PAP Method Research Articles

IgG Deposition in the Lateral Wall of the Endolymphatic Sac in Patients with Menier's Disease

At the end of this series of reports, further information has substantiated the existence of immunoglobuline (IgG, IgA, IgM) in the lateral wall of endolymphatic sacs, obtained during endolymphatic sac surgery in 20 patients with Meniere's disease and one pair of sacs from a cadavers.As an immunohistochemical procedure, the PAP method (peroxidase-anti-peroxidase method) was utilized and IgG was recognized in the epithelium and the subepithelial layer of the sacs of 13 of 20 (65%) patients with Meniere's disease, while IgA and IgM were absent in all of the patients. On the other hand, there were no immunoglobulin deposits in the same part of the sac of the cadaver.Summing up the findings of this series, it can be concluded that there are IgG deposits in the epithelium and the subepithelial layer of the endolymphatic sacs of patients with Meniere's disease, and it is probable that some immunological disorders exist in the endolymphatic space, including the sac, and that they might be closely relataed to one of the causes of endolymphatic hydrops.

Intrarenal localization of angiotensin II specific binding in rat fetuses.

Specific binding sites for angiotensin II were localized in the developing rat kidney (18th day of pregnancy and immediately before birth) by autoradiography using [125I]-ileu-5-angiotensin II either perfused in vivo through the fetal aorta or added in vitro to frozen sections in an incubation mixture. Specific binding was localized in the walls of the afferent and efferent arterioles, in the intraglomerular cells and in the peritubular arterioles of the subcapsular cortical zone. The immunohistochemical analysis, carried out on receptors saturated with unlabelled angiotensin II perfused through the mother's aorta, confirmed the autoradiographical localization. Antisera against ileu-5-angiotensin II were used in the indirect immunofluorescence technique and in the PAP method. Immunolocalization of angiotensin II was also found in the proximal tubule and in the thick ascending limb of Henle's loop.

唾液腺多形性腺腫におけるリゾチームとラクトフェリンの局在についての研究

It has been reported that lysozyme and lactoferrin as antibacterial factors havedefence mechanisms against oral microrganisms. The present study describes that immunohistochemical localization and origin of these substances in salivary glands, in addition their distribution patterns in salivary pleomorphic adenomas. Histogenesis of pleomorphic adenoma was also evaluated. A total 147 cases of salivary pleomorphic adenomas were examined immunohistochemically with the use of the PAP method, as well as histologically with H & E stain.1. Lysozyme staining was expressed only in intercalated duct cells of major salivary gland, whereas it was positive in demilune and serous cells and intralobular duct cells of minor gland. Distribution of lysozymes was different in major and minor salivary glands. Lactoferrin reaction was found in serous acinor cells and ductal segments with irregular fashions.2. Pleomorphic adenomas showed that 14%(21/147) of the cases showed positive staining for lysozyme, and lysozyme was confined to luminal tumor cells. Lactoferrin reaction in the tumor indicated 3 different distribution patterns; 1) Luminal cells positive type 51%(75/147), 2) outer cell positive 3%(4/147) and both luminal and outer cells positive 5%(7/147). Plasmacytoid cells of modified myoepithelium were occasionally positive for lactoferrin (5%). In the stromal cells, histiocytes showed positive reactions for both lysozyme and lactoferrin.3. Modified or neoplastic myoepithelial cells were usually devoid of both the substances, however in a few instances, strong lactoferrin reactivity was found in myoepithelioma cells.4. Histomorphological features and immunohistochemical detectable lysozyme and lactoferrin were evaluated and compared in pleomorphic adenomas and in the normal salivary glands, and histogenesis of pleomorphic adenomas was discussed in terms of immunohistochemical patterns.

Actin as a Component of Cytoskeleton in the Collecting Tubule System of the Mouse Kidney

Actin as a component of the cytoskeleton is familiar as a protein which possibly mediates contraction in nonmuscular cells. In 20 mouse kidneys, the distribution of intracellular actin in the collecting tubule system was investigated by means of the anti-actin PAP method. It could be shown that the positive PAP reaction increases from the proximal to distal up to the collecting tubules in the region of papilla. Stiffening, stabilization and contraction functions are discussed with regard to the biology of the collecting tubule actin.

Demonstration of calcitonin gene-related peptide immunoreactive axons contacting dynorphin A(1–8) immunoreactive spinal neurons in a rat model of peripheral inflammation and hyperalgesia

In a rat model of peripheral inflammation and hyperalgesia, dynorphin A(1–8)-like immunoreactive (DYN-LIr) spinal neurons were examined for contacts from calcitonin gene-related peptide-like immunoreactive (CGRP-LIr) varicosities using a double-label PAP method. Ipsilateral to the inflammation, CGRP-LIr varicosities contacted somata of DYN-LIr neurons in lumbar laminae I, II and V. Few such contacts were found on the contralateral side. The results suggest that opioid neurons which exhibit a dynamic change in dynorphin associated with inflammation, represent a subpopulation of neurons that receive contacts from presumptive nociceptive primary afferents.

A reliable method combining horseradish peroxidase histochemistry with immuno-beta-galactosidase staining.

A sensitive combination of horseradish peroxidase (HRP) tracing and immunohistochemistry was used by Rye et al. [J Histochem Cytochem (1984) 32:1145] in a search for the origins of neurotransmitter- and neuromodulator-containing nerve fibers in brain. In this combination, peroxidase as a marker in immunohistochemistry was thought to yield a homogeneous brown immunoreaction product of diaminobenzidine, different from the black granular reaction product of retrogradely transported HRP, which is visualized by the tetramethylbenzidine (TMB) reaction and subsequent stabilization. A neuron that exhibits both kinds of reaction products in its cytoplasm in sections subjected to combination staining is referred to as a double-labeled cell. With a combined HRP and corticotropin-releasing factor (CRF) immunoperoxidase-antiperoxidase (PAP) method, the first set of experiments showed "false" double-labeled cells in the pyramidal cell layer of rat cerebral cortex, but only rarely in the subcortical areas, possibly because of the use of one enzyme system in two different histochemical procedures. This limitation of the double-staining technique prompted us to demonstrate an alternate combination of HRP tracing and immunohistochemistry in the second set of experiments by employing two previously described independent enzyme systems: HRP as a retrograde tracer and beta-galactosidase as a marker for immunohistochemical demonstration of CRF. A homogeneous blue reaction product indicated immuno-beta-galactosidase staining, and a granular black or brown reaction product labeled retrogradely transported HRP in double-labeled cells in subcortical regions. Neither double labeling nor "false" double labeling was seen in pyramidal cells of cerebral cortex. These findings suggest that application of two independent enzyme systems in a combined HRP and immunohistochemical method may be useful for investigating in origins of peptidergic fibers in brain when the combination of HRP histochemistry and the PAP method appears to be inappropriate.

HEPATIC ANGIOMYOLIPOMA

Hepatic angiomyolipoma in a 59‐year‐old woman Is reported. The tumor was located at the lower edge of the left lobe of the liver, measured 3 × 2 × 2 cm, and appeared solid. Histologically, It consisted of abundant blood vessels of varying caliber, mature fat cells, and round, spindle‐shaped or epithelioid interstitial cells. The latter were considered to be of smooth muscle origin, because myofilamentous structures were demonstrated ultrastructurally in the cytoplasm and because they were positive for desmin by the PAP method. Based on these findings, the diagnosis of angiomyolipoma was confirmed. A review of the literature revealed only 15 cases of hepatic angiomyolipoma. From a clinlcopathological viewpoint, a comparison was made between hepatic angiomyolipoma and its more frequent renal counterpart. The hepatic tumor is considered to be a kind of hamartoma.

Immunocytochemical studies on immunoglobulin‐containing cells in the epidermis of rainbow trout Salmo gairdneri Richardson: influence of bath vaccination*

The presence of immunoglobulin–containing cells in the epidermis of rainbow trout was traced, using post–embedding immunocytochemical techniques, in resin–embedded fragments of skin. Light microscopy observations (for which the PAP method was used)revealed the presence of immunoglobulin–containing cells in the epidermis, although no accurate identification was possible. Electron microscopy observations (employing gold–labelling) showed that, in spite of the poor preservation thought unavoidable to protect the antigenicity of the immunoglobulins, the positive cells were either lymphocyte–like cells or mucous cells. Anti–Vibrio anguillarium antibody was detected in the epidermal mucous cells of rainbow trout juveniles vaccinated by immersion, using an immunoenzymatic sandwich technique, modified for resin–embedded tissue. The possibility of the existence of a local immune system is discussed, considering the involvement of the mucous cells.

Cytology and arrangement of enterochromaffin (EC) cells in the human stomach.

Serotonin containing enterochromaffin (EC) cells in the human gastric mucosa were observed using serial semithin sections immunostained by Sternberger's PAP method and reconstructed by computer-assisted methods. In oxyntic glands, EC cells displayed a marked pleomorphism which suggests their plasticity or active movement. They sometimes possessed multipolar cytoplasmic processes directly contacting the neighboring epithelial cells and/or gastric lumen. In the antropyloric glands, they are exclusively the "closed-type," which fails to contact the lumen, and are often arranged touching other EC cells (cluster formation), apparently exhibiting polynuclear enterochromaffin syncytia. This syncitium-like arrangement is interpreted as the morphological counterpart of a possibly synchronized function of these cells. The morphological differences of EC cells in their shape, luminal endings and arrangement between both regions may be indicative of regional differences in their functions. Furthermore, the present study provides the first three-dimensional visualization of EC cells in the human stomach.

Phylogenetic study of the arginine-vasotocin/arginine-vasopressin-like immunoreactive system in invertebrates

1. 1. A phylogenetic study of arg-vasotocin (AVT)/arg-vasopressin (AVP)-like immunoreactive cells was performed by the PAP method in the central nervous system of invertebrates. 2. 2. The immunoreactivity was detected in the nerve cells of Hydra magnipapillata of the Coelenterata: Neanthes japonica and Pheretima communissima of the Annelida; Pomacea canaliculata, Aplysia kurodai, Oncidium verrucosum, Bradybaena similaris, Achatina fulica, Limax marginatus and Meretrix lamarckii of the Mollusca; Gnorimosphaeroma rayi, Hemigrapsus sanguineus, Gryllus bimaculatus and Baratha brassicae of the Arthropoda; Asterina pectinifera of the Echinodermata; and Halocynthia roretzi of the Protochordata. 3. 3. No immunoreactivity was detected in Bipalium sp. of the Platyhelminths, or in Procambarus clarkii and Helice tridens of the Arthropoda. 4. 4. From these results, it appears that AVT/AVP is a phylogenetically ancient peptide which is present in a wide variety of invertebrates. 5. 5. The actions of AVT/AVP and its presence in invertebrates are discussed.

Phylogenetic study of the oxytocin-like immunoreactive system in invertebrates

1. 1. A phylogenetic study of oxytocin (OXT)-like immunoreactive cells was performed by the PAP method in the central nervous system of invertebrates. 2. 2. The immunoreactivity was detected in the nerve cells of Hydra magnipapillata of the Coelenterata; Neanthes japonica and Pheretima communissima of the Annelida; Oncidium verrucosum, Limax marginatus and Meretrix lamarckii of the Mollusca; and Baratha brassica of the Arthropoda. 3. 3. No immunoreactive cells were found in Bipalium sp. of the Platyhelminthes; Pomacea canaliculata, Aplysia kurodai, Bradybaena similaris and Achatina fulica of the Mollusca; and Gnorimosphaeroma rayi, Procambarus clarkii, Hemigrapsus sanguineus, Helice tridens and Gryllus bimaculatus of the Arthropoda; Asterina pectinifera of the Echinodermata; and Halocynthia roretzi of the Protochordata. 4. 4. These results demonstrate that an OXT-immunoreactive substance is widely present not only in vertebrates but also in invertebrates. 5. 5. OXT seems to have been introduced into these invertebrates at an early stage of their phylogenetic history.

Immunohistochemical hormone content in medullary and undifferentiated thyroid carcinoma and prognosis after surgery

Immunohistochemical determinations in tissue specimens of medullary and “undifferentiated” thyroid carcinomas were carried out with antisera against calcitonin, calcitonin-gene related peptide (CGRP), somatostatin, and also thyroglobulin, using the PAP method. All 8 samples of medullary carcinoma stained positive with antisera against calcitonin and CGRP, 7 samples were also positive for somatostatin. Out of 22 cases initially diagnosed as “undifferentiated thyroid carcinoma” 3 revealed positivity for calcitonin, 2 for CGRP, and 1 for somatostatin. Congo red stain for amyloid, performed several years ago in 2 of these 3 cases, had been negative. The patients with medullary carcinoma survived longer than those with “undifferentiated” carcinoma. One patient of the latter group, but with calcitonin, CGRP, and somatostatin immunoreactivity in the tumour tissue, is now alive and well, more than 4a after initial treatment.

TWO CASES OF PAPILLARY AND CYSTIC TUMOR OF THE PANCREAS

Papillary and cystic tumors of the pancreas are rare and although there seems to be a growing awareness of them, there are still points of ambiguity concerning them such as lack of a classification system. Two cases were encountered and are reported here.Case 1 is a of a 42-year-old woman in whom an abnormal abdominal calcification shadown was found during examination, and case 2 is of a 15-year-old girl with left upper abdominal pain. In each case a postoperative patholoigcal diagnosis of a papillary and cystic tumor of the pancreas was arrived at.In case 1 there was a positive result for alph-1-antitrypsin, insulin and glucagon tested by the PAP method and origin in a stem cell with a multipotential ability to differentiate as a histological origin is suspected.

Clinicopathological and immunohistological study of salivary gland tumors

A total of 126 cases of salivary gland tumors were studied from clinicopathological view point. A particular attention was paid to the reason for the recurrence in pleomorphic adenoma and to the prognosis of adenoid cystic carcinoma. Immunohistological reaction for many markers including lysozyme, lactoferrin, Al-p, keratin, S-100, actin, vimentin, CEA, etc., using PAP method was performed to investigate the origin of tumor cells and to obtain an indicator for malignancy.In recurrent cases with plemorphic adenoma, myxochondromatous pattern was more frequently observed than other patterns, even if no correlation to the numbers of atypical cells and of mitotic figures. A relation between the recurrence and proliferation of tumor cells was not speculated because of an equal frequency of Ki-67 positive cells irrespectively histological subtypes of pleomorphic adenoma. In adenoid cystic carcinoma, the cases with solid pattern, occupying only a part of tumor, had poor prognosis. It was presumably considered that the solid pattern in adenoid cystic carcinoma might reflect high grade malignancy.Immunohistologically, salivary gland tumors, benign or malignant, are composed of lines of ductal epithelial cells. In malignant tumors, however, it was rather difficult to determine the cell origin because of a poor immunohistological reaction. A localization of CEA was different in benign tumors from malignant tumors, showing that it was positive for luminar border of ductal epithelium in former and positive for cytoplasma of tumor cells in latter, particalurly in mucoepidermoid carcinoma and acinic cell carcinoma. CEA might be useful for differential diagnosis in certain histologic types. Ki-67 is known as antigen positive for nuclei of proliferating cells, but there has been no report on salivary gland tumors. In this study, Ki-67 positive cells in malignant tumors were higher than those of normal gland benign tumors. A high frequency of Ki-67 positive cells in salivary gland tumors may give a clue for malignancy.

Functional and morphological analysis on GH producing adenomas with quantitative immunohistochemical method

We studied functioning cells of GH-producing pituitary adenoma quantitatively by the immunohistochemical method using anti GH antibody and compared the result with clinical hormonal data by discussing a clinical significance of the immunohistochemical study of pituitary adenoma. Fifteen acromegalics with pituitary adenoma, who underwent surgical operation followed by immunohistochemical study, were subjected. Volume of the adenoma was calculated on the basis of thin coronal cut CT scanning. The specimen was stained after PAP method. The quantitative analysis was done by investigating the ratio of anti GH antibody positive area in 30 fields (SAR). Using the value obtained, stained volume (SV) was calculated by multiplying total volume by SAR. The SV was regarded as total volume of GH positive cells. These values were compared to the preoperative serum GH levels. The results were as follows: (1) Immunohistochemically, the adenomas were classified into two types, the sporadic type and the diffuse type, by incidence of GH positive cells. SAR ranged from 0.2% to 52.4%, and means of the types were 3.0% and 24.8%, respectively. (2) All the eosinophilic adenomas demonstrated to be the diffuse type and the mean SAR was 23.7%. On the contrary, 75% of chromophobe adenomas were the sporadic type and the mean SAR was 4.9%. (3) There was no significant correlation between the serum GH level and the volume of the adenoma. There was an especially high correlation between the serum GH level and SV with a correlation constant of 0.971. (4) In fibrous adenomas the serum GH level was extremely low for their total volume.

Differential effects of p-chlorophenylalanine on indoleamines in brainstem nuclei and spinal cord of rats. II. Identification of immunohistochemically stained structures using computer-assisted image enhancement techniques

Following intraperitoneal injection of p-chlorophenylalanine (PCPA, 400 and 600 mg/kg) on 3 consecutive days, the brainstem and lumbar cord of rats were removed, frozen-sectioned and immunohistochemically stained (PAP method) for serotonin (5-HT). Using computer-assisted image analysis, the density of 5-HT staining in control, 400 and 600 mg/kg PCPA groups was determined. The mean number of pixels (representing 5-HT staining) was determined in 6 areas in the brainstem containing 5-HT cell bodies (nuclei raphe pallidus, raphe obscurus, rostral and caudal raphe magnus, raphe dorsalis and paragigantocellularis lateralis) and in the dorsal and ventral spinal cord. The resultssuggest a differential depletion of 5-HT within brainstem nuclei following PCPA treatment in that the most marked dose-related reductions were observed in nucleus raphe obscurus and caudal magnus. Furthermore, a computer program designed to isolate terminal structures in the spinal cord identified a differential depletion of 5-HT terminals in the dorsal horn versus the ventral horn. The present study describes 3 analytical approaches combining immunohistochemistry with the computer-assisted image analysis technique and allows comparison between groups of animals which received the same or different drug treatments.

Immunohistochemical study of carbonic anhydrase in mixed tumours and adenomas of sweat and sebaceous glands

Immunohistochemical distribution of carbonic anhydrase II (CA) in mixed tumours and adenomas of sweat gland origin and in sebaceous adenomas was demonstrated by the PAP method. Normal sweat glands, both eccrine and apocrine, clear cells of the secretory coils, and ductal epithelial cells all showed conspicuous staining for CA, and sebaceous glands were also positive. Mixed tumours of the skin indicated strongly positive staining for CA in the luminal cells of tubular and duct-like or cystic structures, while most of the other tumour cells were negative. In solid or massive foci, CA positive cells were found scattered among the cellular mass. Sebaceous adenomas were usually moderately positive for CA throughout the tumour.

Failure of luteinizing hormone-releasing hormone (LHRH) to affect the differentiation of LH cells in the rat hypophysial primordium in serum-free culture.

The aims of this study were to investigate the differentiating capacity of adenohypophysial LH cells in a serum-free culture medium and to test whether cytogenesis is affected by synthetic LHRH. The adenohypophysial primordia of fetal rats were isolated on days 11.5 and 12.5 of gestation and cultured without serum for 10 and 9 days, respectively, in synthetic Medium 199 or alpha MEM. Immunohistochemical examination using the PAP method revealed that most culture explants, apart from a few degenerate ones, contained LH cells. In comparison with Medium 199, which has been widely used as a culture medium for hypophysial explants, alpha MEM gave far better results and the primordia cultured in this medium showed better tissue growth and contained a greater number of LH cells. Administration of synthetic LHRH (10 ng/ml) on the first day of culturing had no effect on the number of LH cells, no matter whether or not the culture medium was supplemented with insulin, transferrin or thyroxine. These results suggest that at the early developmental stage LHRH is not essential for the differentiation and/or proliferation of LH cells.

DERMATOPATHIC LYMPHADENOPATHY

Dermatopathic lymphadenopathy (DPL) is characterized by lymph node enlargement with reactive process and is generally caused by chronic inflammatory skin disease. Interdigitating reticulum cells (IDCs) with broad cytoplasm, innumerable cytoplasmic interdigitation, and bizarre shaped nuclei are the most striking cell type in DPL. According to the shape of the nucleus, arrangement of the tubulovesicular complexes (TVC) and amount of melanin granules, these cells fall into two types: type I has a marked indented nucleus, innumerable TVC and some desmosomal junctions and fewer melanin granules, while type II has a large amount of melanin granules. On the other hand, LCs contain a few loose melanin granules. It appears that IDCs and Langer‐hans cells (LCs), surrounded by T‐lymphocytes, are similar in morphology and function, but both of them can be characterized by the positive of S‐100 protein and Leu 6, absence of lysozymes and CEA by PAP method, and positive of ATP‐ase and ACP‐ase, and they differ only by having or not having Birbeck granules. The mechanism of the proliferation of IDCs in DPL and delineation of the functional relationship between the accessory cells and the T‐lymphocytes remains to be investigated. ACTA PATHOL. JPN. 37:887–900, 1987.

Immunohistochemical demonstration of estrogen, progesterone, CEA and ferritin in breast cancer and their clinical value for the prediction of early postoperative recurrence.

The aim of this paper is to determine whether the incidence of estrogen-, progesterone-, CEA-, and ferritin- positive staining of primary tumors, by using the PAP method, is related to the prognosis of breast cancer status. A significantly higher incidence (71 per cent) of CEA-positive tumors was observed in patients who had a recurrence of breast cancer within 2 years after radical operation. Patients, whose tumors were positive in estrogen or negative in CEA, showed a relatively good prognosis, even after a recurrence of the disease. Distant metastases were seen in most of the patients positive in CEA (78 per cent). Before the recurrence of breast cancer, those patients positive in progesterone had a good prognosis. After the recurrence, however, there was no relationship between the prognosis of recurrent disease and the progesterone-staining of primary tumors. Our data suggest that the immunohistological staining of estrogen, progesterone and CEA might offer the effective prognostic indices in breast cancer patients.