Proteomic Analysis of Lipid Rafts from RBL-2H3 Mast Cells.

Lipid rafts are highly ordered membrane microdomains enriched in cholesterol, glycosphingolipids, and certain proteins. They are involved in the regulation of cellular processes in diverse cell types, including mast cells (MCs). The MC lipid raft protein composition was assessed using qualitative mass spectrometric characterization of the proteome from detergent-resistant membrane fractions from RBL-2H3 MCs. Using two different post-isolation treatment methods, a total of 949 lipid raft associated proteins were identified. The majority of these MC lipid raft proteins had already been described in the RaftProtV2 database and are among highest cited/experimentally validated lipid raft proteins. Additionally, more than half of the identified proteins had lipid modifications and/or transmembrane domains. Classification of identified proteins into functional categories showed that the proteins were associated with cellular membrane compartments, and with some biological and molecular functions, such as regulation, localization, binding, catalytic activity, and response to stimulus. Furthermore, functional enrichment analysis demonstrated an intimate involvement of identified proteins with various aspects of MC biological processes, especially those related to regulated secretion, organization/stabilization of macromolecules complexes, and signal transduction. This study represents the first comprehensive proteomic profile of MC lipid rafts and provides additional information to elucidate immunoregulatory functions coordinated by raft proteins in MCs.

Large Accumulation of Collagen and Increased Activation of Mast Cells in Hearts of Mice with Hyperlipidemia / Grande Acúmulo de Colágeno e Aumento da Ativação de Mastócitos nos Corações de Ratos com Hiperlipidemia

Abstract Background: Hyperlipidemia, which is characterized by an elevation of lipids in the bloodstream, is a major risk factor for cardiac disease. Objectives: The present study investigated the role of fibrosis in the progression of hyperlipidemia in the mice heart, and whether mast cell activation was associated with the fibrosis process. Methods: Hyperlipidemia was produced in C57BL / 6 mice by feeding them on a high-fat diet for 8 weeks.To assess tissue fibrosis, picrosirius red staining was performed. Hematoxylin & eosin (H&E) staining was performed to identify the histopathological changes in the hearts. Immunohistochemistry was also accomplished to determine the localization of transforming growth factor (TGF)-β and α-smooth muscle actin (α-SMA). Western blotting was performed to analyze the expression of chymase, tryptase, TGF-β, α-SMA and activity of Wnt/β-catenin pathway. At the end, serum total cholesterol (TC) and triglycerides (TG) levels were measured. All the values were expressed as means ± SD, the statistical significance level adopted was 5%. Results: Hyperlipidemia mice showed significantly increased collagen deposition in the hearts compared with normal mice. In addition, H&E staining showed significant cellular degeneration. Cardiac muscle was arranged in disorder with fracture in mice of the model group. Immunohistochemistry and western blot analysis revealed that expression levels of tryptase, chymase, β-catenin, TGF-β and α-SMA were significantly increased in the hyperlipidemia mice compared with the control group. Conclusions: The results indicated that mast cell activation might induce cardiac fibrosis by tryptase and chymase in hyperlipidemia, which had a close relationship with the increased activity of TGF-β/Wnt/β-catenin pathway.

Resumo Fundamentos: A hiperlipidemia, que se caracteriza por uma elevação dos lipídeos na corrente sanguínea, é um importante fator de risco para a doença cardíaca. Objetivos: O presente estudo investigou o papel da fibrose na progressão da hiperlipidemia no coração do rato e se a ativação dos mastócitos estava associada ao processo de fibrose. Método: A hiperlipidemia foi produzida em ratos C57BL/6 alimentando-os com uma dieta rica em gordura durante 8 semanas. Para avaliar a fibrose tecidual, foi realizada coloração vermelha picro-Sirius. A coloração com hematoxilina e eosina (H & E) foi feita para identificar as alterações histopatológicas nos corações. A imuno-histoquímica também foi levada a cabo para determinar a localização do fator de crescimento transformante (TGF) -β e α-actina do músculo liso (α-SMA). O Western Blot foi realizado para analisar as expressões de quimase, triptase, TGF-β, α-SMA e a atividade da via Wnt / β-catenina. Finalmente, se mediram os níveis séricos de colesterol total (TC) e triglicerídeos (TG). Todos os valores foram expressos como média ± DP, o nível de significância estatística adotado foi de 5%. Resultados: Os ratos hiperlipidêmicos mostraram aumento significativo da deposição de colágeno nos corações em comparação com ratos normais. Além disso, a coloração de H & E mostrou degeneração celular significativa. O músculo cardíaco estava em desordem com ruptura de fibras em ratos do grupo modelo. A análise imuno-histoquímica e o Western Blot revelaram que os níveis de expressão de triptase, quimase, β-catenina, TGF-β e α-SMA estavam significativamente aumentados nos ratos hiperlipidêmicos em comparação com o grupo controle. Conclusões: Os resultados indicaram que a ativação de mastócitos pode induzir fibrose cardíaca por triptase e quimase em hiperlipidemia, a qual teve uma relação estreita com a atividade aumentada da via TGF-β / Wnt / β-catenina.

Large Accumulation of Collagen and Increased Activation of Mast Cells in Hearts of Mice with Hyperlipidemia.

BACKGROUND: Hyperlipidemia, which is characterized by an elevation of lipids in the bloodstream, is a major risk factor for cardiac disease. OBJECTIVES: The present study investigated the role of fibrosis in the progression of hyperlipidemia in the mice heart, and whether mast cell activation was associated with the fibrosis process. METHODS: Hyperlipidemia was produced in C57BL / 6 mice by feeding them on a high-fat diet for 8 weeks.To assess tissue fibrosis, picrosirius red staining was performed. Hematoxylin & eosin (H&E) staining was performed to identify the histopathological changes in the hearts. Immunohistochemistry was also accomplished to determine the localization of transforming growth factor (TGF)-ß and α-smooth muscle actin (α-SMA). Western blotting was performed to analyze the expression of chymase, tryptase, TGF-ß, α-SMA and activity of Wnt/ß-catenin pathway. At the end, serum total cholesterol (TC) and triglycerides (TG) levels were measured. All the values were expressed as means ± SD, the statistical significance level adopted was 5%. RESULTS: Hyperlipidemia mice showed significantly increased collagen deposition in the hearts compared with normal mice. In addition, H&E staining showed significant cellular degeneration. Cardiac muscle was arranged in disorder with fracture in mice of the model group. Immunohistochemistry and western blot analysis revealed that expression levels of tryptase, chymase, ß-catenin, TGF-ß and α-SMA were significantly increased in the hyperlipidemia mice compared with the control group. CONCLUSIONS: The results indicated that mast cell activation might induce cardiac fibrosis by tryptase and chymase in hyperlipidemia, which had a close relationship with the increased activity of TGF-ß/Wnt/ß-catenin pathway.

Heparan sulfate and heparin interactions with proteins.

Heparan sulfate (HS) polysaccharides are ubiquitous components of the cell surface and extracellular matrix of all multicellular animals, whereas heparin is present within mast cells and can be viewed as a more sulfated, tissue-specific, HS variant. HS and heparin regulate biological processes through interactions with a large repertoire of proteins. Owing to these interactions and diverse effects observed during in vitro, ex vivo and in vivo experiments, manifold biological/pharmacological activities have been attributed to them. The properties that have been thought to bestow protein binding and biological activity upon HS and heparin vary from high levels of sequence specificity to a dependence on charge. In contrast to these opposing opinions, we will argue that the evidence supports both a level of redundancy and a degree of selectivity in the structure-activity relationship. The relationship between this apparent redundancy, the multi-dentate nature of heparin and HS polysaccharide chains, their involvement in protein networks and the multiple binding sites on proteins, each possessing different properties, will also be considered. Finally, the role of cations in modulating HS/heparin activity will be reviewed and some of the implications for structure-activity relationships and regulation will be discussed.

Sialic acid residues are essential for the anaphylactic activity of murine IgG1 antibodies.

Glycosylation of the Ab molecule is essential for maintaining the functional structure of Fc region and consequently for Ab-mediated effector functions, such as binding to cells or complement system activation. Alterations in the composition of the sugar moiety can dramatically influence Ab activity; however, it is not completely clear how differences in the N-linked oligosaccharide structure impact the biological function of Abs. We have described that murine IgG1 Abs can be separated according to their ability to elicit in vivo anaphylaxis in a fraction of anaphylactic and other of non-anaphylactic molecules. Furthermore, we showed that the N-linked oligosaccharide chain is essential for the structural conformation of the anaphylactic IgG1, the binding to FcgammaRIII on mast cells, and, consequently, for the ability to mediate anaphylactic reactions. In this study, we evaluated the contribution of individual sugar residues to this biological function. Differences in the glycan composition were observed when we analyzed oligosaccharide chains from anaphylactic or non-anaphylactic IgG1, mainly the presence of more sialic acid and fucose residues in anaphylactic molecules. Interestingly, the enzymatic removal of terminal sialic acid residues in anaphylactic IgG1 resulted in loss of the ability to trigger mast cell degranulation and in vivo anaphylactic reaction, similarly to the deglycosylated IgG1 Ab. In contrast, fucose removal did not affect the anaphylactic function. Therefore, we demonstrated that the ability of murine IgG1 Abs to mediate anaphylaxis is directly dependent on the amount of sialic acid residues associated to the oligosaccharide chain attached to the Fc region of these molecules.

Increased glial-derived neurotrophic factor in the small intestine of rats infected with the tapeworm, Hymenolepis diminuta.

The neurotrophin, glial-derived neurotrophic factor (GDNF), is essential for the development of the enteric nervous system (ENS) in both the embryo and neonate and may be important for maintenance and plasticity of ENS. The tapeworm, Hymenolepis diminuta, altered the number of cells containing GNDF in the host's jejunum and ileum. Numbers and locations of GDNF-containing cells were determined by applying monoclonal anti-GDNF antibody to intestinal segments collected from infected and uninfected age-matched rats during the initial 34 days post-infection (dpi). Most cells staining positive for GDNF were present in the lamina propria of the jejunum and ileum from both infected and uninfected rats. The co-localization of staining by the antibodies, anti-GDNF and anti-ED2 (a nuclear specific antibody for resident macrophages) indicated that at least 74% of the cells staining for GDNF were macrophages. Mast cells did not stain with the anti-GDNF antibody. The increased number of GDNF+ cells in the infected rat intestine suggests that this neurotrophin may play a role in the neural and mucosal responses to lumenal tapeworm infection.

A quantitative and morphometric study of tryptase-positive mast cells in cutaneous leprosy lesions.

While mast cells are known to induce differences in the matrix structures, microvascular patterns, and immune responses in a number of diseases, the possible role of mast cells in these same processes over the spectrum of leprosy has not yet been investigated. Thus, ascertaining the possible influence of mast cells in the outcome of the anti-leprosy response to Mycobacterium leprae is of major importance. In this study, 51 cutaneous biopsies of leprosy patients were stained with anti-tryptase antibody in order to quantify mast cells in leprosy lesions and compare the number and size of these cells in all the forms of leprosy. Biopsies were grouped according to an adapted Ridley-Jopling clinical-immunological classification (17 T, 17 B and 17 L). It was found that the L (lepromatous leprosy) group had the lowest dermal mast cell density values among the three groups studied. Furthermore, the average mast cell cross-sectional area was significantly higher in the L in comparison to the B (borderline-borderline) and T (tuberculoid) biopsies, suggesting mast cell functional differences within the groups. The higher mast cell density in the T and B groups was considered indirect evidence of the role of mast cells in the activated immune response to M. leprae infection.

Mastocytosis in children: clinicopathological study based on 35 cases.

Immunohistochemical staining is useful in the diagnosis of bone marrow infiltration in systemic mastocytosis. However, it is not clear if antibody staining may be helpful in the diagnosis of cutaneous mastocytosis (CM). We studied the histological appearance of CM in 35 pediatric patients. Cases were assigned to three basic clinical groups: I--Urticaria pigmentosa (UP, n=29); II--Mastocytomas (n=4); and III--Diffuse Cutaneous Mastocytosis (DCM, n=2). The analysis of clinical information revealed an association between the presence of diarrhea and a higher number of cells/field. Nine doubtful cases, all of them macules, were selected based on the scarcity of mast cells (MC) and the absence or rarity of other inflammatory cells. We compared the number of cells identified in Giemsa and immunohistochemical stains in definite and doubtful cases. The intraclass correlation statistic tested the concordance between each staining method. All 9 dubious cases according to the Giemsa stain had their CM diagnosis confirmed by the immunohistochemistry analysis. The intraclass correlation between Giemsa and c-kit was good (0.7) when the number of MC was high. However, there was no correlation between the mast cells counts in the two different stains in the dubious cases. The immunohistochemistry with c-kit might make CM diagnosis easier, especially in the macular cases, when there is a lower number of MC.

Mast cell-specific gangliosides and FcepsilonRI follow the same endocytic pathway from lipid rafts in RBL-2H3 cells.

Recent studies have shown that, in mast cells, membrane microdomains rich in cholesterol and glycosphingolipids called lipid rafts play an important role in FcepsilonRI signaling. The present study demonstrates that, in RBL-2H3 cells following stimulation, the mast cell-specific gangliosides associated with FcepsilonRI are internalized from lipid rafts along with the receptor. When the cells are labeled with iodinated antibodies against the gangliosides or against FcepsilonRI and the cell components are then fractionated on Percoll density gradients, in stimulated cells the gangliosides are internalized with the same kinetics as FcepsilonRI and at 3 hr are present in the dense lysosome fraction. Using transmission electron microscopy, with antibody against the gangliosides conjugated to horseradish peroxidase and antibody against FcepsilonRI conjugated to colloidal gold, it was possible to demonstrate that the gangliosides and FcepsilonRI are internalized in the same coated vesicles. At 5 min, the gangliosides and FcepsilonRI can be identified in early endosomes and at 3 hr are found together in acid phosphatase-positive lysosomes. This study demonstrates that the mast cell-specific gangliosides are internalized from lipid rafts in the same vesicles and traffic intracellularly with the same kinetics as FcepsilonRI. This study contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Mast cell heterogeneity between two different species of Hoplias sp. (Characiformes: Erythrinidae): response to fixatives, anatomical distribution, histochemical contents and ultrastructural features.

Mast cells from two erythrinid species: Hoplias malabaricus and Hoplias lacerdae, were studied in several tissues throughout the body using light and electron microscopy. Mast cells were found in all organs studied, but were especially abundant in the gastrointestinal tract, and were always in association with connective tissue. These cells showed different characteristics between the two species studied, like varied morphology, anatomical distribution, density, basophilic/eosinophilic staining and heparin content. In H. malabaricus, the tissues fixed with Helly's solution contained mast cells that were basophilic, metachromatic and had heparin in their cytoplasmic granules, while the tissues fixed with Karnovsky's solution contained eosinophilic and orthochromatic mast cells in which heparin was not detected. In H. lacerdae, the use of both fixatives resulted in mast cells that were eosinophilic, orthochromatic, with no identifiable heparin content. Exclusively in H. malabaricus oesophagus, the mast cells were additionally seen among the epithelial cells. The ultrastructural studies performed in hindgut fixed with Karnovsky's solution revealed that the cytoplasmic granules seen in H. lacerdae mast cells were better preserved than in H. malabaricus mast cells. The latter had electron-lucent granules that were often merged, forming channels. The present study demonstrated that mast cells from two species belonging to the same genus or even mast cells from the same species but under different fixatives can present heterogeneous characteristics, possibly due to their functional properties or to their sensitivity to fixatives.

Inflammatory cell mapping of the respiratory tract in fatal asthma.

BACKGROUND: The site and distribution of inflammation in the airways of asthmatic patients has been largely investigated. Inflammatory cells are distributed in both large and small airways in asthma. It has been demonstrated that distal lung inflammation in asthma may significantly contribute to the pathophysiology of the disease. The upper airways have also been implicated in the overall asthmatic inflammation. Although it is now accepted that lung inflammation is not restricted to the intrapulmonary airways in asthma, little is known about cell distribution in the other lung compartments and their relation to the intrapulmonary airways. OBJECTIVE: We aimed to map the inflammatory process in fatal asthma (FA), from the upper airways to the lung parenchyma. METHODS: Eosinophil, neutrophil, mast cell and lymphocyte content were determined in nasal mucosa, the trachea, intrapulmonary airways and parenchyma (peribronchiolar and distal) of 20 patients with FA and 10 controls. RESULTS: Eosinophil content was higher in all studied areas in FA compared with controls (P<0.02). Mast cell content was higher in the outer area of larger airways, small membranous bronchioles and in peribronchiolar parenchyma of FA compared with controls (P<0.04). CD3+, CD4+and CD20+cells showed increased content in FA intrapulmonary airways compared with controls (P<0.05). There was a positive correlation between CD4+cell content in nasal mucosa and larger airways in asthmatics. Increased neutrophil content was observed only in peribronchiolar parenchyma of FA (P=0.028). CONCLUSION: Eosinophils present a widespread distribution within the respiratory tract in FA, from the nasal mucosa to the distal lung. The outer wall of small membranous bronchioles is the main site of inflammatory changes in FA. There is a localized distribution of alveolar inflammation at the peribronchiolar region for mast cells and neutrophils. Our findings provide further evidence of the importance of the lung periphery in the pathophysiology of FA.

Identification and characterization of undifferentiated mast cells in mouse bone marrow.

Sequential immunomagnetic isolation with 2 monoclonal antibodies was used to purify and characterize an undifferentiated mast cell in adult mouse bone marrow that had not been previously recognized. This cell represents 0.02% of the cells in the bone marrow, is CD34(+), CD13(+), and c-kit(+), and does not express FcepsilonRI. However, by polymerase chain reaction (PCR) the cell contains message for the alpha and beta subunits of FcepsilonRI, mast cell-specific proteases, and carboxypeptidase A. Morphologically, this cell has a large nucleus, little cytoplasm, few cytoplasmic organelles, and no cytoplasmic granules. In vitro, in the presence of interleukin-3 (IL-3) and stem cell factor (SCF) these cells differentiate only into a granulated mast cell that now expresses CD13, c-kit, mast cell-specific gangliosides, FcepsilonRI, and binds immunoglobulin E (IgE). When injected into lethally irradiated mice, these cells are able to reconstitute the mast cell population in the spleen.

Inflammatory mediators in nasal lavage among school-age children from urban and rural areas in São Paulo, Brazil.

CONTEXT: Some studies have shown that inflammatory processes in the nasal air passages may reflect or affect those in the lower airways. We decided to indirectly assess the inflammatory status of the nasal airways in two groups of children with different sensitization rates to aeroallergens. OBJECTIVE: To compare the inflammatory activity in the nasal airways, through the determination of mediators in nasal lavage fluid in two distinct populations. TYPE OF STUDY: Cross-sectional study. SETTING: Two public elementary schools, one in an urban setting and the other in a rural setting of the State of São Paulo, Brazil. METHODS: Two groups of 40 elementary school children with different sensitization rates to aeroallergens were formed. Samples of nasal lavage fluid were assessed for eosinophil cationic protein (ECP) and tryptase. Non-parametric tests were used for statistical analysis. RESULTS: Significantly higher levels of ECP were observed among students living in the urban area than those in the rural area (p < 0.05). No significant difference in the tryptase levels was observed. Also, the urban children who were sensitized to aeroallergens presented higher levels of ECP in nasal mucosa than the non-sensitized children, while this difference was not observed among the rural children. DISCUSSION: The lack of mast cell activity and increased eosinophil degranulation revealed a chronic inflammatory state in the nasal air passages. The higher eosinophil activity in the urban area, coinciding with higher sensitization to aeroallergens, suggests that there must be some factors in the urban area that can modulate airway inflammation by influencing the activation of inflammatory cells. CONCLUSION: Our findings showed that there was no difference in the concentrations of tryptase in nasal lavage fluids between the two studied groups. However, the children from the urban area presented with higher concentrations of eosinophil cationic protein than did those from the rural area. Also, the urban children who were sensitized to aeroallergens presented with greater concentrations of eosinophil cationic protein in nasal mucosa than the non-sensitized children, while this difference was not observed among the rural children.

Presence of SNAP-25 in rat mast cells.

Mast cells participate in inflammation and allergies by releasing active mediators stored in numerous cytoplasmic granules. Degranulation implies compound exocytosis which involves a combination of granule-granule and granule-plasma membrane fusions. One of the most important proteins in the exocytotic process in neural and endocrine cells is the synaptosomal associated protein of 25 kDa (SNAP-25). In the present study, using a highly specific monoclonal antibody against SNAP-25, we have demonstrated by immunocytochemistry, western blot and immunoelectron microscopy the presence of SNAP-25 in rat peritoneal mast cells. Likewise we localized the protein mainly on the membrane of the secretory granules. Thus while the precise function of SNAP-25 in mast cells remains to be elucidated, it may be envolved in granule-granule fusion needed in degranulation.

Inflammatory mediators in nasal lavage among school-age children from urban and rural areas in São Paulo, Brazil

CONTEXTO: Alguns estudos demonstram que o processo inflamatório nas vias aéreas nasais poderia refletir ou mesmo afetar as vias aéreas inferiores. Decidimos avaliar indiretamente o estado inflamatório das vias aéreas nasais de dois grupos de escolares com diferente sensibilização aos aeroalérgenos mais comuns. OBJETIVO: Comparar a atividade inflamatória nas vias aéreas nasais, através da determinação de mediadores inflamatórios no lavado nasal em duas populações distintas de crianças em idade escolar. TIPO DE ESTUDO: Estudo transversal. LOCAL: O estudo foi realizado em duas escolas públicas de ensino fundamental, uma em zona urbana e outra em zona rural, no Estado de São Paulo. MÉTODOS: Foram constituídos dois grupos de 40 escolares que apresentam diferentes taxas de sensibilização a aeroalérgenos comuns. Amostras do lavado nasal foram colhidas para determinação de proteína catiônica eosinofílica (ECP) e triptase. Testes não-paramétricos foram usados na análise estatística. RESULTADOS: Níveis significativamente maiores de proteína catiônica eosinofílica foram encontrados nos estudantes da área urbana (p < 0,05). Não houve diferença estatística nos níveis de triptase entre os dois grupos. Observou-se ainda que, na área urbana, as crianças sensibilizadas aos aeroalérgenos apresentaram maiores concentrações de proteína catiônica eosinofílica, o que não foi observado nas crianças da zona rural. DISCUSSAO: A ausência de atividade de mastócitos e a degranulação aumentada de eosinófilos revelaram uma inflamação crônica nas vias aéreas das crianças estudadas. A maior atividade de eosinófilos na zona urbana, coincidindo com a maior sensibilização aos aeroalérgenos, sugere que deve haver algum fator a mais na área urbana que modula a resposta das vias aéreas influenciando a ativação das células inflamatórias locais. CONCLUSAO: Nossos achados não mostraram diferenças nos níveis de triptase no lavado nasal entre os dois grupos estudados. Por outro lado, as crianças da area urbana apresentaram maiores concentrações de proteína catiônica eosinofílica do que aquelas da zona rural. Observamos ainda que, na area urbana, as crianças sensibilizadas por aeroalérgenos apresentaram maiores concentrações de proteína catiônica eosinofílica do que aquelas não sensibilizadas, enquanto esta diferença não foi observada nas crianças da area rural.

Differential expression of integrin subunits on adherent and nonadherent mast cells.

Mast cell progenitors arise in bone marrow and then migrate to peripheral tissues where they mature. It is presumed that integrin receptors are involved in their migration and homing. In the present study, the expression of various integrin subunits was investigated in three systems of adherent and nonadherent mast cells. Mesentery mast cells, freshly isolated bone marrow-derived mast cells (BMMC) and RBL-2H3 cells grown attached to tissue culture flasks are all adherent mast cells and peritoneal mast cells, and cultured BMMC and RBL-2H3 cells grown in suspension represent nonadherent mast cell populations. Pure populations of mast cells were immunomagnetically isolated from bone marrow, mesentery and peritoneal lavage using the mast cell-specific monoclonal antibody AA4. By immunomicroscopy, we could demonstrate that all of these mast cells expressed alpha 4, alpha 5, alpha 6, beta 1 and beta 7 integrin subunits. The expression of the alpha 4 integrin subunit was 25% higher in freshly isolated mesentery mast cells and BMMC. Consistent with the results obtained by immunomicroscopy, mesentery mast cells expressed 65% more mRNA for the alpha 4 integrin subunit than peritoneal mast cells. In vitro studies were also conducted using the rat mast cell line RBL-2H3. RBL-2H3 cells grown attached to the tissue culture flasks or as suspension cultures expressed the same integrin subunits identified in bone marrow, mesenteric and peritoneal mast cells ex vivo. Similarly, the expression of alpha 4 integrin was higher in adherent cells. Therefore, alpha 4 integrins may play a critical role in the anchorage of mast cells to the extracellular matrix in bone marrow and in peripheral tissues.

Mast cell subsets and neuropeptides in leprosy reactions.

The immunohistochemical identification of neuropeptides (calcitonin gene-related peptide, vasoactive intestinal polypeptide, substance P, alpha-melanocyte stimulating hormone and gamma-melanocyte stimulating hormone) quantification of mast cells and their subsets (tryptase/chymase-immunoreactive mast cells = TCMC and tryptase-immunoreactive mast cells = TMC) were determined in biopsies of six patients with leprosy reactions (three patients with type I reaction and three with type II). Biopsies were compared with those taken from the same body site in the remission stage of the same patient. We found a relative increase of TMC in the inflammatory infiltrate of the reactional biopsies compared to the post-reactional biopsy. Also, the total number of mast cells and the TMC/TCMC ratio in the inflammatory infiltrate was significantly higher than in the intervening dermis of the biopsies of both periods. No significant difference was found regarding neuroptide expression in the reactional and post-reactional biopsies. The relative increase of TMC in the reactional infiltrates could implicate this mast cell subset in the reported increase of the immune response in leprosy reactions.

A new technique for staining mast cells using ferroin.

We describe here a new method for specific staining of mast cells using ferroin. Different hamster tissues were fixed in 4% formalin and processed for paraffin embedding. Sections were stained with hematoxylin followed by ferroin acidified with 2.5 N sulfuric acid to pH 4.0. Mast cells stained an intense orange color that contrasted markedly with bluish violet nuclei. High contrast was also observed when ferroin colored sections were counterstained with light green instead of hematoxylin. To evaluate the specificity of the stain, hamster cheek pouch sections were stained with toluidine blue, alcian blue-safranin O, and ferroin. Quantitative evaluation of mast cells stained with the three techniques showed no statistical difference. The simplicity and selectivity of this method is sufficient for image analysis of mast cells.

Mast cell subsets and neuropeptides in leprosy reactions

The immunohistochemical identification of neuropeptides (calcitonin gene-related peptide, vasoactive intestinal polypeptide, substance P, alpha-melanocyte stimulating hormone and gamma-melanocyte stimulating hormone) quantification of mast cells and their subsets (tryptase/chymase-immunoreactive mast cells = TCMC and tryptase-immunoreactive mast cells = TMC) were determined in biopsies of six patients with leprosy reactions (three patients with type I reaction and three with type II). Biopsies were compared with those taken from the same body site in the remission stage of the same patient. We found a relative increase of TMC in the inflammatory infiltrate of the reactional biopsies compared to the post-reactional biopsy. Also, the total number of mast cells and the TMC/TCMC ratio in the inflammatory infiltrate was significantly higher than in the intervening dermis of the biopsies of both periods. No significant difference was found regarding neuroptide expression in the reactional and post-reactional biopsies. The relative increase of TMC in the reactional infiltrates could implicate this mast cell subset in the reported increase of the immune response in leprosy reactions.