spiralis infection from the small intestine was not altered. Tumors eventually developed 25-30 wk after the final injection of DMH only in the distal portions of the colon. These results suggest that early stages of DMH-induced colon carcinogenesis manipulate the microenvironment such that mucosal immune function, as measured by immune-regulated Cl$\sp-$ secretion, is suppressed in the distal colon, but not in other regions of the gut. Future elucidation of the mechanisms by which this localized inhibition of immune-mediated ion transport occurs may provide possible clues to the microenvironmental changes necessary for tumor progression in the distal colon. ^

Designed cell penetrating peptide dendrimers efficiently internalize cargo into cells

Redesigning linear cell penetrating peptides (CPPs) into a multi-branched topology with short dipeptide branches gave cell penetrating peptide dendrimers (CPPDs) with higher cell penetration, lower toxicity and hemolysis and higher serum stability than linear CPPs. Their use is demonstrated by delivering a cytotoxic peptide and paclitaxel into cells.