Localization of nitric oxide synthase immunoreactivity in mast cells of human nasal mucosa

Nitric oxide (NO)-synthase immunoreactivity has been detected for the first time in mast cells of human normal nasal mucosa, with an antibody specific for neuronal NO-synthase. Intense immunoreactivity was revealed in secretion granules of mast cells but was found in mast cell granules free in the extracellular matrix only in some instances; no reactivity was found in the cytoplasm of this or other cell types. These findings suggest that human nasal mast cells contain a particulate isoform of NO-synthase, which shares epitopes with neuronal NO-synthase and is rapidly removed from granules upon exocytosis.     

Phenotypic expression of human hepatoma cells in culture

Hepatomas thrive in a hypoxic environment resulting in the induction of a cluster of hypoxia related genes. The protein phenotypic expression include hypoxia inducible factor-alpha, prolyl-4-hydroxylase, vascular endothelear growth factor and erythropoietin. The present study was undertaken to determine if human hepatoma cells when cultured for 72 h in the presence of serum under normoxia would maintain their cancerous phenotypic expression of certain hypoxia inducible genes. Our positive results affords an in vitro model system to test hypoxia inhibitors on the expression and the intracellular compartmentalization or the secretion of these hypoxia-inducible proteins.     

Olfactory neuron-specific expression of NeuroD in mouse and human nasal mucosa

Human olfactory neuroepithelium (OE) is situated within the olfactory cleft of the nasal cavity and has the characteristic property of continually regenerating neurons during the lifetime of the individual. This regenerative ability of OE provides a unique model for neuronal differentiation, but little is known about the structure and biology of human olfactory mucosa. Thus, to better understand neurogenesis in human OE, we studied the expression of olfactory marker protein (OMP), TrkB and NeuroD in human nasal biopsies and autopsy specimens and compared these data with those obtained from normal and regenerating mouse OE. We show that NeuroD and TrkB are coordinately expressed in human OE. Thus, by using these markers we have been able to extend the known boundaries of the human OE to include the inferior middle turbinate. In normal mouse OE, TrkB and OMP expression overlap in cells closest to the superficial layer, but TrkB is expressed more strongly in the lower region of this layer. In contrast, NeuroD expression is more basally restricted in a region just above the globose basal cells. These characteristic expression patterns of OMP, TrkB and NeuroD were also observed in the regenerating mouse OE induced by axotomy. These results support a role of NeuroD and brain-derived neurotrophic actor (BDNF), the preferred ligand for TrkB, in the maintenance of the olfactory neuroepithelium in humans and mice.     

人鼻粘膜微血管的构筑

鼻腔粘膜对净化空气,调节空气温度和湿度具有重要作用。本文采用甲基丙烯酸甲酯制作血管铸型,通过扫描电镜对人鼻粘膜微血管三维构筑进行观察。     

Serglycin proteoglycan promotes apoptotic versus necrotic cell death in mast cells

The mechanisms that govern whether a cell dies by apoptosis or necrosis are not fully understood. Here we show that serglycin, a secretory granule proteoglycan of hematopoietic cells, can have a major impact on this decision. Wild type and serglycin(-/-) mast cells were equally sensitive to a range of cell death-inducing regimens. However, whereas wild type mast cells underwent apoptotic cell death, serglycin(-/-) cells died predominantly by necrosis. Investigations of the underlying mechanism revealed that cell death was accompanied by leakage of secretory granule compounds into the cytosol and that the necrotic phenotype of serglycin(-/-) mast cells was linked to defective degradation of poly(ADP-ribose) polymerase-1. Cells lacking mouse mast cell protease 6, a major serglycin-associated protease, exhibited similar defects in apoptosis as observed in serglycin(-/-) cells, indicating that the pro-apoptotic function of serglycin is due to downstream effects of proteases that are complex-bound to serglycin. Together, these findings implicate serglycin in promoting apoptotic versus necrotic cell death.     

Neuroendocrine cells in the nasal mucosa - preliminary report

The role of neuroendocrine cells (NC) in physiology and pathology of the human's respiratory tract is not fully understood. The aim of the study was the quantitative and morphometric assessment of NC in nasal mucosa in some pathological states. PATIENTS AND METHOD: 40 patients, aged 28-63 years, with clinical signs of chronic, hypertrophic rhinosinusitis were qualified for the study. Rhinitis chronica hypertrophica coexisted with aspirin triad or asthma in 10 patients (group I), with advanced obstructive sleep apnea syndrome (OSAS) in 10 patients (respiratory disturbance index, RDI>40, group II). Group III consisted of 10 patients with simple rhinitis chronica hypertrophica who habitually smoked cigarettes (at least 20 cigarettes a day) while 10 non-smoking patients with simple rhinitis chronica hypertrophica were qualified to the control group. Fragments of nasal mucosa of approximately 0.5 cm(2) were collected from medial or inferior turbinate during mucoplasty procedures. NC were detected immunohistochemically using antibodies against chromogranin A (DAKO). The microscopic sections were evaluated in the light microscopy. RESULTS: The study did not reveal the increased number of NC in examined fragments of nasal mucosa. Scattered NC were detected in single preparations of nasal mucous membrane in some patients in all groups. The number of detected neuroendocrine cells did not differ statistically between groups.     

Frequency of mast cells in the olfactory mucosa of rhesus monkeys

During studies of the olfactory mucosa and its response to the different levels of circulating sex hormones, considerable numbers of mast cells have been observed in its epithelia and subepithelial regions. The number of these cells in the olfactory mucosa of male monkeys differs greatly from that found in females. The frequency of these cells in the olfactory mucosa of females fluctuates significantly during the menstrual cycle. These fluctations stimultaneously correspond to the well known changes in olfactory sensitivity: around ovulation, when the olfactory sensitivity for certain odorants is high, the number of mast cells in the olfactory mucosa also increases.     

Gelatin sponge-supported histoculture of human nasal mucosa

Summary Considerable progress has recently been made in the understanding of airway inflammation by cell culture assays and in vivo provocation studies. Inasmuch as ethical considerations limit experimental work in humans, physiologically relevant in vitro models are required to better understand cellular and molecular tissue interactions in human nasal mucosa. Here we describe a human nasal mucosa culture model utilizing a simple gelatin sponge-supported histoculture system at the air-liquid interface. Viable mucosa was preserved for at least 48 h, as shown by morphology and immunohistochemical staining with Ki-67 as marker for proliferation. Pro-inflammatory mediators (kinins, histamine, thromboxane B₂, prostaglandin F_2α, and substance P) are detectable in serum-containing as well as serum-free culture medium. Incubation with 10⁻⁸ M substance P increases the number of degranulated mast cells after 48 h by 26% (P<0.01). In this model, biochemical responses can be correlated with histologic alterations of the target tissue. Inflammatory parameters can be examined and compared in various patient groups and different stimulators/inhibitors. This culture method provides a valuable research tool for analyzing all compartments present in nasal mucosa under physiologically relevant conditions, and for studying complex interactions and responses of mucosal cell populations in their natural tissue environment.     

Serglycin proteoglycan is required for secretory granule integrity in mucosal mast cells

SG (serglycin) PGs (proteoglycans) are strongly implicated in the assembly of MC (mast cell) granules. However, this notion has mainly been on the basis of studies of MCs of the connective tissue subtype, whereas the role of SG PG in mucosal MCs has not been explored. In the present study, we have addressed the latter issue by using mice with an inactivated SG gene. Bone marrow cells were differentiated in vitro into the mucosal MC phenotype, expressing the markers mMCP (mouse MC protease) -1 and -2. Biosynthetic labelling experiments performed on these cells revealed an approximately 80% reduction of 35SO4(2-) incorporation into PGs recovered from SG-/- cells as compared with SG+/+ counterparts, indicating that SG is the dominating cell-associated PG of mucosal MCs. Moreover, the absence of SG led to defective metachromatic staining of mucosal MCs, both in vivo and in the in vitro-derived mucosal MCs. Ultrastructural analysis showed that granules were present in similar numbers in SG+/+ and SG-/- cells, but that their morphology was markedly affected by the absence of SG, e.g. with electron-dense core formation only seen in SG+/+ granules. Analysis of the MC-specific proteases showed that mMCP-1 and mMCP-7 were completely independent of SG for storage, whereas mMCP-2 showed a partial dependence. In contrast, mMCP-4 and -6, and carboxypeptidase A were strongly dependent on SG for storage. Together, our data indicate that SG PG is of crucial importance for assembly of mature mucosal MC granules, but that the specific dependence on SG for storage varies between individual granule constituents.     

Thymosin beta 4 expression in normal skin,colon mucosa and in tumor infiltrating mast cells

Mast cells (MCs) are metachromatic cells that originate from multipotential hemopoietic stem cells in the bone marrow. Two distinct populations of MCs have been characterized: mucosal MCs are tryptase-positive while mast cells in skin contain tryptase and chymase. We now show that a sub-population of MCs is highly immunoreactive for thymosin β₄, as revealed by immunohistochemical analyses of normal skin, normal colon mucosa and salivary gland tumors. Four consecutive serial sections from each case were immunostained for thymosin β₄ (Tβ₄), chymase, tryptase and stained for toluidine blue. In skin biopsies, MCs showed a comparable immunoreactivity for Tβ₄, chymase and tryptase. In normal colon mucosa the vast majority of mucosal MCs expressed a strong cytoplasmic immunoreactivity for tryptase and for Tβ₄, in the absence of chymase reactivity. A robust expression of Tβ₄ was detected in tumor-infiltrating and peritumoral mast cells in salivary gland tumors and breast ductal infiltrating carcinomas. Tumorinfiltrating MCs also showed a strong immunoreactivity for chymase and tryptase. In this paper, we first demonstrate that normal dermal and mucosal mast cells exhibit strong expression of thymosin β₄, which could be considered a new marker for the identification of mast cells in skin biopsies as well as in human tumors. The possible relationship between the degree of Tβ₄ expression in tumor-infiltrating mast cells and tumor behaviour warrants further consideration in future investigations.Key words: mast cells, thymosin β₄, tryptase, chymase.     

Distributional characteristics of lymphatic vessels in normal human nasal mucosa and sinus mucosa

An immunohistochemical staining technique with the D2-40 antibody was undertaken to examine the functional and morphological features of lymphatic networks in tissue sections and whole-mount preparations of normal nasal mucosa and ethmoid sinus mucosa. In normal nasal mucosa, most lymphatic vessels were found in the superficial mucosa beneath the epithelial layer. Some of these vessels were dilated, whereas others were compressed and had a slit-like lumen. Whole-mount preparations revealed the extent of lymphatic vessels in normal ethmoid sinus mucosa. A network of lymphatic vessels was mainly found in the subepithelial layer, where lymphatic vessels represented rich networks, possessing antler-like branches and typical blind ends. However, these lymphatic networks were not arranged in the form of lymphangion chains, with each lymphangion consisting of a contractile compartment and valve. Thus, recognition of the distinctive features of the lymphatic network in normal nasal and sinus mucosa might aid investigations of lymphatic involvement in sinonasal diseases, such as rhinitis, sinusitis, and malignancy. This work was supported by a Grant-in-Aid for Scientific Research from the Communication Disorders Center, Korea University, Korea.     

CFTR expression analysis in human nasal epithelial cells by flow cytometry

Rationale

Unbiased approaches that study aberrant protein expression in primary airway epithelial cells at single cell level may profoundly improve diagnosis and understanding of airway diseases. We here present a flow cytometric procedure to study CFTR expression in human primary nasal epithelial cells from patients with Cystic Fibrosis (CF). Our novel approach may be important in monitoring of therapeutic responses, and better understanding of CF disease at the molecular level.

Objectives

Validation of a panel of CFTR-directed monoclonal antibodies for flow cytometry and CFTR expression analysis in nasal epithelial cells from healthy controls and CF patients.

Methods

We analyzed CFTR expression in primary nasal epithelial cells at single cell level using flow cytometry. Nasal cells were stained for pan-Cytokeratin, E cadherin, and CD45 (to discriminate epithelial cells and leukocytes) in combination with intracellular staining of CFTR. Healthy individuals and CF patients were compared.

Measurements and Main Results

We observed various cellular populations present in nasal brushings that expressed CFTR protein at different levels. Our data indicated that CF patients homozygous for F508del express varying levels of CFTR protein in nasal epithelial cells, although at a lower level than healthy controls.

Conclusion

CFTR protein is expressed in CF patients harboring F508del mutations but at lower levels than in healthy controls. Multicolor flow cytometry of nasal cells is a relatively simple procedure to analyze the composition of cellular subpopulations and protein expression at single cell level.     

Synaptotagmin I expression in mast cells of normal human tissues, systemic mast cell disease, and a human mast cell leukemia cell line.

Synaptotagmin I (STG I) is a Ca(2+) sensor and one of the synaptic vesicle proteins that mediate exocytosis. To determine the mechanism of release of large granules from mast cells, we studied by immunohistochemistry the presence of STG I in mast cells in normal human tissues simultaneously with the mast cell markers mast cell tryptase (tryptase) and c-kit. The tumor cells of systemic mast cell disease (SMCD) and a human mast cell leukemia cell line (HMC-1) were also examined. Human mast cells in normal tissues and the tumor cells of SMCD expressed STG I as well as mast cell tryptase (tryptase) and c-kit. STG I mRNA and its products in HMC-1 were examined by RT-PCR analysis and immunocytochemistry, respectively. STG I expression in HMC-1 cells was compared with that in cells stimulated and non-stimulated by phorbol 12-myristate 13-acetate and also with that in NB-1 and PC12 cells, known to express STG I. STG I mRNA was detected in both non-stimulated and stimulated HMC-1 cells and in NB-1 and PC12 cells. STG I immunoreactivity was weaker than NB-1 or PC12 immunoreactivity. However, it increased in the stimulated HMC-1 cells. Mast cells expressed STG I in various states. STG I may mediate exocytosis of large granules in mast cells.