Detection Of Mast Cells Research Articles

Maggot alleviates imiquimod-induced psoriasis-like skin lesions in mice by inhibiting immune stress and complement activation

To explore the therapeutic mechanism of maggot for psoriasis-like lesions in mice from the perspective of immune stress and complement activation regulation. Thirty-six male C57BL/6 mice were randomly divided into control group, model group, maggot (1.25%, 2.5%, and 5%) groups, and Benvitimod (1%) group. Psoriasis-like lesions were induced by application of imiquimod cream, and the severity of skin lesions was assessed using the modified Psoriasis Area and Severity Index (MPASI) score. Auricular swelling of the mice was observed, and histopathological changes of the skin lesions were examined with HE staining. Scratching behavior of the mice was observed and the spleen index was calculated. Toluidine blue staining was used to detect mast cells in the skin lesions, and serum levels of IgG, IgM, the complements CH50, C1s, C3, C3a, C5 and C5a, and the inflammatory factors IL-23, IL-17A and TNF-α were determined with ELISA. In mice with imiquimod-induced psoriasis-like skin lesions, treatment with the maggot at the 3 doses significantly decreased MPASI score, alleviated auricular swelling and pathologies in the skin lesions, reduced scratching behaviors, spleen index, and the number of mast cells in the lesions. Treatment with high-dose maggot significantly lowered serum levels of IgG, C1s, C3a, C5a, IL-23, IL-17A and TNF- α and the levels of C1s, C3, C3a, C5 and C5a in the lesion tissue, and increased serum levels of CH50, C3, and C5. The therapeutic effect of maggot showed a dose-effect dependence. Maggot can alleviate psoriasislike skin lesions in mice by inhibiting immune stress and complement activation.

Expression of NMU and NMUR1 in tryptase-positive mast cells and PBLs in allergic rhinitis patients' nasal mucosa.

The neuropeptide U (NMU) has been proven to elicit the release of mediators from mast cells (MCs) through its receptor NMUR1 in allergic inflammatory models. However, little is known about the correlations between NMU and MCs in human allergic rhinitis (AR). The objective of this study is to investigate the expressions of NMU and NMUR1 in the tryptase + MCs and the peripheral blood leukocytes (PBLs) in human nasal mucosa with AR. Specimens of nasal mucosa from patients with AR (n = 10) and control patients without AR (n = 8) were collected and soaked in frozen tissue liquid solution (OCT) in tum. Cryostat sections were prepared for immunofluorescence staining. Tryptase was used as a marker to detect mast cells and other tryptase + immune cells. The expression of NMU and NMUR1 was respectively determined by double staining using a confocal microscope. Neither NMU nor NMUR1 were detected in the tryptase + mast cells in the human nasal mucosa. To our surprise, both NMU and NMUR1 were co-expressed with tryptase in the PBLs within peripheral blood vessels in AR and controls. Our findings showed that NMU could not influence human nasal tryptase + mast cells directly through NMUR1 in AR. The co-expression of both NMU and NMUR1 with tryptase in the PBLs provided new insight into the potential roles of NMU and tryptase in the circulation PBLs, and the infiltrated PBLs may promote nasal allergic inflammation by producing tryptase and NMU.

Quantitative estimation of TNF-α and IL-3 by using ELISA from human lung tissue in fatal asphyxial deaths

Asphyxia-related deaths have always been a challenging task in the specialty of forensic pathology. Apart from helpful macroscopical signs (e.g., strangulation marks, cyanosis, petechial haemorrhage, and lung edema), recent literature indicates that prolonged asphyxia is sufficient to induce an increase in mast cells (MC). Inflammatory cells migrate from the bone marrow to the lungs, aiding in the diagnosis of fatal asphyxial death. The present study analyzed human lung tissue samples from 90 medico-legal autopsy cases, including 45 asphyxial deaths and 45 controls (non-asphyxial deaths). The cases ranged from 2 to 68 years, with a mean age of 33.23 years. In 90 cases, 74 cases were of males, and 16 were of females. Human lung tissue samples were analyzed by using the sandwich ELISA method. The results indicated a statistically significant increase in TNF-α and IL-3 concentration in fatal asphyxial deaths, including those caused by hanging, drowning, and smothering. Mean ± SD in asphyxial and non-asphyxial cases for the TNF-α and IL-3 concentration statistically analysed. In asphyxial cases, the average IL-3 concentration (Conc.) was 1558.50 ± 350.53 pg/ml, and the average TNF-α concentration (Conc.) was 499.75 ± 479.41 pg/ml. In contrast, in non-asphyxial cases, the average IL-3 concentration (Conc.) was found to be 849.73 ± 484.99 pg/ml, and the average TNF-α concentration (Conc.) was 208.08 ± 81.23 pg/ml. The mean change in IL-3 and TNF-α (Conc.) values are found to significant (<0.01) in asphyxial cases as compared to non-asphyxial cases. The ROC (Receiver operating characteristic curve) analysis revealed that TNF-α (AUC = 0.89) and IL-3 (AUC = 0.87) concentration (conc.) were stronger predictors of asphyxial deaths with an optimal cut-off value of 455.20 pg/ml for TNF-alpha and 1700.62 pg/ml for IL-3 respectively. Our findings imply that mast cells (MC) are critical in fatal hypoxia-related mortality and that TNF-α and IL-3 can be reliable markers for detecting mast cells in asphyxial deaths. It could be very beneficial to forensic pathologists tasked with differentiating fatal asphyxial fatalities from other causes of death.

RMBP-NAP Suppresses OXA-induced Allergic Dermatitis by Regulating the Th1/Th2 Balance.

Allergic dermatitis (AD) is an inflammatory skin disease that arises from abnormal T lymphocyte activation. A recombinant fusion protein comprising Helicobacter pylori neutrophil-activating protein and maltose binding protein, rMBP-NAP, has been documented as a novel immunomodulatory TLR agonist. To explore the effect of the rMBP-NAP on the OXA-induced AD in a mouse model and clarify the possible action mechanism. The AD animal model was induced by repeated administration of oxazolone (OXA) in BALB/c mice. H&E staining was used to analyze the ear epidermis thickness and the number of infiltrating inflammatory cells. TB staining was used to detect mast cell infiltration in the ear tissue. ELISA was used to analyze the secretion of cytokines IL-4 and IFN-γ in peripheral blood. qRT-PCR was used to determine the expression levels of IL-4, IFN-γ, and IL-13 in ear tissue. OXA induced the establishment of an AD model. After the rMBP-NAP treatment, the thickness of the ear tissue and the number of mast cells infiltrated in AD mice reduced, and the serum and ear tissue levels of IL-4 and IFN-γ increased, but the ratio of IFN-γ (rMBP-NAP group)/IL-4 (rMBP-NAP group) was greater than the ratio of IFN-γ (sensitized group)/IL-4 (sensitized group). The rMBP-NAP improved the disease symptoms including skin lesions in AD, alleviated the inflammation in ear tissue, and restored the Th1/2 balance by inducing a shift from the Th2 to the Th1 response. The results of our work support the use of rMBP-NAP as an immunomodulator for AD treatment in future investigations.

Role of mast cells in the pathogenesis of severe lung damage in COVID-19 patients

BackgroundThere is still insufficient knowledge with regard to the potential involvement of mast cells (MCs) and their mediators in the pathology of coronavirus disease-2019 (COVID-19). Therefore, our study aimed to investigate the role of MCs, their activation and protease profiles in the pathogenesis of early and late lung damage in COVID-19 patients.MethodsFormalin-fixed and paraffin embedded lung specimens from 30 patients who died from COVID-19 and 9 controls were used for histological detection of MCs and their proteases (tryptase, chymase) followed by morphometric quantification.ResultsOur results demonstrated increased numbers of MCs at early stage and further augmentation of MCs number during the late stage of alveolar damage in COVID-19 patients, as compared to the control group. Importantly, the percentage of degranulated (activated) MCs was higher during both stages of alveolar lesions in comparison to the controls. While there was no prominent alteration in the profile of tryptase-positive MCs, our data revealed a significant elevation in the number of chymase-positive MCs in the lungs of COVID-19 patients, compared to the controls.ConclusionsMCs are characterized by dysregulated accumulation and increased activation in the lungs of patients suffering from COVID-19. However, future profound studies are needed for precise analysis of the role of these immune cells in the context of novel coronavirus disease.

LB1030 Automated image analysis methods to detect mast cells in skin

Mast cells are a key cell in the innate immune system. These cells are tissue resident and are considered to act as sentinels for the detection of a multitude of different ligands. Activation of mast cells via stimulation of IgE receptors underlies allergic reactions to food, pollen, pet dander, and many other allergens. Stimulation of mast cells results in their activation leading to degranulation – a process in which the cell releases extracellular mediators like histamine, proteases, and cytokines. These mediators can then go on to activate other cell types nearby or can recruit more immune cells to the tissue. Some chronic pruritic diseases are thought to involve mast cells in the skin and many studies have demonstrated an increase in total mast cell number in skin from atopic dermatitis (AD) or allergic contact dermatitis (CAD). Previous methods relied on staining of skin biopsy sections with a chromogenic stain (like toulidine blue), then manual counting of the mast cells in small sections of the biopsy. More recently, the use of fluorescently-tagged avidin has been used to detect mast cells in skin biopsies, which represents a more specific detection method. A challenge still remains in the differentiation between quiescent (resting) and active (or degranulated) mast cells. Here we describe a method for the automated detection and quantification of quiescent and resting mast cells based on cell morphology, using commercially available image software analysis tools. Our method was applied to human skin biopsies that were treated to induce degranulation and to skin biopsies collected from AD patients. In contrast to earlier results, we do not observe an increase in total mast cell number nor in the number of active mast cells in the skin of AD patients. This method allows for the unbiased detection of mast cells in human skin with out the need to develop de novo proprietary algorithms.

Design and optimization of ROS-reactive nanocarriers for controlled release of dexamethasone and real-time inhibition of mast cell activation

Upon activation, mast cells release many mediators, including reactive oxygen species (ROS) and tumor necrosis factor (TNF) orchestrating allergic inflammation. Nanocarriers (NCs) that can detect mast cell activation and respond by releasing anti-inflammatory compounds in real-time, would and effective therapy.

Modern Imaging Technologies of Mast Cells for Biology and Medicine (Review).

Mast cells play an important role in the body defense against allergens, pathogens, and parasites by participating in inflammation development. However, there is evidence for their contributing to the pathogenesis of a number of atopic, autoimmune, as well as cardiovascular, oncologic, neurologic, and other diseases (allergy, asthma, eczema, rhinitis, anaphylaxis, mastocytosis, multiple sclerosis, rheumatoid arthritis, inflammatory gastrointestinal and pulmonary diseases, migraine, etc.). The diagnosis of many diseases and the study of mast cell functions in health and disease require their identification; so, the knowledge on adequate imaging techniques for mast cells in humans and different species of animals is of particular importance.The present review summarizes the data on major methods of mast cell imaging: enzyme histochemistry, immunohistochemistry, as well as histochemistry using histological stains. The main histological stains bind to heparin and other acidic mucopolysaccharides contained in mast cells and stain them metachromatically. Among these are toluidine blue, methylene blue (including that contained in May-Grünwald–Giemsa stain), thionin, pinacyanol, and others. Safranin and fluorescent dyes: berberine and avidin — also bind to heparin. Longer staining with histological dyes or alcian blue staining is needed to label mucosal and immature mast cells.Advanced techniques — enzyme histochemistry and especially immunohistochemistry — enable to detect mast cells high-selectively using a reaction to tryptases and chymases (specific proteases of these cells). In the immunohistochemical study of tryptases and chymases, species-specific differences in the distribution of the proteases in mast cells of humans and animals should be taken into account for their adequate detection. The immunohistochemical reaction to immunoglobulin E receptor (FcεRI) and c-kit receptor is not specific to mast cells, although the latter is important to demonstrate their proliferation in normal and malignant growth.Correct fixation of biological material is also discussed in the review as it is of great significance for histochemical and immunohistochemical mast cell detection.Fluorescent methods of immunohistochemistry and a multimarker analysis in combination with confocal microscopy are reported to be new technological approaches currently used to study various mast cell populations.

Tongxie Anchang Decoction Relieves Visceral Hypersensitivity in Diarrhea-Predominant Irritable Bowel Syndrome Rats by Regulating the NGF/TrkA Signaling Pathway.

Irritable bowel syndrome (IBS) is a functional gastrointestinal disease characterized by visceral hypersensitivity-related abdominal pain, in which diarrhea-predominant IBS (IBS-D) is the main subtype and has a high clinical incidence. Tongxie Anchang Decoction (TXACD) has been proved to significantly improve abdominal pain in patients with IBS-D, but its underlying therapeutic mechanism still remains unclear. In the present study, IBS-D model rats were induced by neonatal maternal separation (NMS) combined with restraint stress (RS). The therapeutic effect of TXACD was evaluated by fecal characteristics and abdominal withdrawal reflex (AWR) scores. After 14 days of intragastric administration, the colonic tissues of rats were collected to detect the protein and gene level of the NGF, TrkA, and TRPV1 using Western blotting and real-time polymerase chain reaction, respectively, and detect mast cells infiltration using toluidine blue staining. The abdominal aorta blood centrifuged was collected for detecting serum levels of SP, 5-HT, and CGRP with ELISA. The results revealed that TXACD could significantly improve visceral hypersensitivity in IBS-D rats, reflected in the decrease of AWR score and the serum levels of SP, 5-HT, and CGRP. In addition, TXACD treatment could alleviate mast cells infiltration. Moreover, the expression levels of the NGF, TrkA, and TRPV1 were repressed by TXACD. The findings of the present study indicated that the therapeutic effect of TXACD on visceral hypersensitivity might be closely related to the downregulation of the NGF/TrkA signaling pathway, the reversal of TRPV1 expression and mast cells infiltration, and the decreased release of neuroendocrine factors SP, 5-HT, and CGRP.

Mast cells and Kurloff cells - Their detection throughout the oestrous cycle in normal guinea pig ovaries and in guinea pigs with cystic rete ovarii

Mast cells (MCs) and Kurloff cells (KCs) were detected in guinea pig ovaries in the follicular and luteal phases of the oestrous cycle. The samples of ovaries were fixed in Mota's basic lead acetate. Toluidine blue was used for detection of MCs and periodic acid-Schiff for detection of KCs. The percentage of KCs in a differential leukocyte count was determined in blood smears stained according to the Pappenheim method. Non-pregnant females with normal ovaries and with cystic rete ovarii were included in the study and the numbers of MCs and KCs were compared in these two groups and in follicular and luteal phases of the oestrous cycle. MCs' distribution in ovaries was different in the guinea pig in comparison to previously studied species: MCs were found exclusively in the superficial layers of cortical stroma and no significant difference was found between the number of MCs in the follicular and luteal phases, neither in normal ovaries, nor in ovaries with cystic rete ovarii. Significantly lower numbers of MCs were found in ovaries with cystic rete ovarii (P < 0.01) in contrast to normal ovaries. A significantly higher percentage of KCs in the peripheral blood was found in the follicular phase (P < 0.05), whereas no significant difference was found in relation to the presence of cystic rete ovarii. Interestingly, no KCs were found in the samples of ovaries (either in the follicular or luteal phase, and with or without cysts). Thus, the expected role of KCs in ovarian physiology or in the aetiology of the cystic rete ovarii can be excluded.

Paeoniflorin suppresses allergic and inflammatory responses by promoting autophagy in rats with urticaria

Paeoniflorin (PF) has been reported to be effective against several skin disorders, such as allergic contact dermatitis and psoriasis; however, it remains unclear whether PF can protect against urticarial lesions. Herein, the effects of PF on rats with urticarial lesions and the possible underlying mechanism were investigated. The effects of PF administration on a rat model of ovalbumin-induced urticarial-like lesions were evaluated via pathological analysis using hematoxylin-eosin staining. Toluidine blue staining was performed to detect mast cells and ELISA was performed to determine serum histamine levels. PF-induced regulatory effects on autophagic activity and the potential underlying mechanism of this were also investigated using transmission electron microscopy, immunohistochemistry and reverse transcription-quantitative PCR. It was demonstrated that PF suppressed allergic and inflammatory responses to improve urticarial lesions, as evidenced by the attenuation of pathological abnormalities, mast cell infiltration and histamine secretion. Mechanistically, PF treatment was found to markedly limit the production and release of inflammatory cytokine interleukin (IL)-23, while the levels of IL-17 remained unchanged. PF intervention led to an increased number of autophagosomes, along with higher levels of light chain 3B (LC3B) and Beclin-1, and lower levels of P62, indicating that PF could augment autophagic activity in urticarial lesions. PF treatment increased the expression of liver kinase B1 (LKB1) and AMP-activated protein kinase-α (AMPK-α), contributing to the PF-enhanced autophagic activity. In conclusion, PF could effectively improve urticarial lesions by inhibiting inflammatory cytokine IL-23 and increasing the autophagic activity via the LKB1/AMPK-α pathway.

Tryptase Profile of the Rat Skin Mast Cell Population During the Wound Healing

Mast cells cyclically synthesize and excrete a wide range of biogenesis products with different biological activities into the extracellular matrix and are regulators of local homeostasis both in normal conditions and in pathology – inflammation, oncogenesis, etc. The relative specificity of classical histochemical methods for detecting mast cells in relation to chromogenic to substrates causes certain difficulties in the selective study of the components of the secretome of mast cells, for example, heparin, histamine, chymase or tryptase. Therefore, immunomorphological techniques have become very popular, which identify specific substrates and allow differentiation of the components of the mast cell secretome. Mediators produced by mast cells promote neoangiogenesis, fibrillogenesis and re-epithelialization during the repair process.The aim of our work was to study the tryptase profile of the mast cell population of rat skin during the wound processusing an original combined method of immunohistochemical staining.Material and methods. The experiment involved 12 Wistar rats divided into two groups – intact (n=6) and with the existing wound process of the skin in the withers (n=6). The tryptase profile of mast cells was assessed on the 7th day of the wound process in comparison with the control group.Results. The results obtained showed a significant increase in the number of tryptase-positive mast cells on the 7th day of the wound process in the skin against the background of a general increase in the population of mast cells. Intragranular tryptase reserve was significantly increased. In contrast to the control, where mast cells with single tryptase-positive granules dominated, during the wound process, cells of this type were practically not detected in the skin (43.69±2.9% and 8.55±0.9%). The content of tryptase-positive mast cells with complete filling of the cytoplasm in the control group and the group of animals with a wound process was 14.24±1.2% and 38.03±2.9%, respectively.Conclusion. Thus, when modeling a wound, an increase in tryptase synthesis is detected both in individual MCs and within the entire MC population. This fact indicates that mast cell proteases can become a potential therapeutic target for improving wound regeneration by correcting immunogenesis, inflammation and fiber formation.

Effects of combined treatment with Indomethacin and Juglone on AOM/DSS induced colon carcinogenesis in Balb/c mice: Roles of inflammation and apoptosis

AimIndomethacin [IND] is reported to treat colon cancer. However, continuous exposure to IND causes gastric ulceration, an adverse side effect in humans. This study implies the therapeutic effect of IND and juglone [JUG] against colon carcinogenesis, without gastric ulceration – an adverse side effect of IND. Materials and methodsAdult male Balb/C mice were divided into six groups randomly: control, AOM/DSS-induced, IND-treated, JUG-treated, IND + JUG-treated and drug-control. Levels of serum markers, haematoxylin & eosin staining to observe tissue architecture, toluidine blue staining to detect mast cells expression, Masson's trichrome and sirius-red staining were used to detect the collagen deposition. RT-PCR and western blot analysis were carried out to detect inflammation and apoptosis. Key findingsIND + JUG effectively decreased the levels of serum markers: CEA, AFP, LDH, AST and ALT. Although, IND restored colonic architecture by regulating the accumulation of mast cell and collagen content, it causes gastric ulceration. To address this adverse effect of IND, JUG was given along with IND and was shown to alleviate IND-induced gastric ulceration. AOM/DSS induced animals showed increased expression of inflammatory molecules - TNFα, NFκB and Cox-2, apoptosis regulator - Bcl-2 and decreased expression of pro-apoptotic molecules - Bad, Bax and caspase3; whereas, IND and JUG treated groups showed decreased inflammatory expression with increased expression of pro-apoptotic molecules. SignificanceIND and JUG reduce the inflammatory activity and induce apoptotic cell death, while JUG effectively prevents IND induced gastric ulceration. These findings establish that a combination of IND + JUG may serve as a promising treatment regimen for colon cancer.

Quantitative Analysis of Intramucosal Mast Cells in Irritable Bowel Syndrome: A Comparison With Inflammatory Bowel Disease in Remission.

We aimed to study the density of intramucosal mast cells in histologically normal colonic mucosa biopsied from patients with a clinical diagnosis of irritable bowel syndrome (IBS). Mast cell activation has been thought to implicate in the pathogenesis of inflammatory bowel disease (IBD). Whether it serves a role in the pathogenesis of IBS remains controversial. A total of 127 colonoscopic mucosal biopsies were immunohistochemically stained, including 51 IBS, 66 IBD, and 10 normal control samples. Intact mast cells were quantified in 3 high power fields (HPF) in areas showing the highest density. CD117 was sensitive in detecting mast cells in colonic mucosa. The mast cell counts in all biopsies ranged from 2 to 60 per HPF (mean=17.5±7.2). The density of intramucosal mast cells were similar among IBS, IBD and normal control groups (P=0.6733). IBD in remission versus IBS (17.1±8.0 vs. 18.1±7.0; P=0.4804), Crohn disease versus ulcerative colitis (17.1±10.4 vs. 17.2±5.2; P=0.9463), IBS with diarrhea versus without diarrhea (19.5±6.3 vs. 16.8±6.9; P=0.1404). Forty biopsies (31.5%) showing ≥20 mast cells per HPF appeared to equally distribute among various disease groups (P=0.7283). There is no significant difference in the number of intramucosal mast cells between IBS and IBD that show normal colonic biopsies. In IBS patients, the number of intramucosal mast cell does not correlate with symptoms. The mast cell count (≥20/HPF) is not a reliable criterion for the diagnosis of IBS or for the distinction between patients with IBS and those with IBD in remission.

Different fixative solutions in the detection of mast cells in the canine and feline reproductive organs.

The aim of the study was to evaluate the usability of different fixative fluids in the detection of mast cells in ovaries and uteri of female dogs and cats. Samples were fixed in 4% formaldehyde, Carnoy's fluid, Mota's basic lead acetate and isotonic formaldehyde-acetic acid (IFAA). Mast cells (MCs) were detected by acidified toluidine blue staining and counted for various parts of the ovaries and uteri. In the ovaries of both species, the numbers of MCs were significantly (p < 0.05) higher in Carnoy than in formalin. No significant differences were found between Carnoy and Mota (tested only in cats). In the uterus, numbers of MCs were significantly (p < 0.05) higher in Carnoy, Mota and IFAA compared to formalin (canine endometrium, feline endometrium and feline myometrium), in Carnoy and Mota compared to formalin (canine myometrium) and in Mota compared to IFAA (feline myometrium). The majority of MCs were formalin-sensitive in the canine and feline uterus, in the canine ovary and in the feline cortex ovarii. In the feline medulla ovarii, the majority of MCs were formalin-resistant. No formalin-resistant MCs were detected in the feline tunica albuginea ovarii. Thus, using Mota's or Carnoy's fluid in the canine or feline female reproductive organs is recommended. This study improves methodology for all studies which clarify the role of MCs in the reproductive organs of the domestic and laboratory animals.

Detection of mast cells in ameloblastomas and odontogenic keratocysts.

BackgroundMCs (MCs) have been ascribed to mediating several diseases, including malignant neoplasms. These cells can play a role in angiogenesis, tissue remodeling and immune modulation and favor neoplasm progression. Despite the studies analyzing the contribution of MCs in odontogenic lesions, its biological behavior in ameloblastomas (AMBs) and odontogenic keratocysts (OKCs) remains unclear. This study aims to detect MCs in OKCs and AMBs and clarify the role of MCs in these lesions. Material and MethodsA total of 40 odontogenic lesions were analyzed. This included 20 OKCs and 20 AMBs, 10 being the solid type and the other 10 being the unicystic type of AMB. All cases were histologically reviewed in hematoxylin-eosin. Clinical data, such as age, gender, location, size, radiographic presentation and, histologic patterns were collected from the clinical charts. The Mann–Whitney U test (MWU) was used verify the hypothesis, through inferential statistics. The level of significance used in the statistical test was 0.5%. ResultsMCs were observed in 60% of OKCs, and 35% of AMBs. The ratio of MCs observed in OKCs was 0.37, 0.48 in solid AMBs and 0.01 in unicystic AMBs. There was no significant difference between number of MCs in AMBs and OKCs, however, a significant difference was observed between solid and unicystic AMBs (p ≤ 0.01). ConclusionsMCs may play an important role in the biological behavior of AMBs and OKCs. However, in this study it was not possible to confirm the contribution of MCs in the biological behavior of these lesions and more studies are needed to clarify this relation. Key words:AMB, OKC, MCs, histochemistry, toluidine blue.

Presence of langerhans cells, regulatory T cells (Treg) and mast cells in asymptomatic apical periodontitis.

Asymptomatic Apical Periodontitis is essentially an inflammatory disease of microbial aetiology. Association and function of the cell components involved, or specific inductive factors and growth mediators associated with development, maintenance and resolution of the periapical lesions are still unknown. The objective of this study was to evaluate the concentration of Regulatory T cells (FoxP3+; Treg), Langerhans cells (CD1a+; LC) and mast cells in asymptomatic apical periodontitis. 73 cases were selected: 30 periapical granulomas, 29 radicular cysts and 14 residual cysts. All groups were submitted to morphological analysis for classification of inflammatory infiltrate and thickness of the epithelial lining as well as to immunohistochemical analysis for detection of LC and Treg cells. Toluidine blue staining was used for detecting mast cells. Analysis showed higher mean numbers of LC (8.2 cells/0.2mm2), and Treg cells in radicular cysts (5.910 cells/0.2mm2). As for mast cells, it was found that radicular cysts had a higher mean number of these cells compared to other periapical lesions (12.68 cells/0.2mm2). The association between thickness of the epithelial lining and inflammatory cells showed that the presence of hypertrophic epithelium in radicular cysts presented higher density of LC. The number of LC and Treg cells play an important role in the control of the inflammatory micro-environment in periapical granulomas and radicular cysts, respectively. The presence of mast cells in radicular cysts may be associated with progression of the lesion. Knowledge regarding the inflammatory cell profile is therefore essential for a better understanding of the pathogenesis of asymptomatic periapical periodontitis.

Detection of Mast Cells and Basophils by Immunohistochemistry.

Staining cells or tissues with basic dyes was the mainstay of mast cell and basophil detection methods for more than a century following the first identification of these cell types using such methods. These techniques have now been largely supplanted by immunohistochemical procedures with monoclonal antibodies directed against unique constituents of these cell types. Immunohistochemistry with antibodies specific for the granule protease tryptase provides a more sensitive and discriminating means for detecting mast cells than using the classical histochemical procedures, and using antibodies specific for products of basophils (2D7 antigen and basogranulin) has allowed detection of basophils that infiltrate into tissues. The application of immunohistochemistry to detect more than one marker in the same cell has underpinned concepts of mast cell heterogeneity based on differential expression of chymase and other proteases. The double labeling procedures employed have also provided a means for investigating the expression of cytokines and a range of other products. Protocols are here set out that have been used for immunohistochemical detection of mast cells and basophils and their subpopulations in human tissues. Consideration is given to pitfalls to avoid and to a range of alternative approaches.

Comparison of histochemical staining techniques for detecting mast cells in oral lesions

ABSTRACTMast cells are large cells with granular cytoplasm that participate in wound healing, angiogenesis and defense against pathogens. They also contribute to inflammation by initiating innate and acquired immunity. The granules of these cells exhibit characteristic staining properties. We investigated toluidine blue, astra blue, Alcian blue-pyronin Y and May-Grunwald Giemsa stains for mast cells in various oral lesions and assessed the efficacy of each for identifying mast cells. Sections were obtained from 10 each of diagnosed cases of inflammatory fibrous hyperplasia, periapical cyst, mild dysplasia, oral submucous fibrosis and oral squamous cell carcinoma and stained using the stains listed above. Mast cells were assessed for their presence, contrast of the mast cell in the connective tissue background and number. We found that May-Grunwald Giemsa stain was the best for identification of mast cells, although toluidine blue staining is less time-consuming. Overall we obtained better results using May-Grunwald Giemsa and toluidine blue for staining mast cells.

Detection of Mast Cells Expressing c-Kit Using Antibody Covalently Bound to Gelatin Elongated from Surface of Immunosensor Based on Surface Plasmon Resonance.

An immunosensor based on surface plasmon resonance was applied to detect mast cells expressing c-Kit. Sufficient detection of the mast cells was achieved by covalent immobilization of gelatin firstly on the sensor surface and followed by covalent binding of the anti-c-Kit antibody to lysine residues in the gelatin molecules through bis(sulfosuccinimidyl)suberate (BS3) treatment. By using BS3, which is a homo-bifunctional reagent, the lysine residues of the anti-c-Kit antibody easily bound to the lysine residues of the gelatin in the physiological condition. The lower limit of detection was 104 cells/mL.