PAP Method Research Articles

下顎歯肉部に初発したＢ細胞型非ホジキン悪性リンパ腫の１例

Malignant lymphoma occurs rarely in the oral cavity.We experienced a case of malignant lymphoma arising from the mandibular gingiva in 69-year-old man. His chief complaint was swelling on the gingiva of the right side molar region. At first, we suspected periostitis of the mandible, so we administrated anti-biotics. We palpated fluctuation at the region and incised, but it was solid tumor and not discharged pus. Histopathological examination found chronic inflammation of gingiva. After incision, the clinical course was bad, so we performed biopsy again.Histopathological examination indicated non-Hodgkin's lymphoma, diffuse large cell type. The tumor cell demonstrated B-cell type by PAP method. The immunologic study demonstrated IgM and kappa.Patient received chemotherapy with VCR and ADM. After 3 courses of therapy, the tumor disappeared.

An immunocytochemical study using the PAP method for tyrosine hydroxylase and serotonin in alternate sections, and in situ hybridization to detect tryptophan hydroxylase mRNA in the rat's locus ceruleus

In situ hybridization (LARRSON and HOUGAARD 1990), using the APAAP complex, shows that many small and medium-sized neurons with signals of tryptophan hydroxylase mRNA are uniformly scattered throughout the rat's locus ceruleus, and that a few extrinsic neurons just ventro-medial to it also shows the hybridization signals. The specificity of this technique has been established by Northern blot hybridization. Synthesis of serotonin in intrinsic neurons is indicated not only by the results obtained from in situ hybridization, but also by the fact that, in distribution, masked serotonin cells, immunocytochemically revealed after pargyline and 5-hydroxytryptophan loading, correspond to the neurons showing mRNA hybridization signals. Identification of the same neurons in adjacent cryostat sections, immunostained alternately for serotonin or tyrosine hydroxylase after loading, provides evidence for the coexistence of serotonin and noradrenaline in a single neuron of this center. A few extrinsic, non-specific indoleamine cells located just ventro-medial to the center may be related to the lateralization of the raphe's neurons. The expression of tryptophan hydroxylase in CNS appears to be restricted to the specific regions such as the locus ceruleus and raphe's nuclei.

A Case of Epidural Granulocytic Sarcoma Preceding Acute Leukemia

A 20-year-old male developed both coccygeal and leg pain and followed by rectocystic disturbance. Disc herniation between L5 and S was suspected and laminectomy was performed. At surgery, an easily curretable tumor occupied the epidural space from L5 to the end of the sacrum. In part, the tumor spread out of the vertebral canal and invaded the surrounding muscle tissue. This muscle tissue and part of the lamina were checked histologically. Initial blood analysis revealed 5% blast-like cells, but failed to confirm them as leukemic cells. Histologically, the tumor cells had round or oval nuclei with large nucleoli and scanty cytoplasm without granulocytic differentiation. Malignant lymphoma or Ewing's sarcoma was initially suspected, but the definite diagnosis was uncertain. Immunohistochemical staining with the PAP method and enzyme histochemistry revealed that the tumor cells were positive for lysozyme and naphthol ASD chloracetate esterase. Thus, granulocytic sarcoma was finally diagnosed. Electron microscopic findings supported this diagnosis. Subsequent karyotyping of bone marrow cells revealed 8; 21 translocation, thus the final diagnosis of this patient was myelodysplastic syndrome, refractory anemia with excess blast cells in transformation or acute myelogenous leukemia, M2, by the FAB classification.

The crab-eating frog Rana cancrivora: Serum mineral concentrations and histology of the ultimobranchial gland and the parathyroid gland

In the crab-eating frog Rana cancrivora, taken from a full-strength seawater pond, the in situ levels of serum Na, Cl, K, Ca, Mg, Pi, urea, and osmolarity were examined. The levels were higher than those usually reported for freshwater anurans. However, values for the monovalent salts, urea, and osmolarity were lower than those reported by Gordon et al. (1961, J. Exp. Biol. 38, 659–678) following immersion of this species in 80% seawater for 7 days. The histological features of the ultimobranchial gland and parathyroid gland were coincident with those known in usual freshwater frogs, in spite of the peculiarity of the habitat of this species. The ultimobranchial gland was composed of a single follicle or multiple follicles. In most parenchymal cells, immunoreactive calcitonin was detected using the PAP method with anti-salmon calcitonin antiserum. In the parathyroid gland, cells in the central part of the gland were smaller than those in the peripheral part.

Ultrastructural Localization Study of Carcinoembryonic Antigen (CEA) in Gastric Cancer: Immunoelectron Microscopic Observation

On the basis of light microscopic observation, the ultrastructural localization of CEA in gastric cancer was studied by immunoelectron microscopic technique. The distribution of CEA in gastric cancer and its biological significance and the mechanism of abnormal distribution of CEA were further discussed.Among 104 surgically resected specimens of gastric cancer with PAP method at light microscopic level, the incidence of CEA(+) was 85.58%. All of mucinous carcinoma exhibited CEA(+). In tubular adenocarcinoma the incidence of CEA(+) showed a tendency to rising with the increase of degree of differentiation. In normal epithelia and intestinal metaplasia CEA was faintly present and was found only in the luminal surface. The CEA staining patterns in cancer cells were of three types--- cytoplasmic, membranous and weak reactive type. The ultrastructural localization of CEA in 14 cases of gastric cancer was studied by immunoelectron microscopic technique.There was a little or no CEA in the microvilli of normal epithelia. In intestinal metaplasia CEA was found on the microvilli of absorptive cells and among the mucus particles of goblet cells. In gastric cancer CEA was also distributed on the lateral and basal surface or even over the entire surface of cancer cells and lost their polarity completely. Many studies had proved that the alterations in surface glycoprotein were characteristic changes of tumor cells. The antigenic determinant of CEA was glycoprotein, so the alterations of tumor-associated surface glycoprotein opened up a new way for the diagnosis of tumors.

Observation of basal cells in the olfactory epithelium after axotomy

In the olfactory epithelium of mice, cytokeratin was present in the basal cells but not in the olfactory cells. Our previous study using antikeratin antibodies showed that the basal cells were columnar or pyramidal in shape in the early postnatal period, but became flat in adult mice. In this study, structural changes of the basal cells after axotomy were investigated by immunohistochemistry, transmission electron microscopy (TEM), and scanning electron microscopy (SEM).The unilateral olfactory nerves of adult mice were sectioned at the level of the lamina cribrosa. The mice were sacrificed 4, 8-10, and 14 days postoperatively. The olfactory mucosae were removed, frozen with freon 22, and cut on a cryostat at 10 μm. The sections were stained by the PAP method using a PAP kit. The antikeratin antibody (PKK 2) against pig kidney epithelial cell line, which reacts with 40, 46, 48, and 54 kd subunits (Immunotech), was used. To label the dividing cells, bromodeoxyuridine (BrdU) was administered to mice each postoperative day 1 hour before sacrificing. The presence of BrdU was detected imrnunohistochemically using anti-BrdU antibody (Becton-Dickinson).

Opioid-like immunoreactants in human cervical cancer

Cervical carcinoma cell lines produce several polypeptide hormones. While screening human cervical cancers for such factors we have found many tumors to be immunoreactive for opioid peptides. The tumors were collected at surgery or prior to actinotherapy, fixed in buffered formalin, paraffin-embedded, and immunocytochemically stained using the PAP method. The localization of opioid-like immunoreactivity was investigated using a primary antiserum recognizing the Tyr-Gly-Gly-Phe sequence common to all known opioid peptides. The presence of neuropeptide Y (NPY) and peptide histidine methionine (PHM), both known to be present in normal human cervical tissue, was also investigated using specific antisera. Controls included staining controls and absorption controls, using Sepharose-coupled antigen and were all negative. Out of 40 cervical carcinomas, 27 displayed varying numbers of opioid-immunoreactive cells. In 12 normal cervical specimens no specific staining was observed. None of the tumors displayed any NPY-like or PHM-like immunoreactivity. The growth of cervical carcinoma seems therefore to be accompanied by a destruction of the local peptidergic innervation of blood vessels and smooth muscle cells. The demonstration of opioid-like immunoreactants and the known immunoregulatory properties of these peptides, indicate that opioid peptides may be important in the regulation of growth of human cervical carcinoma.

酵素抗体法（ＰＡＰ）による内耳組織の免疫グロブリンの検出

The distribution of immunoglobulins IgG, IgA and IgM in the inner ear tissue from a patient who died of lung bleeding followed after sepsis was studied, and also the normal guinea pig inner ears and the inner ear disorders induced by Kanamycin injection were studied for the distribution of IgG. The temporal bones were fixed in formaldehyde, decalcified in EDTA and embedded in paraffin. The PAP method was used for the demonstration of the immunoglobulins. In both the human inner ear tissue and the normal control inner ear tissue of the guinea pigs deposits of IgG were found in the sensory organs and the endolymphatic sac, however, in the stria vascularis was slight. The severe damaged inner ears induced by Kanamycin the remarkable decreased deposits of IgG were found in the cochlea, but in the endolymphatic sac the remarkable increased deposits of IgG were found. No IgA and IgM were found in the human inner ear tissue.

カンジダの菌要素が毛内に認められたカンジダ性毛そうの１例

カンジダの菌要素が毛内に認められたカンジダ性毛そうの１例

The effect of ionizing radiation on epidermal langerhans cells. A quantitative analysis of autopsy cases with radiation therapy.

Langerhans cells (LCs) are dendritic cells located in the epiderm is with antigen-presenting capacities. We performed a quantitative analysis of LC density in the anterior chest skin of 286 autopsy cases, including 31 cases treated with radiation therapy. Skin specimens were stained by immunoperoxidase technique (PAP method) with an anti-S-100 protein antiserum. S-100 positive LCs were counted for comparison between non-irradiated and irradiated cases. In this study we noted that, 1) The decline in density of the LCs was age-related and dendritic processes were more prominent in younger groups. 2) The cases irradiated within one month before autopsy showed a reduction in LC density compared with age-matched controls. 3) The cases irradiated more than one month before autopsy demonstrated no consistent or definite tendency. It is suggested that ionizing irradiation as well as ultraviolet light may deplete the LC density in an acute phase. The possibility that radiation therapy alters immunological surveillance in the human skin is discussed.

下顎骨に発生した好酸球肉芽腫の１症例とその電顕的，組織化学的観察

A case of eosinophilic granuloma occuring in the mandibular of a 6-years old boy was reported.In the histopathological examination of extirpated material, eosinocyte infiltrates and increased histiocytes in the connective tissue and Langerhans cell granules were observed by electronmicroscopy. Histiocytosis-X cells showed a positive reaction to S-100 protein by PAP method. These results suggested that the histiocyte of eosinophilic granuloma was derived from T-zone histiocyte.We performed both surgical therapy and combination chemotherapy of prednisolone and Vinblastin. No recurrence was observed in 2 years and 6 months follow up, but a long term follow up study at least for 5 years would be necessary for early detection and adequate treatment of recurrence.

Immunohistochemical Characterization of Rat Central and Peripheral Nerve Tumors Induced by Ethylnitrosourea

Ethylnitrosourea-induced central and peripheral nerve tumors in Sprague-Dawley rats were tested for GFAP (Glial Fibrillary Acidic Protein), S-100 protein, NSE (Neuron Specific Enolase) and Anti-Leu 7 (HNK-1) immunoreactivity utilizing the ABC method (avidin-biotin-complex) for GFAP, S-100 protein and NSE, and the PAP method (peroxidase-antiperoxidase) for Anti-Leu 7. Peripheral nerve neurinomas were consistently positive for S-100 protein and consistently negative for GFAP and Anti-Leu 7. Neurinomas would occasionally exhibit positive staining for NSE (2 of 55 tumors). The staining intensity for S-100 protein varied from strongly positive in differentiated neurinomas to weakly positive in anaplastic tumors. Neoplastic and reactive astrocytes exhibited positive staining for both S-100 protein and GFAP. Variation in the GFAP staining intensity of glial tumors correlated with the degree of differentiation as anaplastic tumors did not stain with the same intensity as their more differentiated counterparts. Oligodendrogliomas exhibited occasional immunoreactivity to S-100 protein (3 of 36 tumors). NSE reactivity in oligodendrogliomas was rarely observed (1 tumor in 36) and immunoreactivity against GFAP or Anti-Leu 7 was consistently absent. Anti-Leu 7 and NSE proved to be of little value in the classification of ENU-induced neural tumors.

Immunocytochemical study of the endocrine pancreas in the grey kangaroo (Macropus fuliginosus)

The endocrine pancreas of the grey kangaroo, Macropus fuliginosus, was investigated by means of immunocytochemistry using the PAP method on the same section at the light- and electron-microscopic levels. Semithin plastic sections were stained individually with primary antibodies for insulin, glucagon, somatostatin and pancreatic polypeptide (PP), and then photographed. Sections were osmicated, re-embedded in BEEM capsules, and ultrathin sections made and examined. The same labelled cells as in the semithin sections were localised in the thin sections, photographs taken and the morphology of secretory granules studied. The insulin cells were pleomorphic; their secretory granules displayed an electron-dense core surrounded by an empty halo. The glucagon cells possessed granules with an electron-dense core usually surrounded by a halo of less dense granular material. Somatostatin cells had larger, less dense secretory granules. The PP cells showed small, dense secretory granules. In order for an ultrastructural study to be considered reliable for the definite identification of endocrine cell types, it is essential that it be corroborated by correlated immunocytochemical data at the light- and electron-microscopic levels.

THREE CASES OF CARCINOID TUMOR ASSOSIATED WITH GASTROINTESTINAL CANCER

Three cases of carcinoid tumor associated with gastrointestinal cancer have been reported. Case 1 had eraly gastric cancer and carcinoid tumor of the transvers colon, for which radical operations were performed. Case 2 has an advanced gastric cancer and pancreatic carcinoid, for which palliative excision of the stomach and transverese colon was done. After 6 months the case 2 died from cancer, and the autopsy revealed a further complication of multiple endocrine neoplasia of I (MENI). Case 3 was found to have a carcinoid which looked like a hematoma of the omentum najus at radical operation for sigmoid colon cancer. The case 3 can have a disease-free period for 2 years after surgery. In the three carcinoid tissues PAP method could demonstrated serotonin, somatostatin and gastrin, and serotonin, respectively, however, these substances could not be detected in each tissues.

Benign Cephalic Histiocytosis: Report of Four Cases

We cared for four patients with benign cephalic histiocytosis, a self-healing non-X, nonlipid cutaneous histiocytosis of children. The age of onset of the disease was 5 to 9 months, with papules and erythematous macules involving the head (mainly the cheeks), and posterior spread to the trunk and limbs in three patients. Microscopic examination of skin biopsies revealed a histiocytic infiltrate in the superficial dermis that was S100 protein-negative by immunoperoxidase (PAP method). One patient showed comma-shaped bodies and desmosomelike junctions on electron microscopy. No Birbeck's granules were present. Benign cephalic histiocytosis is a self-limiting condition that requires no treatment.

Immunohistochemical study of alpha-1-antichymotrypsin in glioma

GFAP is a specific antigen of glial element, but Alpha-1-antichymotrypsin has not been reported in the literature. Alpha-1-antichymotrypsin was guided by GFAP using PAP method to the astrocytes of 137 gliomas. 120 (87%) gliomas were positive for Alpha-1-antichymotrypsin. Of these 120 gliomas, 86 (72%) gave diffuse distribution, 17 (14%) gave focal distribution, and 17 (14%) gave scattered distributions. Alpha-1-antichymotrypsin in glioma tissue may be an important tumor marker for diagnosis.

A Malignant Tumor of the Pancreas Producing Glucagonoma Syndrome

In this study, liver metastases from a patient with a pancreatic glucagonoma producing the syndrome have been investigated histologically, ultrastructurally, and immunocytochemically. A comparison has also been made between the metastases and the primary pancreatic tumor investigated in a parallel study. In the metastatic tissue, glucagon-, pancreatic polypeptide (PP)-, and somatostatin-containing cells were found together with a majority of cells without any immunoreactivity. Glucagon-positive cells were much more numerous than PP- and somatostatin-immunoreactive cells. As in the primary tumor, double immunogold staining of ultrathin sections demonstrated the co-existence of glucagon and PP immunoreactivities in most of the granulated cells, but PP immunolabeling was often faint, so that it probably could not be revealed by the PAP method in light microscopical sections. Such a finding, together with the histological and ultrastructural features, is consistent with an ontogenic and phylogenetic primitiveness of the metastatic cell population.

Solid-phase adsorption method for removing undesired antibodies from polyclonal antiserum.

We have developed a novel and simple method, requiring only a small amount of antigen, for removal of undesired antibodies from antiserum. The method was established using a well-characterized antiserum against rat luteinizing hormone (anti-rLH). Wells of polystyrene tissue culture plates were coated with rat LH (rLH). Anti-rLH diluted 1:3000 was added to rLH-coated wells and shaken to remove LH antibodies. Control anti-rLH was treated in a similar manner in non-rLH-coated wells. Both antisera were tested by immunocytochemistry on rat pituitaries. Antiserum from rLH-coated wells stained no cells, whereas the control serum stained cells that were morphologically typical of LH cells. The effectiveness of this antibody removal was also confirmed in a modified ELISA. In another experiment, anti-rLH and anti-hTSH beta sera were mixed. The final dilution of both antisera was 1:10,000. Anti-rLH was removed by the purification method described. Completeness of antibody removal was confirmed by a double-immunohistochemical staining of rat pituitary in which sections were first stained by the PAP method and then stained with an immunofluorescence procedure after elution of the first antigen-antibody complex. The mixed antiserum incubated in rLH-coated wells did not stain LH cells. There was no co-localization between the LH immunopositivity demonstrated by an anti-rLH serum using immunofluorescence and cells immunostained with the purified antiserum using the PAP method. As indicated in ELISA, the titer of the TSH beta antiserum was not decreased compared to that of the untreated, mixed control antiserum, and the LH antibodies were eliminated by the treatment. This new purification method has four distinct advantages: (a) antiserum is not treated chemically; (b) it requires only a small amount of antigen compared with the amount required for affinity chromatography; (c) neither the undesired antigen-antibody complex(es) nor an excess amount of antigen is present in the purified antiserum; and (d) removal of undesired antibodies can be monitored by ELISA.

A case of gallbladder carcinoma producing both α-fetoprotein (AFP) and carcinoembryonic antigen (CEA)

A 73-year-old woman with gallbladder carcinoma infiltrating to the liver presenting high serum values of AFP and CEA was reported. Serum values of AFP and CEA were remarkably high (165,000 ng/ml and 1,070 ng/ml). Immunohistochemically the tumor cells were stained for AFP and CEA by the PAP method. Serum AFP subfraction was analyzed by crossed immunoaffinoelectrophoresis with lentil lectin and concanavalin A, which showed most of the serum AFP bound to both lentil lectin and concanavalin A. As a case of gallbladder carcinoma presenting a high serum value of AFP is rare, it may imply a diagnostic challenge.

Initial experiences with a monoclonal antibody to the progesterone receptor complex

Today, the immuncytochemical demonstration of estrogen receptors can be regarded as established. Recently, a monoclonal antibody (mab) against the progesterone receptor-complex became commercially available. The antibody Mi 60-10, developed by Milgrom and co-workers, is murine and principally directed against the progesterone receptor of the rabbit, but it also attacks an "epitop" of the human receptor. The Western-Blot analysis shows the antibody having several specific bands against T-47-D cells in the region of 110,000 dalton. To estimate the practical performance of this antibody, 73 primary breastcarcinomas were incubated with both the ER-ICA-Kit and the new "mab". The PAP method was used for visualisation. In a frozen section the mab Mi 60-10 proved as successful as the ER-ICA-Kit. However, in contrast to the ER-ICA-Kit, the mab Mi 60-10 is also successful in locating the receptor, also in formalin-fixed samples embedded in paraffin. This is also the case with blocks, which were stored for several years. In aspirated cytological samples the immunocytochemical demonstration of progesterone was just as successful as that of estrogen and comparable with results on the histological section. A satisfactory validity can be obtained today through the development of highly specific monoclonal antibodies against the progesterone receptor complex, with the complete receptor diagnosis of mammary carcinoma both immunocytochemically in frozen sections and additionally, of progesterone receptors in paraffin wax sections. The only current problems involve the exact quantification of the immunocytochemical results. It is predictable today, that the DCC-method, when compared to the immunocytochemical receptor analysis, will reduce in significance.