Neutrophil virucidal activity against SARS-CoV-2 is mediated by NETs.

BACKGROUND: The inflammation in the lungs and other vital organs in COVID-19 are characterized by the presence of neutrophils and high concentration of neutrophil extracellular traps (NETs), which also seems to mediate host tissue damage. However, it is not known whether NETs could have virucidal activity against SARS-CoV-2. METHODS: We investigated whether NETs could prevent SARS-CoV-2 replication in neutrophils and epithelial cells, and what the consequence of NETs degradation in K18-humanized ACE2 transgenic mice infected with SARS-CoV-2. RESULTS: Here, by immunofluorescence microscopy we observed that viral particles co-localize with NETs in neutrophils isolated from COVID-19 patients or from healthy individuals and infected in vitro. The inhibition of NETs production increased virus replication in neutrophils. In parallel, we observed that NETs inhibited virus abilities to infect and replicate in epithelial cells after 24 h of infection. Degradation of NETs with DNase I prevented their virucidal effect in vitro. Using K18-humanized ACE2 transgenic mice we observed a higher viral load in animals treated with DNase I. On the other hand, the virucidal effect of NETs was not dependent on neutrophil elastase or myeloperoxidase activity. CONCLUSION: Our results provide evidence of the role of NETosis as a mechanism of SARS-CoV-2 viral capture and inhibition.

RACK1 plays a critical role in mast cell secretion and Ca2+ mobilization by modulating F-actin dynamics.

Although RACK1 is known to act as a signaling hub in immune cells, its presence and role in mast cells (MCs) is undetermined. MC activation via antigen stimulation results in mediator release and is preceded by cytoskeleton reorganization and Ca2+ mobilization. In this study, we found that RACK1 was distributed throughout the MC cytoplasm both in vivo and in vitro. After RACK1 knockdown (KD), MCs were rounded, and the cortical F-actin was fragmented. Following antigen stimulation, in RACK1 KD MCs, there was a reduction in cortical F-actin, an increase in monomeric G-actin and a failure to organize F-actin. RACK1 KD also increased and accelerated degranulation. CD63+ secretory granules were localized in F-actin-free cortical regions in non-stimulated RACK1 KD MCs. Additionally, RACK1 KD increased antigen-stimulated Ca2+ mobilization, but attenuated antigen-stimulated depletion of ER Ca2+ stores and thapsigargin-induced Ca2+ entry. Following MC activation there was also an increase in interaction of RACK1 with Orai1 Ca2+-channels, ß-actin and the actin-binding proteins vinculin and MyoVa. These results show that RACK1 is a critical regulator of actin dynamics, affecting mediator secretion and Ca2+ signaling in MCs. This article has an associated First Person interview with the first author of the paper.

Relative expression of KLK5 to LEKTI is associated with aggressiveness of oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) remains a challenging cancer to treat despite all the advances of the last 50 years. Kallikrein 5 (KLK5) is among the serine proteases implicated in OSCC development. However, whether the activity of KLK5 promotes carcinogenesis is still controversial. Moreover, knowledge regarding the role of the KLK5 cognate inhibitor, Lympho-Epithelial Kazal-Type related Inhibitor (LEKTI), in OSCC is scarce. We have, thus, sought to investigate the importance of KLK5 and LEKTI expression in premalignant and malignant lesions of the oral cavity. METHODS: KLK5 and LEKTI protein expression was evaluated in 301 human samples, which were comprised of non-malignant and malignant lesions of the oral cavity. Moreover, a bioinformatic analysis of the overall survival rate from 517 head and neck squamous cell carcinoma (HNSCC) samples was performed. Additionally, to mimic the uncovered KLK5 to serine peptidase inhibitor (SPINK5) imbalance, the KLK5 gene was abrogated in an OSCC cell line using CRISPR-Cas9 technology. The generated cell line was then used for in vivo and in vitro carcinogenesis related experiments. RESULTS: LEKTI was found to be statistically downregulated in OSCCs, with increased KLK5/SPINK5 mRNA ratio being associated with a shorter overall survival (p = 0.091). Indeed, disruption of KLK5 to SPINK5 balance through the generation of KLK5 null OSCC cells led to smaller xenografted tumors and statistically decreased proliferation rates following multiple time points of BrdU treatment in vitro. CONCLUSION: The association of increased enzyme/inhibitor ratio with poor prognosis indicates KLK5 to SPINK5 relative expression as an important prognostic marker in OSCC.

The lectin ArtinM activates RBL-2H3 mast cells without inducing degranulation.

Mast cells are connective tissue resident cells with morphological and functional characteristics that contribute to their role in allergic and inflammatory processes, host defense and maintenance of tissue homeostasis. Mast cell activation results in the release of pro-inflammatory mediators which are largely responsible for the physiological functions of mast cells. The lectin ArtinM, extracted from Artocarpus heterophyllus (jackfruit), binds to D-manose, thus inducing degranulation of mast cells. ArtinM has several immunomodulatory properties including acceleration of wound healing, and induction of cytokine release. The aim of the present study was to investigate the role of ArtinM in the activation and proliferation of mast cells. The rat mast cell line RBL-2H3 was used throughout this study. At a low concentration (0.25µg/mL), ArtinM induced mast cell activation and the release of IL-6 without stimulating the release of pre-formed or newly formed mediators. Additionally, when the cells were activated by ArtinM protein tyrosine phosphorylation was stimulated. The low concentration of ArtinM also activated the transcription factor NFkB, but not NFAT. ArtinM also affected the cell cycle and stimulated cell proliferation. Therefore, ArtinM may have therapeutic applications by modulating immune responses due to its ability to activate mast cells and promote the release of newly synthesized mediators. Additionally, ArtinM could have beneficial effects at low concentrations without degranulating mast cells and inducing allergic reactions.

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.

Mast Cell Protease 7 Promotes Angiogenesis by Degradation of Integrin Subunits.

Previous studies from our laboratory have shown that during angiogenesis in vitro, rmMCP-7 (recombinant mouse mast cell protease-7) stimulates endothelial cell spreading and induces their penetration into the matrix. The ability of rmMCP-7 to induce angiogenesis in vivo was assessed in the present study using a directed in vivo angiogenesis assay (DIVAA™). Vessel invasion of the angioreactor was observed in the presence of rmMCP-7 but was not seen in the control. Since integrins are involved in endothelial cell migration, the relationship between rmMCP-7 and integrins during angiogenesis was investigated. Incubation with rmMCP-7 resulted in a reduction in the levels of integrin subunits αv and ß1 on SVEC4-10 endothelial cells during angiogenesis in vitro. Furthermore, the degradation of integrin subunits occurs both through the direct action of rmMCP-7 and indirectly via the ubiquitin/proteasome system. Even in the presence of a proteasome inhibitor, incubation of endothelial cells with rmMCP-7 induced cell migration and tube formation as well as the beginning of loop formation. These data indicate that the direct degradation of the integrin subunits by rmMCP-7 is sufficient to initiate angiogenesis. The results demonstrate, for the first time, that mMCP-7 acts in angiogenesis through integrin degradation.

The role of TCPTP on leptin effects on astrocyte morphology.

Leptin and LPS has been implicated in the development of hypothalamic astrogliosis in rodents. Astrocytes, which are interconnected by gap junction proteins, have emerged as important players in the control of energy homeostasis exerted by the hypothalamus. To investigate the hypothesis of action of T-cell protein tyrosine phosphatase (TCPTP) on the astrocyte morphology, astrocytes from the hypothalamus of one-day-old rats were stimulated with leptin and LPS (used as a positive control). Leptin and LPS induced a marked increase in astrocyte size, an increase in Ptpn2 (TCPTP gene) and gap junction alpha-1 protein, - Gja1 (connexin 43 - CX43 gene) mRNA expression and a decrease in gap junction protein, alpha 6 - Gja6 (CX30 gene) mRNA expression. Remarkably, these effects on astrocytes morphology and connexins were prevented by Ptpn2 siRNA. Astrocytes are known to produce cytokines; here we show that TCPTP acts as an important regulator of the cytokines and it possesses a reciprocal interplay with protein tyrosine phosphatase 1B (PTP1B). Our findings demonstrate that leptin and LPS alter astrocyte morphology by increasing TCPTP, which in turn modulates connexin 30 (CX30) and connexin 43 (CX43) expression. TCPTP and PTP1B seem to act in the regulation of cytokine production in astrocytes.

Fungal biofilms in the hemodialysis environment.

Hemodialysis, which is a kidney failure treatment that uses hemodialysis machine, dialyzer, dialysis solution, catheters, and needles, favors biofilm formation. This study evaluates whether Aspergillus, Candida, and Fusarium can form biofilm in dialysis fluids. Biofilms were grown in 96-well microplates containing solutions (acid and basic) consisting of dialysate, dialysate per se, or dialysate plus glucose as culture medium. The biofilms were incubated at 30 °C for 72 h, quantified by the violet crystal methodology, and visualized by transmission electron microscopy. All the fungi formed biomass in all the tested solutions. However, Bonferroni analysis revealed that the dialysate facilitated Aspergillus biomass development, whereas the dialysate and dialysate with glucose provided similar Fusarium oxysporum biomass development. Candida parapsilosis development was favored in biofilms grown in basic electrolytic solution. Electron micrographs of biofilms that grew on catheters after 72 h showed that Aspergillus formed abundant hyphae; the extracellular matrix was visible on the surface of some hyphae when Aspergillus was grown in the dialysate. A multilayered hyphal structure emerged when F. oxysporum biofilms were incubated in the dialysate with glucose. C. parapsilosis biofilm growth in basic solution elicited a dense network of yeasts and pseudohyphae as well as the extracellular matrix; the biofilm was attached across the catheter length. This study may contribute to the formulation of new strategies to monitor biofilm formation and to increase knowledge associated with fungal biofilms in the dialysis environment.

Mast Cells Interact with Endothelial Cells to Accelerate In Vitro Angiogenesis.

Angiogenesis is a complex process that involves interactions between endothelial cells and various other cell types as well as the tissue microenvironment. Several previous studies have demonstrated that mast cells accumulate at angiogenic sites. In spite of the evidence suggesting a relationship between mast cells and angiogenesis, the association of mast cells and endothelial cells remains poorly understood. The present study aims to investigate the relationship between mast cells and endothelial cells during in vitro angiogenesis. When endothelial cells were co-cultured with mast cells, angiogenesis was stimulated. Furthermore, there was direct intercellular communication via gap junctions between the two cell types. In addition, the presence of mast cells stimulated endothelial cells to release angiogenic factors. Moreover, conditioned medium from the co-cultures also stimulated in vitro angiogenesis. The results from this investigation demonstrate that mast cells have both direct and indirect proangiogenic effects and provide new insights into the role of mast cells in angiogenesis.

Mast Cell Mediators Inhibit Osteoblastic Differentiation and Extracellular Matrix Mineralization.

Mast cells are multifunctional immune cells that participate in many important processes such as defense against pathogens, allergic reactions, and tissue repair. These cells perform their functions through the release of a wide variety of mediators. This release occurs mainly through cross-linking IgE (immunoglobulin E) bound to high affinity IgE receptors by multivalent antigens. The abundance of mast cells in connective tissue, surrounding blood vessels, and their involvement in the early stages of bone repair support the possibility of physiological and pathological interactions between mast cells and osteoblasts. However, the participation of mast cell mediators in osteogenesis is not fully understood. Therefore, the objective of this work was to investigate the role of mast cell mediators in the acquisition of the osteogenic phenotype in vitro. The results show that pooled mast cell mediators can affect proliferation, morphology, and cytoskeleton of osteoblastic cells, and impair the activity and expression of alkaline phosphatase as well as the expression of bone sialoprotein. Also, mast cell mediators inhibit the expression of mRNA for those proteins and inhibit the formation and maturation of calcium nodules and consequently inhibit mineralization. Therefore, mast cell mediators can modulate osteogenesis and are potential therapeutic targets for treatments of bone disorders.

Paracoccin distribution supports its role in Paracoccidioides brasiliensis growth and dimorphic transformation.

Paracoccidioides brasiliensis yeast was reported to express paracoccin, a GlcNAc-binding protein that displays N-acetyl-ß-d-glucosaminidase (NAGase) activity. Highly specific anti-paracoccin antibodies have been previously used to examine the localization of paracoccin in yeast and inhibit its growth in vitro. In the present study, anti-paracoccin antibodies were used to characterize, by scanning confocal microscopy, the distribution of paracoccin in P. brasiliensis hyphae, transition forms from hyphae to yeast, and mature yeast. In the mycelial phase, paracoccin was detected mainly in the hyphae tips, where it demonstrated a punctate distribution, and was associated with the cell wall. During the first 48 hours after a temperature shift from 26°C to 37°C, paracoccin expression in the differentiating hyphae was mainly detected in the budding regions, i.e. lateral protrusions, and inside the new daughter cells. There was an increased number of chlamydoconidia that expressed a high concentration of paracoccin on their surfaces and/or in their interiors 72-96 hours after the temperature shift. After 120 hours, yeast cells were the predominant form and their cytoplasm stained extensively for paracoccin, whereas Wheat Germ Agglutinin (WGA) staining was predominant on their exterior walls. After 10 days at 37°C, the interior of both mother and daughter yeast cells, as well as the budding regions, stained intensely for paracoccin. The comparison of mRNA-expression in the different fungal forms showed that PCN transcripts, although detected in all evaluated morphological forms, were higher in hypha and yeast-to-hypha transition forms. In conclusion, the pattern of paracoccin distribution in all P. brasiliensis morphotypes supports prevalent beliefs that it plays important roles in fungal growth and dimorphic transformation.

Adaptor protein-3: A key player in RBL-2H3 mast cell mediator release.

Mast cell (MC) secretory granules are Lysosome-Related Organelles (LROs) whose biogenesis is associated with the post-Golgi secretory and endocytic pathways in which the sorting of proteins destined for a specific organelle relies on the recognition of sorting signals by adaptor proteins that direct their incorporation into transport vesicles. The adaptor protein 3 (AP-3) complex mediates protein trafficking between the trans-Golgi network (TGN) and late endosomes, lysosomes, and LROs. AP-3 has a recognized role in LROs biogenesis and regulated secretion in several cell types, including many immune cells such as neutrophils, natural killer cells, and cytotoxic T lymphocytes. However, the relevance of AP-3 for these processes in MCs has not been previously investigated. AP-3 was found to be expressed and distributed in a punctate fashion in rat peritoneal mast cells ex vivo. The rat MC line RBL-2H3 was used as a model system to investigate the role of AP-3 in mast cell secretory granule biogenesis and mediator release. By immunofluorescence and immunoelectron microscopy, AP-3 was localized both to the TGN and early endosomes indicating that AP-3 dependent sorting of proteins to MC secretory granules originates in these organelles. ShRNA mediated depletion of the AP-3 δ subunit was shown to destabilize the AP-3 complex in RBL-2H3 MCs. AP-3 knockdown significantly affected MC regulated secretion of ß-hexosaminidase without affecting total cellular enzyme levels. Morphometric evaluation of MC secretory granules by electron microscopy revealed that the area of MC secretory granules in AP-3 knockdown MCs was significantly increased, indicating that AP-3 is involved in MC secretory granule biogenesis. Furthermore, AP-3 knockdown had a selective impact on the secretion of newly formed and newly synthesized mediators. These results show for the first time that AP-3 plays a critical role in secretory granule biogenesis and mediator release in MCs.

Cross-Linking Mast Cell Specific Gangliosides Stimulates the Release of Newly Formed Lipid Mediators and Newly Synthesized Cytokines.

Mast cells are immunoregulatory cells that participate in inflammatory processes. Cross-linking mast cell specific GD1b derived gangliosides by mAbAA4 results in partial activation of mast cells without the release of preformed mediators. The present study examines the release of newly formed and newly synthesized mediators following ganglioside cross-linking. Cross-linking the gangliosides with mAbAA4 released the newly formed lipid mediators, prostaglandins D2 and E2, without release of leukotrienes B4 and C4. The effect of cross-linking these gangliosides on the activation of enzymes in the arachidonate cascade was then investigated. Ganglioside cross-linking resulted in phosphorylation of cytosolic phospholipase A2 and increased expression of cyclooxygenase-2. Translocation of 5-lipoxygenase from the cytosol to the nucleus was not induced by ganglioside cross-linking. Cross-linking of GD1b derived gangliosides also resulted in the release of the newly synthesized mediators, interleukin-4, interleukin-6, and TNF-α. The effect of cross-linking the gangliosides on the MAP kinase pathway was then investigated. Cross-linking the gangliosides induced the phosphorylation of ERK1/2, JNK1/2, and p38 as well as activating both NFκB and NFAT in a Syk-dependent manner. Therefore, cross-linking the mast cell specific GD1b derived gangliosides results in the activation of signaling pathways that culminate with the release of newly formed and newly synthesized mediators.

Recombinant ArtinM activates mast cells.

BACKGROUND: Mast cells are hematopoietically derived cells that play a role in inflammatory processes such as allergy, as well as in the immune response against pathogens by the selective and rapid release of preformed and lipid mediators, and the delayed release of cytokines. The native homotetrameric lectin ArtinM, a D-mannose binding lectin purified from Artocarpus heterophyllus seeds, is one of several lectins that are able to activate mast cells. Besides activating mast cells, ArtinM has been shown to affect several biological responses, including immunomodulation and acceleration of wound healing. Because of the potential pharmacological application of ArtinM, a recombinant ArtinM (rArtinM) was produced in Escherichia coli. The current study evaluated the ability of rArtinM to induce mast cell degranulation and activation. RESULTS: The glycan binding specificity of rArtinM was similar to that of jArtinM. rArtinM, via its CRD, was able to degranulate, releasing ß-hexosaminidase and TNF-α, and to promote morphological changes on the mast cell surface. Moreover, rArtinM induced the release of the newly-synthesized mediator, IL-4. rArtinM does not have a co-stimulatory effect on the FcεRI degranulation via. The IgE-dependent mast cell activation triggered by rArtinM seems to be dependent on NFkB activation. CONCLUSIONS: The lectin rArtinM has the ability to activate and degranulate mast cells via their CRDs. The present study indicates that rArtinM is a suitable substitute for the native form, jArtinM, and that rArtinM may serve as an important and reliable pharmacological agent.

The topical use of insulin accelerates the healing of traumatic tympanic membrane perforations.

OBJECTIVE: In recent years, there has been a tendency to search for regulatory substances that can optimize the healing process of perforated tympanic membranes (TMs). The purpose of this study was to determine the effects of using topical insulin on the healing process of traumatic TMs perforations. STUDY DESIGN: Experimental. METHODS: Tympanic membranes of 20 Wistar rats were perforated in two sections, anterior and posterior to the malleus. The rats were divided into two groups: control and insulin. The insulin group was treated with topical regular insulin. The TMs were histologically examined 3, 5, and 7 days after the perforation through a morphometric analysis of the epithelial layer thickness, perforation size, TM cross-sectional area, semiquantitative and qualitative evaluation of the collagen production by polarization microscopy, and immunohistochemical evaluation of epithelial cells and myofibroblasts markers. RESULTS: Insulin accelerated the healing process of the perforated TMs (P < 0.01); stimulated early thickening of the outer epithelial layer (P < 0.01); contributed to a larger identification of the antipankeratin antibody as epithelial marker (P < 0.05); and increased labeling of smooth muscle anti-alpha-actin antibody (P < 0.05), indicating greater proliferation of myofibroblasts. When the topical insulin was used, it resulted in a greater formation of collagen tissue (P < 0.05), with thicker and better-organized collagen fibers (P < 0.05). CONCLUSION: Insulin accelerated the healing process of TMs traumatic perforations.

Mast Cell Proteases 6 and 7 Stimulate Angiogenesis by Inducing Endothelial Cells to Release Angiogenic Factors.

Mast cell proteases are thought to be involved with tumor progression and neo-vascularization. However, their exact role is still unclear. The present study was undertaken to further elucidate the function of specific subtypes of recombinant mouse mast cell proteases (rmMCP-6 and 7) in neo-vascularization. SVEC4-10 cells were cultured on Geltrex® with either rmMCP-6 or 7 and tube formation was analyzed by fluorescence microscopy and scanning electron microscopy. Additionally, the capacity of these proteases to induce the release of angiogenic factors and pro and anti-angiogenic proteins was analyzed. Both rmMCP-6 and 7 were able to stimulate tube formation. Scanning electron microscopy showed that incubation with the proteases induced SVEC4-10 cells to invade the gel matrix. However, the expression and activity of metalloproteases were not altered by incubation with the mast cell proteases. Furthermore, rmMCP-6 and rmMCP-7 were able to induce the differential release of angiogenic factors from the SVEC4-10 cells. rmMCP-7 was more efficient in stimulating tube formation and release of angiogenic factors than rmMCP-6. These results suggest that the subtypes of proteases released by mast cells may influence endothelial cells during in vivo neo-vascularization.

Phospholipase D2 Modulates the Secretory Pathway in RBL-2H3 Mast Cells.

Phospholipase D (PLD) hydrolyses phosphatidylcholine to produce phosphatidic acid (PA) and choline. It has two isoforms, PLD1 and PLD2, which are differentially expressed depending on the cell type. In mast cells it plays an important role in signal transduction. The aim of the present study was to clarify the role of PLD2 in the secretory pathway. RBL-2H3 cells, a mast cell line, transfected to overexpress catalytically active (PLD2CA) and inactive (PLD2CI) forms of PLD2 were used. Previous observations showed that the Golgi complex was well organized in CA cells, but was disorganized and dispersed in CI cells. Furthermore, in CI cells, the microtubule organizing center was difficult to identify and the microtubules were disorganized. These previous observations demonstrated that PLD2 is important for maintaining the morphology and organization of the Golgi complex. To further understand the role of PLD2 in secretory and vesicular trafficking, the role of PLD2 in the secretory process was investigated. Incorporation of sialic acid was used to follow the synthesis and transport of glycoconjugates in the cell lines. The modified sialic acid was subsequently detected by labeling with a fluorophore or biotin to visualize the localization of the molecule after a pulse-chase for various times. Glycoconjugate trafficking was slower in the CI cells and labeled glycans took longer to reach the plasma membrane. Furthermore, in CI cells sialic acid glycans remained at the plasma membrane for longer periods of time compared to RBL-2H3 cells. These results suggest that PLD2 activity plays an important role in regulating glycoconjugate trafficking in mast cells.

rPbPga1 from Paracoccidioides brasiliensis Activates Mast Cells and Macrophages via NFkB.

BACKGROUND: The fungus Paracoccidioides brasiliensis is the leading etiological agent of paracoccidioidomycosis (PCM), a systemic granulomatous disease that typically affects the lungs. Cell wall components of P. brasiliensis interact with host cells and influence the pathogenesis of PCM. In yeast, many glycosylphosphatidylinositol (GPI)-anchored proteins are important in the initial contact with the host, mediating host-yeast interactions that culminate with the disease. PbPga1 is a GPI anchored protein located on the surface of the yeast P. brasiliensis that is recognized by sera from PCM patients. METHODOLOGY/PRINCIPAL FINDINGS: Endogenous PbPga1 was localized to the surface of P. brasiliensis yeast cells in the lungs of infected mice using a polyclonal anti-rPbPga1 antibody. Furthermore, macrophages stained with anti-CD38 were associated with P. brasiliensis containing granulomas. Additionally, rPbPga1 activated the transcription factor NFkB in the macrophage cell line Raw 264.7 Luc cells, containing the luciferase gene downstream of the NFkB promoter. After 24 h of incubation with rPbPga1, alveolar macrophages from BALB/c mice were stimulated to release TNF-α, IL-4 and NO. Mast cells, identified by toluidine blue staining, were also associated with P. brasiliensis containing granulomas. Co-culture of P. Brasiliensis yeast cells with RBL-2H3 mast cells induced morphological changes on the surface of the mast cells. Furthermore, RBL-2H3 mast cells were degranulated by P. brasiliensis yeast cells, but not by rPbPga1, as determined by the release of beta-hexosaminidase. However, RBL-2H3 cells activated by rPbPga1 released the inflammatory interleukin IL-6 and also activated the transcription factor NFkB in GFP-reporter mast cells. The transcription factor NFAT was not activated when the mast cells were incubated with rPbPga1. CONCLUSIONS/SIGNIFICANCE: The results indicate that PbPga1 may act as a modulator protein in PCM pathogenesis and serve as a useful target for additional studies on the pathogenesis of P. brasiliensis.

The Role of Mast Cell Specific Chymases and Tryptases in Tumor Angiogenesis.

An association between mast cells and tumor angiogenesis is known to exist, but the exact role that mast cells play in this process is still unclear. It is thought that the mediators released by mast cells are important in neovascularization. However, it is not known how individual mediators are involved in this process. The major constituents of mast cell secretory granules are the mast cell specific proteases chymase, tryptase, and carboxypeptidase A3. Several previous studies aimed to understand the way in which specific mast cell granule constituents act to induce tumor angiogenesis. A body of evidence indicates that mast cell proteases are the pivotal players in inducing tumor angiogenesis. In this review, the likely mechanisms by which tryptase and chymase can act directly or indirectly to induce tumor angiogenesis are discussed. Finally, information presented here in this review indicates that mast cell proteases significantly influence angiogenesis thus affecting tumor growth and progression. This also suggests that these proteases could serve as novel therapeutic targets for the treatment of various types of cancer.

Spontaneous healing of the tympanic membrane after traumatic perforation in rats.

UNLABELLED: The most common etiologies of tympanic membrane perforation are infections and trauma. OBJECTIVE: The objective of the present study was to assess the healing of traumatic tympanic membrane perforation in rats. METHODS: The tympanic membrane from male Wistar rats was perforated in the anterior and posterior portions to the handle of the malleus. Five tympanic membranes were evaluated 3 days after tympanic perforation; 5 after 5 days; 5 after 7 days; 3 after 10 days; and 4 after 14 days. The tympanic membranes were submitted to histopathological evaluation after hematoxylin-eosin staining. RESULTS: Tympanic membrane closure occurred at about 7-10 days after injury and the healing process was complete by day 14. The proliferative activity of the outer epithelial layer was present close to the handle of the malleus and to the tympanic annulus. CONCLUSION: The spontaneous healing process of the tympanic membrane starts from the outer epithelial layer, with later healing of the lamina propria and the mucosal layer.