Ocular defense mechanisms with special reference to the demonstration and functional morphology of the conjunctiva-associated lymphoid tissue in Japanese monkeys

In order to define the histological components of ocular defense, the conjunctiva in Japanese monkeys was studied using a whole mount method, light microscopy, and electron microscopy. We investigated the distribution of the conjunctiva-associated lymphoid tissue (CALT) using stereoscopic observations of the conjunctiva immunostained with human leukocyte antigen (HLA)-DR antibody and /or stained with alcian-blue. The outer surface of the conjunctival fornix was lined by sheets of mucus secreting goblet cells, with small epithelial patches without goblet cells, scattered among them. The patches, termed CALT, consisted of flattened epithelial cells, intraepithelial lymphocytes and dendritic cells, and lymphoid follicles with a germinal center. The CALT in Japanese monkeys was fundamentally similar in structure to those found in other animal species. CALT patches ranged in size ranging from 200 microm to 300 microm in diameter. The number of patches varied from 20 to 40 in the superior eyelid and 10 to 20 in the inferior eyelid. Latex microspheres administrated as eye drops were selectively taken up first by flattened associated epithelial cells covering the surfaces of CALT patches and then by intraepithelial dendritic cells of the CALT. These morphological findings show that CALT patches in the eyelids of primates are focal sites for particulate uptake and contact with lymphoid constituents, indicating that they are inductive sites for the common mucosal immune system as well as important components in ocular defense.     

Biological aspects of the tongue morphology of wild-captive WWCPS rats: a histological,histochemical and ultrastructural study

The aim of this study was to characterise the tongue in wild-type rats using several microscopic techniques. Warsaw Wild Captive Pisula Stryjek (WWCPS) rats belong to a lineage of wild-caught rats. The study was carried out on tongues of 15 male and 15 female WWCPS rats. Histological, histochemical and ultrastructural studies were carried out. There were no significant differences between the male and female WWCPS rat tongues. There was a median groove approximately 1 cm long in the apex of the tongue that faded caudally. The intermolar prominence was clearly marked in the distal part of the lingual body. Lingual mechanical papillae located on the surface of the tongue formed four subtypes based on their shape: small filiform papillae, giant filiform papillae, thin elongated filiform papillae and wide filiform papillae. Gustatory papillae formed the second group of papillae and were divided into bud-shaped fungiform papillae, a single vallate papilla surrounded by an incomplete papillary groove and foliate papillae, which were a well-formed and composed of several pairs of folds divided by longitudinal grooves. In the posterior lingual glands (mucoserous and serous), acidic sulphated mucin-secreting cells gave a strong AB pH 2.5 positive reaction, and a positive reaction with the AB pH 1.0 stain for acidic carboxylated mucin. Double AB/PAS staining showed the presence of the majority of mucous cells with predominant of acidic mucins. Positive PAS staining showed the presence of neutral mucin. HDI staining demonstrated a weak positive reaction within Weber’s glands of the WWCPS rat tongue.     

Prenatal development of the human Brunner's glands

The prenatal development of the human Brunner's glands has been investigated in 23 fetuses from the 10th week of gestation to full-term. At 12 weeks, a few cords of epithelial cells were seen budding from the duodenal mucosa immediately beyond the pyloric sphincter. They represent the initial stage of the development of Brunner's glands. At 16 weeks, Brunner's glands originated as simple tubular downgrowths from the bottoms of the most proximal crypts of the duodenum. The secretory products of the component cells of these primitive tubules contained periodic acid schiff (PAS) positive material which was largely supranuclear in position and resisted digestion by diastase. From 20 weeks to full term, the Brunner's glands developed in a progressive fashion starting in the proximal part of the duodenum near the pyloroduodenal junction. Further tubular downgrowths were added distally, leading to an increase in length of the glandular tissue. The gland showed an increase in size proximally due to elongation and branching of the tubules. At birth, the glandular cells of Brunner's glands resembled those of normal adult in structure and staining reactions. The PAS staining of the cells of the early developed glands (at 12 weeks) was as intense as those of the full-term. The secretory materials of the developed Brunner's glands showed negative reaction with Alcian blue (AB) at pH 2.5 at any stage of development. These results suggest that the mucin secreted by the developed Brunner's glands of human is neutral mucopolysaccharide in nature.     

用AB/PAs、HID、CEA法提高胃粘液细胞癌淋巴结转移检出率的研究

用AB(pH2.5)/PAS、HID及CEA(ABC法)对48例胃粘液细胞癌及其淋巴结进行染色观察,对照HE染色,结果表明应用上述方法染色可提高淋巴结转移检出率33.1%,其中AB(pH2.5)/PAS法显示了很强的特异性和敏感性,较HID和CEA法优越。淋巴结微小转移灶多为PAS阳性细胞,说明幼稚的癌细胞分泌中性粘液、侵袭力强、容易转移并先发生转移。CEA标记结果显示本肿瘤有分化越差、越幼稚其CEA含量越少的现象。     

Do the cardiac glands exist? 7. The cow

The glands distributed in the narrow region of the abomasum contiguous to the omasum of the cow have been described as cardiac glands. We doubted this assertion and therefore performed histological and histochemical investigations of the glands to clarify their characteristics. 1. All glandular cells except the parietal cells in a few glands contiguous to the omasum react strongly to PAS, AB(pH 2.5), and PAS-AB(pH 2.5) staining, and moderately to AB(pH 0.5) staining. 2. Glandular cells at the base of these glands contain fine pepsinogen granules and a few parietal cells are distributed in these glands, indicating that they are undifferentiated gastric glands and that the so-called cardiac glands do not exist in the cow stomach. 3. Glandular cells in undifferentiated gastric glands are filled with PAS, AB(pH 2.5 and 0.5) and PAS-AB(pH 2.5) positive substances. Which gradually decrease and finally disappear with differentiation, remaining only in the neck (mucous neck cells) and the cells in the upper part of the glandular body (immature chief cells), in mature gastric glands. 4. Mature chief cells in differentiated gastric glands are distributed in the middle and lower bodies and base of the glands and contain a number of PAS and PAS-AB(pH 2.5) positive granules and a large number of coarse pepsinogen granules, while pepsinogen granules in the mucous neck cells and immature chief cells are finer. 5. In the cow the region in which undifferentiated gastric glands are located is very narrow. 6. Parietal cells in the cow stomach are numerous.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anatomical and histological structure of the tongue and histochemical characteristics of the lingual salivary glands in the Persian squirrel (<Emphasis Type="Italic">Sciurus anomalus</Emphasis>)

This study was carried out to describe the anatomical, histological and mucinous histochemical characteristics of the tongue in the Persian squirrel. This species is a rodent distributed all over the Middle East and recently has been considered a companion animal. Anatomical observations showed the median sulcus on the apex and absence of a lingual prominence in the body. Light and scanning electron microscopy showed that the filiform papillae cover the entire dorsal surface of the tongue, and their sizes increased approaching the root. The fungiform papillae, which contained 1–4 taste buds, were scattered on the apex, margin, body and root of the tongue. Three vallate papillae were observed on the root, each one surrounded by a groove and crescent pad with taste buds on its lateral walls. The foliate papillae on both margins of the tongue contained several laminae with taste buds. The core of the tongue was composed of lingual glands, skeletal muscles and connective tissues. These glands were confined to the body and root, which were composed of serous cells located anteriorly and mucosal and seromucosal cells placed posteriorly. The mucin histochemistry using the periodic acid-Schiff (PAS), alcian blue (AB) (pH 1.0 and 2.5), PAS–AB (pH 2.5) and aldehyde fuchsin-AB (pH 2.5) techniques showed that the mucosal content included both carboxylated and sulfated acidic mucins with neutral mucins. The results of this study could contribute to the knowledge of the morphological characteristics of the wild animal tongue and provide data for comparison with other rodents.     

奶牛乳腺肥大细胞的粘膜免疫学特征

应用改良甲苯胺蓝染色法 (MTB )和阿尔辛蓝 番红鉴别染色法 (AB S )对静止期和活动期奶牛乳腺肥大细胞的粘膜免疫学性质进行了研究。MTB染色结果显示 ,静止期乳腺中的肥大细胞主要分布于各级输乳管上皮基膜区 ,小血管周围也有肥大细胞分布。在活动期 ,肥大细胞主要分布于腺泡上皮基膜附近 ,上皮内也有极少数肥大细胞分布 ,小叶间结缔组织中的肥大细胞则分布于小叶间导管或输乳管上皮基膜区。AB S染色证实 ,乳腺肥大细胞只被阿尔辛蓝染成蓝色 ,而不被结缔组织型肥大细胞的标志性染料番红着染。这表明 ,乳腺肥大细胞是一种典型的粘膜型肥大细胞 (MMC ) ,并参与构成乳腺粘膜免疫的第一道防线。     

Munc13-2-/- baseline secretion defect reveals source of oligomeric mucins in mouse airways

Since the airways of control mouse lungs contain few alcian blue/periodic acid–Schiff's (AB/PAS)+ staining 'goblet' cells in the absence of an inflammatory stimulus such as allergen sensitization, it was surprising to find that the lungs of mice deficient for the exocytic priming protein Munc13-2 stain prominently with AB/PAS under control conditions. Purinergic agonists (ATP/UTP) stimulated release of accumulated mucins in the Munc13-2-deficient airways, suggesting that the other airway isoform, Munc13-4, supports agonist-regulated secretion. Notably, however, not all of the mucins in Munc13-2-deficient airways were secreted, suggesting a strict Munc13-2 priming requirement for a population of secretory granules. AB/PAS+ staining of Munc13-2-deficient airways was not caused by an inflammatory, metaplastic-like response: bronchial–alveolar lavage leucocyte numbers, Muc5ac and Muc5b mRNA levels, and Clara cell ultrastructure (except for increased secretory granule numbers) were all normal. A Muc5b-specific antibody indicated the presence of this mucin in Clara cells of wildtype (WT) control mice, and increased amounts in Munc13-2-deficient mice. Munc13-2 therefore appears to prime a regulated, baseline secretory pathway, such that Clara cell Muc5b, normally secreted soon after synthesis, accumulates in the gene-deficient animals, making them stain AB/PAS+. The defective priming phenotype is widespread, as goblet cells of several mucosal tissues appear engorged and Clara cells accumulated Clara cell secretory protein (CCSP) in Munc13-2-deficient mice. Additionally, because in the human airways, MUC5AC localizes to the surface epithelium and MUC5B to submucosal glands, the finding that Muc5b is secreted by Clara cells under control conditions may indicate that it is also secreted tonically from human bronchiolar Clara cells.     

子宫颈微偏腺癌6例临床病理分析

目的探讨子宫颈微偏腺癌的形态学、组织化学及免疫表型特征。方法对6例子宫颈微偏腺癌组织学特征进行观察,并行黏液组化及免疫组化染色（S-P法）。结果6例均有子宫颈腺体显著增生,腺体腔缘面呈花边状、锯齿状或乳头状突入到腺管腔内,并有成角状外翻,腺体呈浸润性生长。黏液组织化学：AB（pH1．0、2．5）／PAS染色证实,腺体腔内为混合性黏液,主要含唾液酸黏液,硫酸黏液减少,中性黏液较多。免疫表型：CEA（5／6）阳性,CA125（6／6）阴性。vimentin、SMA浸润性腺体周围纤维母细胞／肌纤维母细胞（6／6）阳性。结论子宫颈微偏腺癌以其特殊的形态结构和细胞轻微的异型、AB／PAS阳性、CEA阳性及腺体周围反应性纤维母细胞／肌纤维母细胞增生为特征。     

人泪腺上皮细胞的体外培养及鉴定

目的　探讨人泪腺上皮细胞原代体外培养、鉴定、冻存和复苏的方法。方法　应用组织块培养法和混合消化液培养法对健康猝死成年人泪腺上皮细胞进行体外原代培养 ,应用免疫组织化学染色方法对培养有第 2代上皮细胞进行Pan keratin蛋白染色 ,以进行鉴定 ,取第 2、4代细胞进行冻存 ,1个月后取出复苏。结果　组织块培养法和混合消化液培养法均可获得较纯的泪腺上皮细胞 ,以组织块培养法接种的细胞早期生长较快 ,但第一代以后两种方法所获得的细胞生长无明显差别。两种方法所获取的泪腺上皮均为贴壁生长 ,未能形成生理状态下泪腺的囊腔结构 ,亦未见分泌小泡的形成。培养的泪腺上皮免疫组化染色Pan keratin蛋白染色阳性。经冻存和复苏 ,细胞保持良好活性。结论　组织块培养法和混合消化液培养法均可获得较纯的泪腺上皮细胞 ,但不能保持其泪腺束腔结构     

Histological and mucin histochemical study of the small intestine of the Persian squirrel (Sciurus anomalus)

This article describes the histological and mucin histochemical properties of the small intestine of the Persian squirrel (Sciurus anomalus). This species is widely distributed in the Middle East and can be found as a companion animal. The histological studies revealed that the plicae circulares were not visible in the tunica mucosa. The maximum height and width of the villi were observed in the duodenum, which then decreased toward the ileum. The muscularis mucosa was scattered, whereas the tunica submucosa was composed of dense connective tissue. The lymphatic nodules were seen in the submucosa of the distal part of the jejunum and ileum, and Brunner’s glands were embedded in the initial portion of the duodenum. The tunica muscularis was significantly thicker in the ileum, and the circular muscle layer was thicker than the longitudinal muscle layer throughout the entire length of the small intestine. The mucin histochemistry, which was examined using the periodic acid-Schiff (PAS) and alcian blue (AB) (pH 1.0 and 2.5) and also PAS–AB (pH 2.5) and aldehyde fuchsin-AB (pH 2.5) techniques coupled with methylation and saponification reaction for some sections, showed that the small intestine mucous content included both carboxylated and sulfated acidic mucins with few neutral mucins. The results of this study contribute to the knowledge of the histological and histochemical characteristics of the gastrointestinal tracts of exotic mammals and provide data for comparison with other mammals.     

Cytodifferentiation of secretory cells in the sublingual gland of the prenatal rat: A histological,histochemical and ultrastructura study

Sublingual and submandibular glands were prepared for light and electron microscopy, and for histochemical staining with periodic acid-Schiff (PAS), Alcian Blue (AB) or both (AB-PAS). Between 15 and 17 days post-conception, the sublingual gland undergoes active morphogenesis from a single, solid bud into a branched glandular tree. At 18 days the first overt signs of secretory differentiation appear in the formation of cells with three kinds of secretion granules; that is, electron-dense serous granules, empty-looking mucous granules with fine thread-like substructures, and granules which have the general appearance of mucous granules but also contain an internal, electron-dense core (“mixed” granules). During the period from 18 to 20 days, all three types of granulated cells increase in number, with mucous cells predominating, and they all border directly on the acinar lumina, in seemingly random combinations in different acini. This diversity is reflected in the histochemical staining, since most acini and cells are both PAS- and AB-positive, but a substantial minority stain only with PAS, indicating that they contain serous granules. By comparison, all secretory cells in the submandibular gland stain with PAS but not with AB after the initial appearance of secretory granules at 18 days. From 20 days to birth (at 22 days), the cells with mixed granules disappear, while the cells with serous granules become fewer in number and displaced to the peripheral outpocketings of the acini. As a result of these changes, the general organization in the newborn is similar to that in the adult, i.e., purely mucous acini with serous demilunes.     

Immunohistochemical detection of the estrogen receptor‐α and progesterone receptor in the canine pregnant uterus and placental labyrinth

The presence of hormone receptors is as important as the amount of hormone to predict hormone action. Therefore, the presence of estrogen receptors of the alpha subtype (ER‐α) and progesterone receptors (PR) was evaluated in six pregnant uteri including the placenta and in three postpartum uteri of dogs. This preliminary study is part of our immunohistochemical research project on steroid hormone receptor distribution in the canine female genital tract. Specific staining for ER‐α or PR was found only in cell nuclei. Staining for ER‐α was rare in the various cell types of pregnant and postpartum uteri. Staining for PR was absent or weak in epithelial cells. Moderate staining for PR was observed in endometrial stromal cells and myometrial smooth muscle cells, two cell types playing an important role in the maintenance of pregnancy. Stromal cells stained more frequently positive for ER‐α and PR than epithelial cells, indicating that both hormones may act on epithelial cells indirectly via stromal cells. In the placental labyrinth, fetal cells showed no evidence of ER‐α or PR. In contrast, both receptors were present in maternal mesenchymal cells that were located around the basement membrane of the maternal blood vessels. These cells showed signs of decidualization. No difference in PR distribution was seen between pregnant and postpartum uterine tissue, suggesting that during parturition the decrease in serum progesterone levels and the concomitant increase in the estrogen/progesterone ratio are probably more important than the decline in receptor availability. Anat Rec 260:42–50, 2000. © 2000 Wiley‐Liss, Inc.     

Development of the airway epithelium and submucosal glands in the pig lung: changes in epithelial glycoprotein profiles

Glycoproteins in the normal airway surface epithelium and submucosal glands of 13 Large White pigs were studied from birth to adult life using alcian blue (AB) staining at pH 2.6 or at pH 1.0, with and without sialidase digestion, and by the combination of AB and PAS (AB/PAS) stains. Immediately after birth the percentage of cells producing acidic and neutral glycoproteins increased in both airway surface epithelium and submucosal glands. In the airway surface epithelium, the percentage of mucus-secreting cells producing sulphated glycoprotein increased with age, whereas in the submucosal glands the glycoproteins were mainly sulfated between birth and 3 days and sialylated between 3 days and adult life. The percentage of cells producing neutral glycoprotein in the airway surface epithelium increased during the first 24 h of life after which there was little change with age. In the submucosal glands, however, the greatest increase in the percentage of cells producing neutral glycoprotein occurred between 7 days and adult life. The rapid increase of intracellular glycoprotein production at birth and the presence of the same types of glycoprotein in the immature pig and mature human lung, suggest that the pig may be a useful model in which to study mechanisms and changes of glycoprotein synthesis and secretion during lung development.     

Development of the airway epithelium and submucosal glands in the pig lung: changes in epithelial glycoprotein profiles.

Glycoproteins in the normal airway surface epithelium and submucosal glands of 13 Large White pigs were studied from birth to adult life using alcian blue (AB) staining at pH 2.6 or at pH 1.0, with and without sialidase digestion, and by the combination of AB and PAS (AB/PAS) stains. Immediately after birth the percentage of cells producing acidic and neutral glycoproteins increased in both airway surface epithelium and submucosal glands. In the airway surface epithelium, the percentage of mucus-secreting cells producing sulphated glycoprotein increased with age, whereas in the submucosal glands the glycoproteins were mainly sulfated between birth and 3 days and sialylated between 3 days and adult life. The percentage of cells producing neutral glycoprotein in the airway surface epithelium increased during the first 24 h of life after which there was little change with age. In the submucosal glands, however, the greatest increase in the percentage of cells producing neutral glycoprotein occurred between 7 days and adult life. The rapid increase of intracellular glycoprotein production at birth and the presence of the same types of glycoprotein in the immature pig and mature human lung, suggest that the pig may be a useful model in which to study mechanisms and changes of glycoprotein synthesis and secretion during lung development.     

p53 expression in normal paraffin-embedded tissue using different antibodies and antigen retrieval buffer systems

AIMS: The study was undertaken to demonstrate wild-type p53 in normal paraffin-embedded tissues using two widely used antibodies, DO7 and 1801 and two different antigen retrieval buffer systems. METHODS AND RESULTS: Formalin-fixed paraffin-embedded normal tissue samples were obtained from the archives of the John Radcliffe Hospital, Oxford. Antigen retrieval was performed by microwaving using two different buffer systems: (i) the commercially available Dako target retrieval solution (Cat. no. 1699) (pH 9.8-9.9), (ii) freshly prepared buffer consisting of 0.1 m EDTA with 0.1% Tween pH 6.0, and (iii) freshly prepared buffer consisting of 0.1 m EDTA with 0.1% Tween pH 8.0. Staining was performed with DO7 and 1801 antibodies using the Dako Envision kit (peroxidase/DAB). DO7 antibody elicited strong nuclear staining in the mucosal cells of the small and large intestine, lymphoid cells, decidua, neurones such as Purkinje cells of the cerebellum, glandular epithelial cells and stromal cells of the prostate, cardiac myocytes and bronchial epithelial cells. Cytoplasmic staining was noted in Purkinje cells, glandular epithelium of prostate, exocrine pancreas and renal tubular epithelium. The 1801 antibody did not produce staining in any of these tissues. CONCLUSIONS: Our study demonstrates the presence of p53 in normal paraffin-embedded tissue with nuclear and/or cytoplasmic localization in some instances. In our view, DO7 appears to be better suited for such detection.     

Unexpected Results of Trichrome Staining of Quenched Epithelial Tissue Following Delayed Fixation

Abstract

The histological effects of freezing and thawing unfixed tissue before experimental diffusion studies across the mucosal barrier were investigated in the face of claims that such treatment was of little significance. Following fixation, tissue sections were stained by periodic acid-Schiff (PAS), alcian blue (pH 1.0 & 2.5)-PAS, Masson and Mallory trichromes, acid-picro-Mallory phosphotungstic acid-hematoxylin, Martius-scarlet-blue, luxol-fast-blue, and Sudan black B. Trichrome type staining revealed a previously unrecorded artifact, particularly evident in quenched tissues. Three distinct epithelial cell types could be identified on the basis of differential dye uptake. This finding was not evident in PAS, alcian blue, or Sudan black B stained sections. We postulate that distortion of the cellular matrix occurs as a result of the freeze, thaw, diffusion, and fix sequence and that these cells illustrate the well known phenomenon of tissue density/dye molecular size differential staining by acid dyes. (The J Histotechnol 16:343, 1993)     

The proximal border of the human respiratory unit, as shown by scanning and transmission electron microscopy and light microscopical cytochemistry.

The epithelial cell types present in respiratory (= distal alveolarized) and terminal (= distal nonalveolarized) bronchioles in adult human lung were characterized with scanning and transmission electron microscopy (SEM, TEM) and light microscopic cytochemistry, using specific antibodies against surfactant protein SP-A and mucins, and Alcian blue/periodic acid-Schiff (AB/PAS) staining. In the respiratory bronchiole, two epithelial cell populations share the same basal lamina: one pseudostratified columnar with ciliated, secretory, and basal cells and the other predominantly simple cuboid with some interspersed flat (type I) cells. The columnar secretory cells show the ultrastructure of mucous cells. Light microscopically, they react with mucin antibodies and contain primarily periodate-reactive acid mucins. The mucous cells are the distal secretory cells described by Clara (1937). The cuboid cells are identified as type II (precursor) cells based on ultrastructural criteria for embryonic type II cells (Ten Have-Opbroek et al., 1988a, 1990a), including a cuboid cell shape, a large and roundish nucleus, rough and smooth endoplasmic reticulum (ER), osmiophilic multivesicular bodies, and dense bodies. These dense bodies in turn frequently exhibit--like those in embryonic type II cells--internal vesicles or lamellae, variability in size and shape, a specific relationship to ER and a widespread cytoplasmic distribution. Finally, the cuboid cells show a cytoplasmic staining pattern for SP-A. The terminal bronchiole is lined by the columnar cell population. In the respiratory bronchiole, the columnar (bronchial) and cuboid (alveolar) cell populations occupy distinctly different zones (pulmonary artery zone versus remaining wall). The alveolar part of the respiratory bronchiole (called alveolar tubule) defines the proximal border of a true respiratory unit.     

The role of eye-associated lymphoid tissue in corneal immune protection

Because the cornea is optimized for refraction, it relies on supporting tissues for moistening and nutrition and in particular for immune protection. Its main support tissue is the conjunctiva, in addition to the lacrimal gland, the latter which provides soluble mediators via the tear film. The cornea and conjunctiva constitute a moist mucosal surface and there is increasing evidence that apart from innate defence mechanisms, also lymphoid cells contribute to the normal homeostasis of the corneal surface. A Medline-based literature search was performed in order to review the existing literature on the existence, composition and functions of mucosa-associated lymphoid tissue (MALT) at the ocular surface for corneal protection. The existence of lymphoid cells at the ocular surface and appendage has been known for many years, but for a long time they were believed erroneously to be inflammatory cells. More recent research has shown that in addition to the known presence of lymphoid cells in the lacrimal gland, they also form MALT in the conjunctiva as conjunctiva-associated lymphoid tissue (CALT) and in the lacrimal drainage system as lacrimal drainage-associated lymphoid tissue (LDALT). Together this constitutes an eye-associated lymphoid tissue (EALT), which is a new component of the mucosal immune system of the body. When the topographical distribution of CALT is projected onto the ocular surface, it overlies the cornea during eye closure and is hence in a suitable position to assist the corneal immune protection during blinking and overnight. It can detect corneal antigens and prime respective effector cells, or distribute protective factors as secretory IgA.