Eosinophils and mast cell homogeneity of the guinea pig eyelid skin, conjunctiva, and ileum

Mast cell heterogeneity has been described on the basis of differential staining reactions, light microscopic morphology, anatomic location, degranulation after polyamines, biochemical contents, growth requirements, and reactions to lymphokines. We have demonstrated typical "connective-tissue mast cells" by using anatomic criteria, histological staining reactions, electron microscopy, and reaction to compound 48/80 in the guinea pig conjunctiva, eyelid skin, and ileum. A second, much larger population of cells in the ileal mucosa and the conjunctiva, and rarely in the eyelid skin stained reddish-blue with acid toluidine blue in tissue fixed in ethanol-acetate-lead subacetate (BLA) and with alkaline Giemsa in formaldehyde-fixed tissue, did not stain with ethanolic or acid toluidine blue in formaldehyde-fixed tissue or with alkaline Giemsa in BLA-fixed tissue, and did not degranulate after 48/80 treatment. These are features of the rat intestinal "mucosal mast cells"; however, ultrastructural and light microscopic studies with the orcein Giemsa stain demonstrated these cells in the guinea pig to be eosinophils. Tissue culture, biochemical, and immunological studies indicate the existence of a second type of mast cell (bone-marrow-derived mast cell), ultrastructurally almost indistinguishable from the connective tissue mast cell. Our studies demonstrate only one mast cell type in the guinea pig and support the contention that other forms of mast cells are immature forms or variants of the connective-tissue mast cell.     

Staining Mast Cells for Morphometric Evaluation on an Image Analysis System

Multiple skin sections from three nonhuman primates (Macaca mulatta) and three hairless guinea pigs (Cavia porcellus) were stained with 12 different histologic stains to determine whether mast cells could be selectively stained for morphometric analysis using an image analysis system (IAS). Sections were first evaluated with routine light microscopy for mast cell granule staining and the intensity of background staining. Methylene blue-basic fuchsin and Unna's method for mast cells (polychrome methylene blue with differentiation in glycerin-ether) stained mast cell granules more intensely than background in both species. Toluidine blue-stained sections in the guinea pig yielded similar results. Staining of the nuclei of dermal connective tissue was enhanced with the methylene blue-basic fuchsin and toluidine blue stains. These two stains, along with the Unna's stain, were further evaluated on an IAS with and without various interference filters (400.5-700.5 nm wavelengths). In both the methylene blue-basic fuchsin and toluidine blue stained sections, mast cell granules and other cell nuclei were detected together by the IAS. The use of interference filters with these two stains did not distinguish mast cell granules from stained nuclei. Unna's stain was the best of the 12 stains evaluated because mast cell granule staining was strong and background staining was faint. This contrast was further enhanced by interference filters (500.5-539.5 nm) and allowed morphometric measurements of mast cells to be taken on the IAS without background interference.     

Mast cell "disappearance" in chronic murine graft-vs-host disease (GVHD)-ultrastructural demonstration of "phantom mast cells"

Mast cells were studied during the induction of chronic graft-vs-host disease (GVHD) induced in mice across minor histocompatibility barriers. B10.D2 spleen cells (or control BALB/c cells) were injected into irradiated (600 rad) BALB/c recipients. Serial skin biopsies were taken over 26 days, during which time changes occurred resembling scleroderma, namely, dermal fibrosis, a mononuclear cell infiltrate, and loss of fat and appendages. Mast cells, when stained with toluidine blue, "disappeared" from GVHD, but not from control skin. Ultrastructural analysis showed that mast cells in GVHD skin were indeed present but underwent degranulation. Some mast cells showed only pale expanded sacs, indicating granule depletion. Because these cells could not be seen by toluidine blue staining but were plainly present, we have called them "phantom mast cells." Cellular activation occurred in many GVHD mast cells as shown by increased cytoplasmic activity, with numerous Golgi complexes, ribosomes, granular endoplasmic reticulum, and small vesicles. No identifiable mast cells were seen after day 19. No significant changes were seen in the mast cells of syngeneic control mice. We believe that immunologic processes in chronic GVHD cause a slow release of mast cell granule contents, which is different from anaphylactic degranulation. The depleted mast cells (invisible by toluidine blue staining) are also activated, perhaps in an attempt to replete their stores of granule contents. We discuss the relation of mast cell changes to fibrosis.     

鸡中枢淋巴器官肥大细胞的组织化学与形态学

对哺乳动物的,特别是啮齿动物和人类肥大细胞已有了比较深入的研究, 但关于家禽肥大细胞的研究很少.本研究旨在阐明鸡中枢淋巴器官中肥大细胞的组织化学与形态学特征.本研究证实Carnoy 氏液是鸡肥大细胞的优良的固定液,而中性缓冲福尔马林(NBF) 却阻断了大多数肥大细胞的着染力.甲苯胺蓝和阿尔新蓝是鸡肥大细胞的良好的染料,但阿尔新蓝能使更多的肥大细胞着染,虽然其也可使杯状细胞着染.作者的一种新的染色法, 长时间阿尔新蓝染色(LAB-S)可用于NBF固定的组织中肥大细胞的染色,因为其着染的细胞数与Carnoy 氏液固定甲苯胺蓝染色的细胞数无显著差异(P<0.001).在胸腺髓质中见有大量的肥大细胞,而胸腺皮质仅可见个别肥大细胞位于血管周围及小叶间结缔组织中.腔上囊的皮质与髓质中很少见有肥大细胞.肥大细胞有血管周围分布的倾向,但一个有趣的发现是血管内偶尔也有个别肥大细胞.电镜下可见肥大细胞的胞浆颗粒内充满无定形的颗粒状基质,但其电子密度有的较高,有的较低.少数胞浆颗粒内有旋涡状及网状亚微结构.但未见有人类肥大细胞胞浆颗粒内特征性的晶格状和卷轴状的亚微结构,也未见到在绵羊肥大细胞中描述过的特殊亚微结构.     

Mast cells in human odontogenic cysts

Abstract

Mast cells are granule-containing cells in mucosal and connective tissues that are known to play a central role in allergic and inflammatory responses owing to pro-inflammatory mediators. Cysts in jaws are among the most common expansive, benign and destructive bone lesions; at some stage they are associated with chronic inflammation. Earlier studies have identified mast cells in odontogenic cysts (OC). We investigated the presence and distribution of mast cells and compared their number in different types of radicular cysts (RC), dentigerous cysts (DC) and odontogenic keratocysts (OKC). Ten cases each of RC, DC and OKC diagnosed clinically and histopathologically were selected and stained with 1% toluidine blue. The greatest number of mast cells/mm² was found in RC. The fewest mast cells/mm² were found in OKC. The subepithelial zones of all cysts contained more mast cells than the deeper zones.     

Staining properties of duodenal mast cells in 48/80-treated rats

Summary Mast cells in the tongue, mesentery and lamina propria of the duodenal mucosa in normal and 48/80-treated rats were observed at different time intervals. The tissues were studied comparatively after staining with toluidine blue, acridine orange or alcian bluesafranin. Under the experimental conditions used, the mast cells in the tongue and mesentery showed constant positive reactions to toluidine blue and acridine orange, both of which failed to demonstrate the presence of mast cells in the lamina propria of the duodenal mucosa. The combined alcian blue-safranin stain elicited a safranin-positive reaction in the mast cells of the tongue and mesentery and an alcian blue reaction in those of the lamina propria of the duodenal mucosa. This alcianophilia of the duodenal mast cells was not affected by compound 48/80. On the other hand, the safranin stain of the tongue and mesentery mast cells was altered to alcian blue by the drug. The results are discussed in the light of recent developments in mast cell research.This work was supported by grant MA-2236 of the Medical Research Council of Canada.     

Hepatic mast cells in rats infected with Taenia taeniaeformis

Mast cells appeared in the liver around metacestodes of Taenia taeniaeformis by 13 days after infection (DAI) of rats. The cells often occurred in clusters. The population increased until 28 DAI, then gradually declined. These hepatic mast cells (HMC) were compared to intestinal mucosal mast cells (MMC) and connective tissue mast cells (CTMC) histochemically, morphologically and in their response in vivo to Compound (built|48/80) and dexamethasone. Hepatic mast cells were similar to MMC in that they stained strongly blue with Astra blue at pH 1·0, could not be demonstrated with 0·005% toluidine blue, disappeared after treatment with dexamethasone, and were unaffected by 48/80. Immunoglobulin-containing cells in the liver were characterized by immunofluorescence. Immunoglobulin E-positive, and to a lesser extent IgG_2a-, and IgG_2c-positive cells surround the parasites in increasing numbers until 28 DAI, then declined. Many IgE-positive cells were HMC, and the IgE was frequently located intracytoplasmically. These cells were clearly distinguishable from eosinophils which stained characteristically with Giemsa and did not react with the anti-IgE probe. The results suggest that mast cell progenitors may be induced to localize and proliferate at the host-parasite interface in the Taenia-infected livers, giving rise to a cell population comparable to the MMC often seen at parasitized mucosal surfaces.     

Fixation and Staining of Planaria for Histological Study

Fixation and staining of planaria can affect the interpretation of histopathological changes following their exposure to various agents. We assessed several fixation protocols with various stains in planaria to determine an optimal combination. Planaria were fixed in each of the following: 10% neutral buffered formalin, 2.5%, glutaraldehyde, Bouin's, Zenker's, 70% ethanol, and relaxant. In addition, planaria were fixed in relaxant and postfixed in each of the fixatives above. Paraffin embedded sections from each fixation protocol were stained with hematoxylin and eosin (H & E), toluidine blue, periodic acid-Schiff (PAS), or phosphotungstic acld-hematoxylin (PTAH). Relaxant fixed planaria were also stained with Steiner's, Holmes, trichrome, Giemsa, Grocott's methenamine silver (GMS) and antibodies for intermediate filaments (cytokeratin, vimentin and desmin). Relaxant and Zenker's gave the best fixation with minimal artifacts. Formalin, glutaraldehyde, and ethanol were unacceptable because they caused contortions of the body, crenation, and a darkly pigmented epidermis. Gastroderm could be differentiated from stroma best when stained with H & E, toluidine blue and PTAH. Other organ systems differentially stained included the epidermis, marginal adhesion gland, nervous tissue, and muscle. PAS, Steiner's, Holmes, trichrome and the intermediate filament stains were not useful for planaria staining. The most morphological information was obtained with relaxant fixative and a combination of sections stained with H & E and PTAH.     

Concomitant Staining of Mast and Parietal Cells in Human Gastric Mucosa

A simple technique for concomitant staining of mast and parietal cells in the same section is described. Mast cells were stained by alcian blue or astra blue in methanol-formalinacetic acid fixed biopsies of gastric mucosa. Parietal cells were visualized by Dolichos biflorus lectin binding.     

Mast and enterochromaffin-like cells of the gastric mucosa]

An attempt has been made to reveal simultaneously both mast and ECL cells in the fundic mucosa of some laboratory animals and man. In Bouin fluid fixed specimens, toluidine blue pH 5.0 and alcian blue pH 1.0 failed to reveal mucosal mast cells in rats and mice only. In those animals mucosal mast cells became demonstrable in Carnoy fluid fixed tissues after staining with alcian blue pH 1.0. A double staining technique has been applied using Grimelius silver method followed by staining either with toluidine blue after acid hydrolysis or with alcian blue. Both mast and ECL cells became visible showing here and there their close arrangement. The latter might be a point for some functional relations between both cell types.     

Reduction in rat mesentery mast cell staining after degranulation by protamine. A competitive antagonism.

Degranulation of rat mesentery mast cells by increasing concentrations of protamine causes a parallel decrease in the numbers of mast cells stained with toluidine blue or with berberine sulfate. No decrease in mast cell numbers occurs when degranulation is inhibited. Since protamine does not enter into non stimulated mast cells, these results suggest that this reduction in mast cell numbers is caused by the binding of protamine to the anionic sites of heparin of exocytosed granules thereby preventing their staining. There seems to be a competitive antagonism between protamine and toluidine blue at the anionic sites of heparin for increasing concentrations of toluidine blue progressively reverse the reduction in mast cell numbers.     

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.     

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.     

Prevalence and Comparison of Diagnostic Methods for Trichomonas vaginalis Infection in Pregnant Women in Argentina

The objectives of this study were to conduct a prevalence survey of trichomoniasis in pregnant women and to evaluate the utility of different methods for its diagnosis. A total of 597 vaginal exudates from pregnant women who were examined at the Hospital de Clinicas in Buenos Aires, Argentina from 1 August 2005 to 31 January 2007, were prospectively and consecutively evaluated. The investigation of Trichomonas vaginalis was made by different microscopic examinations, and culture on liquid medium. The sensitivity and specificity of the microscopic examinations were assessed considering culture on liquid medium as the "gold standard". The prevalence of T. vaginalis obtained by culture on liquid medium was 4.0% (24/597). The prevalence of T. vaginalis obtained by direct wet smear, prolonged May-Grunwald Giemsa staining, and sodium acetate-formalin (SAF)/methylene blue staining-fixing technique was 1.8%, 2.3% and 2.5%, respectively. The sensitivity of the direct wet smear was 45.8%, that of the prolonged May-Grunwald Giemsa staining was 58.3%, and that of the SAF/methylene blue method was 62.5%. Considering the 3 microscopic examinations altogether, the sensitivity rose to 66.7% and the specificity was 100% for all of them. This is the first time that the prevalence data of T. vaginalis by culture in pregnant women are published in Argentina. Due to the low sensitivity obtained by microscopy in asymptomatic pregnant women, the use of the liquid medium is recommended during pregnancy, in order to provide an early diagnosis and treatment.     

牛蛙肥大细胞的组织化学与形态学

目的鉴定牛蛙组织中肥大细胞的存在。方法用于肥大细胞研究的一些常规组织化学技术与形态学方法。结果牛蛙的舌、肠、肠系膜和脾中肥大细胞数量较多,少量也见于神经、心、肾、肝和皮肤等多种组织中。肥大细胞有沿血管周和神经分布的倾向。脾脏中的肥大细胞形状比较一致,呈圆形或卵圆形,而在其它部位的肥大细胞则形态多样。Bouin氏液及Carnoy氏液是牛蛙肥大细胞优良的固定液。然而,与哺乳动物的黏膜肥大细胞相似的是,中性缓冲福尔马林(NBF)固定显著的阻断了牛蛙肠黏膜肥大细胞(MMC)的染色。有趣的是,甲苯胺蓝是牛蛙肥大细胞的最佳染料,它比阿尔新蓝能很好地显示牛蛙的肥大细胞。透射电镜下证实,牛蛙肥大细胞中含有大量特征性的胞浆颗粒。肥大细胞靠近雪旺氏细胞,并可见于神经束膜间,甚至以其突起与神经束膜相连。结论通过组织化学与形态学研究证实了牛蛙组织中肥大细胞的存在,再次证实肥大细胞与外周神经之间存在密切的解剖学关系。     

Selective labeling of lipid droplets in aldehyde fixed cell monolayers by lipophilic fluorochromes

Abstract

We evaluated a number of lipophilic dyes and fluorochromes, including oxazone and thiazone derivatives of oxazine and thiazine dyes, scintillator agents, a carotenoid and a metal-porphyrin complex for visualization of lipid droplets within aldehyde fixed cultured HeLa and BGC-1 cells. Observation under ultraviolet, blue or green exciting light revealed selective fluorescence of lipid droplets, particularly after treatment with aqueous solutions of Nile blue and brilliant cresyl blue oxazones, toluidine blue thiazone, or propylene glycol solutions of canthaxanthin, ethyl-BAO, and ZnTPyP. Mounting in water was required to maintain the fluorescence of lipids; the use of glycerol, Mowiol or Vectashield was not adequate. The effect of dye structure on staining intensity was assessed with the aid of numerical structure parameters modeling lipophilicity (HI and log P), overall size (MW) and the size of the conjugated system (conjugated bond number; CBN). The best stains for lipid droplets were relatively lipophilic (HI > 4.0, log P > 5.0), of small size overall (MW < 370), with small conjugated systems (CBN < 24), and not significantly amphiphilic. The two hydrophobic-hydrophilic parameters (the classic log P and the hydrophobic index, HI; values calculated by molecular modeling software) were highly correlated; however, HI was a more suitable hydrophobicity index for the dyes studied here.     

Improved demonstration of mast cells using alcian blue tetrakis (methylpyridium) chloride

Many batches of alcian blue dye are incompletely soluble at the low pH used for demonstrating mast cells. An improved technique using alcian blue tetrakis (methylpyridium) chloride (alcian blue pyridine variant) is described here. It produces stronger mast cell staining than other alcian blue stains tested.     

Improved demonstration of mast cells using alcian blue tetrakis (methylpyridium) chloride

Many batches of alcian blue dye are incompletely soluble at the low pH used for demonstrating mast cells. An improved technique using alcian blue tetrakis (methylpyridium) chloride (alcian blue pyridine variant) is described here. It produces stronger mast cell staining than other alcian blue stains tested.     

肥大细胞在大鼠输卵管急性沙眼衣原体感染中的作用

目的　为研究肥大细胞在大鼠输卵管急性沙眼衣原体 (Chlamydialtrachomatis,CT)感染中的作用。方法　选择成年雌性SD大鼠 6 0只 ,通过手术从一侧卵巢囊接种沙眼衣原体D型株 ,对照组接种 2 -SPA缓冲液。分别于感染后第 1/ 2d、第 7d、第 14d将大鼠处死 ,取手术侧的输卵管常规固定、脱水、包埋。结果　S -P法显示 :输卵管局部的CD4 + T细胞和血管内皮细胞粘附分子 (VCAM - 1)的表达均较对照组明显增强 (P <0 0 1)。改良的甲苯胺蓝染色法显示 :感染组肥大细胞较对照组数量有显著性增高 (P <0 0 5 ) ,并且其变化趋势与CD4 + T细胞和VCAM - 1表达的变化趋势一致。结论　可以推测 ,沙眼衣原体感染引起急性输卵管炎时 ,肥大细胞通过促进炎症局部小静脉内皮细胞上VCAM -1的表达 ,诱导CD4 + T细胞的浸润 ;然后分泌IL - 4等细胞因子促进CD4 + T细胞向TH2 细胞方向转化 ,不利于机体清除沙眼衣原体 ,从而使发生局部输卵管病理损伤的可能性增加。     

奥尼罗非鱼胃肠道中肥大细胞的分布特征

本研究采用改良甲苯胺蓝染色法探讨了奥尼罗非鱼(Oreochromis niloticus ♀×O. aureus♂)胃肠道肥大细胞的分布及其形态特点。结果发现,经甲苯胺蓝染色的肥大细胞其核着深蓝色,颗粒被染成紫红色,着色深浅不一。肥大细胞大小不一,形态各异,呈圆形、椭圆形或梭形、菱形,散在或集中分布在黏膜层固有膜和黏膜下层,尤其常见分布于小血管周围。经统计,肥大细胞在奥尼罗非鱼的胃、幽门盲囊、后肠、前肠、中肠的数量依次减少。胃和幽门盲囊内肥大细胞数量显著高于前肠和中肠(P0.05),与后肠无显著差异;前肠、中肠和后肠内肥大细胞数量并无明显差别。