PAP Method Research Articles

Visceral noxious stimulation induced expression of Fos protein in medullary catecholaminergic neurons projecting to nucleus accumbens in the rat: a study with triple labeling method of HRP tracing combined with Fos and TH immunohistochemistry

Horseradish peroxidase (HRP) was stereotaxically injected into the nucleus accumbens (Acb), and visceral noxious stimulation given by injecting formalin into the stomach. Sections of the medulla were subjected to HRP reaction combined with immunohistochemical reactions for Fos protein (ABC method) and tyrosine hydroxylase (TH, PAP method). The catecholaminergic neurons of the medulla (including vagal complex, ventrolateral medulla and reticular formation between them) which expressed Fos protein and projected to Acb were studied. The results showed that HRP retrogradely labeled cells were seen in the medulla bilaterally with apparent ipsilateral predominance and TH-LI and Fos-LI single labeled cells were bilaterally distributed; HRP/TH,TH/Fos double labeled neurons were more numerous than HRP/Fos double-labeled neurons. HRP/TH/Fos triple-labeled neurons were small in number and were mainly distributed in the nucleus tractus solitarii (nTS) and ventrolateral medulla (VLM), but only a few labeled cells were located in RF between nTS and VLM. It is concluded that TH-LI neurons in the medulla projected to Acb and some of them expressed Fos protein after noxious stimulation of the stomach.

Immunocytochemical demonstration of melanotransferrin (p97) in thyroid tumors of follicular cell origin

Using the PAP method on paraffin sections, we investigated the distribution pattern of melanotransferrin (p97) in follicular adenomas (20 cases), atypical adenomas (4 cases), papillary (10 cases) and follicular carcinomas (25 cases) of the thyroid; frozen sections collected from 3 follicular adenomas and 2 follicular carcinomas were also tested. Our results document positivity for p97 in atypical adenomas, papillary and follicular carcinomas, whereas no reactivity is encountered in follicular adenomas and normal portions of thyroid. We suggest that the production of p97 by neoplastic cells of atypical adenomas and differentiated thyroid carcinomas could result in a greater availability of iron for tumor cell DNA synthesis.

AF64A affects septal choline acetyltransferase but not parvalbumin immunoreactive cells

Rats received bilateral intracerebroventricular (ICV) infusions of either AF64A (1.5 nmol/ventricle; n = 9) or vehicle (3.0 μ1/ventricle; n = 7). Four weeks later, the animals were anesthetized and their brains processed to visualize and quantify choline acetyltransferase (ChAT) immunoreactive (IR) and parvalbumin-IR GABAergic neurons in the septal complex by immunocytochemistry (PAP method). AF64A significantly reduced the number of ChAT-IR perikarya in the medial septum (28%), ventral limb of the diagonal band of Broca (30%), and horizontal limb of the diagonal band of Broca (20%), but did not affect the number of parvalbumin-containing GABAergic neurons in any of the septal subdivisions. These results provide further evidence that AF64A is a selective cholinotoxin.

Immunohistochemical study on x antigen of HBV (HBxAg) in primary hepatic carcinoma

The specimens were from 110 patients with primary hepatic carcinoma. The formalin- fixed and paraffin-embedded sections were stained for HBxAg by ABC method and for Bag and Hubcap by PAP method. Of the 110 cases, 64 (58.2%) showed HBxAg-positive reaction in tumor tissue, and 63 (78.8%) of 80 cases displayed positive HBxAg in surrounding non-cancerous hepatic tissue. Among the 64 cases with positive HBxAg in tumor tissue, 15 (23.4%) were associated with Bag and/or Hubcap, while in the 63 cases with positive HBxAg in non-tumor tissue, 45(71.4%) were accompanied with Bag arid /or Hubcap. These findings suggest a close relationship between primay hepatic carcinoma and HBV infection. The high detection rate of HBxAg indicates a very active expression of the integrated HBV- DNA genome in the host cells. However, the action of HBxAg in pathogenesis of hepatocellular carcinoma remains to be further investigated.

Endogenous creatinine clearance measurement of glomerular filtration rate in dogs

Summary Renal clearance procedures were performed on adult mixed-breed dogs with a wide range of renal function. Endogenous creatinine clearance was computed after analyzing plasma and urine for creatinine by use of 2 methods, PAP and kinetic Jaffe. For 20-minute clearance procedures, [14C]inulin clearance was measured simultaneously with endogenous creatinine clearance. For 111 twenty-minute clearance procedures performed on 24 dogs, [14C]inulin clearance was highly correlated with creatinine clearance for both methods of creatinine analysis (R2 = 0.979 for [14C]inulin-PAP; R2 = 0.943 for [14C]inulin-Jaffe). The absolute values for PAP and [14C]inulin clearance were nearly the same (PAP-to-[14C]inulin clearance ratio = 1.03 ± 0.08), but those for Jaffe clearance were substantially less than those for [14C]inulin clearance (Jaffe-to-[14C]inulin clearance ratio = 0.88 ± 0.10). The Jaffe-to-[14C]inulin clearance ratio was inversely correlated with degree of renal function (R2= 0.464), whereas the PAP-to-[14C]inulin clearance ratio was not correlated with degree of renal function (R2 = 0.060). Thus, Jaffe-determined creatinine clearance varied, in relation to [14C]inulin clearance, depending on degree of renal function. In 4 clinically normal dogs, 20-minute and 24-hour sample collections analyzed by use of the PAP method gave clearance values significantly greater, for both periods, than did Jaffe analyses. The PAP-determined creatinine clearance values were less than, but not significantly different from 20-minute exogenous creatinine clearance values determined 10 days after 24-hour collections. For 20-minute and 24-hour collections, the difference in clearance values between the PAP and Jaffe methods was attributable mostly to lower plasma creatinine values for the PAP method (mean ± SEM, plasma PAP-to-Jaffe ratio = 0.798 ± 0.053). However, urine creatinine values also were less by use of the PAP method (urine PAP-to-Jaffe ratio = 0.943 ± 0.103). We conclude that PAP-determined creatinine clearance reliably measured glomerular filtration rate during 20-minute collections, and probably during 24-hour collections as well. By contrast, Jaffe-determined creatinine clearance underestimated glomerular filtration rate by a variable amount.

Zum Risiko endokriner Aktivierung bei Eingriffen an Paragangliomen im Kopf-Hals-Bereich

The intracytoplasmatic glycolytic enzyme neuron-specific enolase (NSE) can be found in cells of endocrine tissue and their derived tumours. The enzyme is not secreted but released if cells are destroyed, i.e. continuously in malignant tumours. The serum level of neuron-specific enolase (NSE) has been used as a marker for small cell carcinomas of the lung, glioblastomas and malignant phaeochromocytomas. In this investigation blood samples were taken prior to and following surgical manipulation of the tumour in 21 patients with paragangliomas of the head and neck region and 6 controls. 22 serum samples were obtained before angiography and two hours after angiography, 41 before surgery, during surgery and after surgery. The serum level of NSE was measured by NSE-RIA test (Diagnostics, Uppsala). In all tumour specimens NSE could be demonstrated by the immunohistological PAP method (Abb. 1). Only two patients had preoperatively elevated serum-NSE levels. An arterial venous shunt had been detected by angiography in one of these patients. No significant effect on NSE serum level could be produced in the majority of patients by an angiography with embolisation or surgical manipulation of the tumour (Fig. 1, 2). A reduction in serum NSE level was observed postoperatively in both cases mentioned before. None of the 6 controls showed any significant change in serum NSE level (Fig. 3). It can be concluded from this study that embolisation does not lead to direct or indirect cell destruction through ischaemia. Manipulation of the tumour does not destroy a significant number of cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The presence of a common antigen between human gastric cancer cells and OK-432.

Twenty two surgical specimens of gastric cancer resected after administration of OK-432 for the skin reaction test were examined to determine whether the cancer cells had the same antigens as OK-432, a product of hemolytic streptococcus cells. When the tissues were stained by the PAP method with anti-Su streptococcus antibody used as the primary antibodies, the common antigens were demonstrated in 10 (45.5%) of the 22. The presence or absence of the common antigens was independent of the degree of skin reaction to OK-432, and the relations of the common antigens to other host responses were not clear in this study. This is the first report for the presence of such common antigens between human gastric cancer and OK-432.

香川県で発生した豚のアメーバ症

Several weanling piglets reared in a piggery of Kagawa Prefecture showed bloody and watery diarrhea. Pathological examination on 3 of the piglets revealed fibrinous and hemorrhagic ceco-colitis with intramural infestation of many amoebic trophozoites measuring 13-18μm in diameter. The amoebas were stained positively with PAS, Heidenhein's iron hematoxylin, and immunostaining (PAP method) using specific antibody against Entamoeba histolytica. Ultrastructurally, the trophozoites contained many and large cytoplasmic vacuoles and numerous ribosomes in the absence of mitochondria and Golgi apparatus. Feces of sows and feeder pigs raised in the farm frequently contained many amoebic cysts, which multiplied and were serially passaged on Tanabe-Chiba medium. Some of the cysts had two or four nuclei. Indirect hemoagglutination reaction of sera from 39 sows and 16 feeder pigs of the farm demonstrated positive antibody titers (<160) in 3 sows (7.7%) and 6 feeder pigs (37.5%).

下顎歯肉に発生したＩｇＡ‐λ型髄外形質細胞腫の１例

A rare case of extramedullary plasmacytoma arising from mandibular gingiva is presented. A 33-year-old man with localized swelling in the gingiva of the mandibular incisor region was referred for consultation. The swelling was diagnosed as plasmacytoma by biopsy, and therefore the patient underwent a series of clinical examinations. The examinations failied to establish a diagnosis of multiple myeloma. The lesion was resected with part of the mandibular bone, because radiographs indicated resorption of the bone adjacent to the gingival lesion. PAP method, which is an immunohistochemical technique for staining, revealed that the tumor was an Ig-λ type of extramedullary plasmacytoma. Recurrence and metastasis of the tumor have not been noted during a 2-year follow-up period after resection.

Detection of Human Papillomavirus DNA in the Normal Cervices of Japanese Women by the Dot‐Blot (Vira Pap™) Method

Exfoliated cervical cells obtained from 9 patients with cervical intraepithelial neoplasia (CIN) I and II, 11 with CIN III, 22 with cervical carcinoma, and 599 with cytologically normal cervices (including 63 pregnant women) were evaluated for the presence of human papillomavirus (HPV) by the dot-blot (Vira Pap) method. HPV infection was detected in 33.3% of the CIN I and II cases, in 54.5% of the CIN III cases, in 68.2% of invasive cervical carcinoma cases, and in 4.2% of the normal group. The rate of detectable HPV infection increased in exfoliated cervical cells during pregnancy. These results suggested that a few Japanese women with cytologically normal cervices were infected with HPV.

Lectin histochemical investigations of fetal cultivated pancreatic tissue.

The terminal glycoproteins of fetal, cultivated (7-12 days), and adult nondiabetic and diabetic pancreatic tissues (Balb c, C3h mice) were investigated by lectin histology (peanut-, phytohemagglutinin, wheat germ agglutinin, Ulex europeus I, concanavalin A and Ricinus communis agglutinin, PaP method +/- neuraminidase). Anti-insulin and -glucagon were used to identify islet cells. S-100 antibody showed dendritic reticulum cells, anti-IAK proved MHC II antigens (C3h). Cultured tissue was partly incubated with anti-IAK and complement for lysis of MHC II antigens. On the 19th gestational day fetal pancreatic tissue did not bind peanut agglutinin, Phytohemagglutinin, or wheat germ agglutinin, whereas concanavalin A and Ricinus communis were weakly bound. Terminal fucose residues were not expressed by C3h fetal islet cells in contrast to Balb c. Following neuraminidase digestion peanut agglutinin and phytohemagglutinin were strongly bound, indicating sialic acid-substituted terminal glycoproteins. Cultivated tissue (Day 7) bound all investigated lectins (except Ulex europeus I in C3h mice), indicating maturation of islet cells. In spite of the peak of insulin concentration in the medium we observed a faint binding of anti-insulin and investigated lectins following 12 days of cultivation. This indicates a disorder of terminal glycoprotein synthesis at this point. There was no difference in lectin binding patterns of adult nondiabetic islet cells compared to the cultivated tissue (7 days), but no Ulex europaeus I binding of the adult Balb c mice was observed. S-100 binding decreased during the cultivation period as dendritic reticulum cells became destroyed by cultivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of specific antibody against L-dopa and its ultrastructural localization of immunoreactivity in the house-shrew (Suncus murinus) lateral habenular nucleus.

An antiserum was raised against L-DOPA bound to bovine serum albumin, purified by affinity chromatography, and its specificities were verified by immunoblotting and enzyme-linked immunosorbent assays. The antiserum did not cross-react with dopamine (DA), tyrosine, tyramine, octopamine, norepinephrine or epinephrine. Immunocytochemical studies using the PAP method revealed that tyrosine hydroxylase- and L-DOPA positive but aromatic L-amino acid decarboxylase- and DA-negative neurons were present in the lateral habenular nucleus of the house-shrew (Suncus murinus) brain. Ultrastructurally L-DOPA immunoreactive products were localized in the cytoplasmic matrix and terminals with vesicles.

Immunochemical and immunohistochemical detection of S-100-like immunoreactivity in spinach tissues.

S-100 proteins represent a group of closely related acidic, calcium binding proteins originally isolated from the mammalian nervous system and later detected in non-neural cell types and in a wide variety of vertebrate and invertebrate species. The present study used immunochemical and immunohistochemical methods to extend the investigation of S-100 during phylogenesis to plant tissues. The presence of S-100-like immunoreactive material was detected in extracts of spinach (Spinacia oleracea L.) terminal buds and young leaves by the ELISA method and by Western blotting using different anti-S-100 rabbit antisera. Using the PAP method, serial sections of young spinach leaves treated with the same antisera exhibited an immunoreaction product that was confined to the cytoplasm and nucleus (but absent from the vacuoles) in meristematic, epidermal, and parenchymal cells. The present data enlarge the field of investigation of S-100 proteins in the search of the function(s) of S-100 in biological organisms.

Observation of the activity of factor VIII in the endometrium of women pre- and post-insertion of three types of IUDs

The endometrial materials were obtained from 90 women who had been randomly inserted with three types of IUDs (Stainless steel ring, SS; copper T 220, TCu 220,and levonorgestrel-releasing device, LNG). An immunoperoxidase reaction, PAP method, with the antiserum of Factor VIII as the primary antibody, was carried out to detect the Factor VIII activity in the endometrial endothelium pre- (control) and post-insertion of the IUDs. The results revealed that: 1. 1. There was a generalized lower Factor VIII activity in the endometrium of women post-insertion of IUDs (except LNG). 2. 2. Comparison of the Factor VIII activity in the endometrium of women using different types of IUDs showed that the TCu type and the SS type decreased the activity significantly whereas the activity remained unchanged after 3–6 months' use of the LNG-IUD. The different types of IUDs seemed to influence the coagulation regulatory system in different ways; the lower Factor VIII activity, the more tendency to bleeding. 3. 3. The Factor VIII activity in the endometrium of women using IUDs was lower in all phases of the menstrual cycle including the proliferative phase when the Factor VIII activity is normally high. It cannot be excluded that this could be a contributing factor to IUD-induced bleedings.

Comparison of culture, peroxidase-antiperoxidase reaction, and serum latex agglutination methods for diagnosis of chlamydiosis in pet birds

Summary Comparison was made among results of cloacal specimen culture, and cloacal swab specimen (cytologic) peroxidase-antiperoxidase (pap), serum latex agglutination (la), and tissue pap assays for diagnosis of chlamydiosis in 144 birds. Swab specimen pap findings correlated poorly with la results and failed to predict the la test result in any bird. Only 1 cloacal swab specimen was regarded as pap-positive and was from the cloaca of a bird from which chlamydiae were isolated in culture. The sensitivity of swab specimen pap, compared with culture results, was 33%, whereas specificity was 94%. In this study, swab specimen pap was a less sensitive test, compared with culture, than was reported in a previous study. The sensitivity of la in identifying birds that were cloacal culture-positive was poor; true-positive results were not detected, compared with culture results. The specificity of the la method was 93%, compared with culture results. Results of the tissue pap method correlated with culture results in the 3 birds for which both tests were performed.

Immunohistological detection of Saruplase (recombinant single-chain urokinase-type plasminogen activator) in normal rat tissue

Saruplase--a recombinant single-chain urokinase-type plasminogen activator was identified immunohistochemically in normal rat tissue after intravenous administration by means of a polyclonal antibody. For this purpose, rat tissues were fixed in various ways (liquid nitrogen, ethanol, formaldehyd solution). Saruplase could be detected by the PAP method, streptavidinbiotin system and indirect immunofluorescence in the kidney (proximal tubule), liver (hepatocytes, Kupffer cells) and spleen (reticular cells). Saruplase was not localized in the rat endothelium. It is discussed that the rat-specific receptors for urokinase-type plasminogen activator on endothelial cells cannot bind Saruplase due to the extreme species specificity.

Septal choline acetyltransferase immunoreactive neurons: Dose-dependent effects of AF64A

Two experiments were performed. In the first, the cholinotoxin. AF64A (0.5, 1.0 or 1.5 nmol/ventricle), or vehicle (3.0 μl) was injected (ICV) bilaterally into male rats ( n = 23). Choline acetyltransferase (ChAT) immunoreactive (IR) perikarya in the four subgroups of the septal complex were visualized by immunocytochemistry (PAP method) 28 days postinjection, and counted using a microprojector (× 40). The 0.5 nmol/ ventricle dose of AF64A significantly reduced (31%) the number of ChAT-IR cell bodies in the intermediate subgroup (rostral extension of the nucleus basalis/substantia innominata). Higher doses did not produce additional reductions. The highest dose (1.5 nmol/ventricle) of AF64A resulted in significant decreases in ChAT-IR cell bodies in the dorsal (51%) and midline (35%) subgroups (medial septum), but did not affect the number of ventral subgroup (diagonal band of Broca) ChAT-IR neurons. In the second experiment, electrolytic lesions were placed in the corpus callosum, cingulum and overlying cingulate gyrus, in order to simulate the nonselective damage seen following the 1.5 nmol/ventricle dose of AF64A. In comparison to the surgical controls ( n = 3), the electrolytic lesions ( n = 6) failed to significantly affect the number of ChAT-IR perikarya in any of the septal subdivisions. Thus the distinct subgroups of septal ChAT-IR neurons are differentially sensitive to the toxic effects of ICV administered AF64A: intermediate 》 dorsal 》 midline 》 ventral subgroup.

Species differences in the coexistence of 5-hydroxytryptamine and substance P in presynaptic boutons in the cervical ventral horn

Substance P (SP)- and 5-hydroxytryptamine (5-HT)-containing presynaptic boutons in the cervical ventral horn were studied in chicken, hamster, rat and monkey spinal cords, using PAP and protein A-gold double-labeling techniques in conjunction with monoclonal antibodies. In the chicken, the PAP method demonstrated that SP-immunoreactive boutons contained large spherical dense-cored vesicles (DCVs) whereas 5-HT-immunoreactive boutons displayed both elongated and spherical DCVs. Using the protein A-gold double-labeling technique, 10-nm gold particles for SP were localized over the spherical DCV-containing boutons whereas 15-nm gold particles for 5-HT were localized on elongated DCV-containing boutons. On the other hand, in the other species investigated, both SP- and 5-HT-immunoreactive boutons had similar morphological features as shown by the PAP method; both contained elongated and spherical DCVs. The two different sized gold particles, each of which labeled either 5-HT or SP, were found together over DCVs in a single bouton. These results indicate that 5-HT and SP are contained in different presynaptic boutons in the chicken, although in the hamster, rat and Japanese macaque, the two neurotransmitters/modulators coexist in the same DCVs in a single bouton. Species differences have thus been demonstrated for the coexistence of 5-HT and SP in the spinal ventral horn.

AN OPERATED CASE OF PRIMARY MALIGNANT FIBROUS HISTIOCYTOMA OF THE LUNG

Malignant fibrous histiocytoma (MFH), which has been clarified in a recent years, commonly arises in the soft tissue of the adult extremities, but rarely in the lung. Here an operated case of primary MFH of the lung is reported. An 80-year-old man visited with the chief complaint of cough. Biopsy revealed no malignant findings, but imaging techniques offered a suspicion of adenocarcinoma in the right lobe. Right upper lobectomy with regional lymph node dissection was carried out. The tumor was located in the S2 area with pleural indentation. In dissected mediastinal lymph nodes, metastatic tumor cells were observed. Light microscopic findings showed predominantly mixed cells of both histiocytic and fibroblastic cells with a storiform pattern. Electron microscopic findings showed multinucleated giant histiocytic cells. The fibroblastic cells also presented with moderately prominent rough endoplasmic reticulum. Immunohistochemical findings by PAP method were as follows: staining for α-1-antitrypsin, α-1-antichmyotrypsin, lysozyme and ferritin were all positively stained, whereas staining for CEA, keratin, secretary component, epithelial membrane antigen, vimentin, actin, myoglobin and factor were not stained. From the above-mentioned histological findings, we diagnosed this lung tumor as primary malignant fibrous histiocytoma of the lung. A review of the 35 cases in the literature indicated: Including this case, the patient ranged in age 12 to 80 years old (median of 57.8 years). Two patients survived for 5 and 10 years, respectively and the median survival period was 1 year and 6 months. Twenty eight cases had surgical therapy: lobectomy in 20 (80%), pneumonecotmy in 4 (14.2%) and partial resection in 4 cases (14.2%). Distant metastasis from lung MFH occurred in 18 cases (50%) in which brain metastasis in 8 cases (44.4%) was most frequently observed. These findings suggested poor prognosis of MFH arising primarily in the lung.

FOUR CASES OF ALPHA-FETOPROTEIN PRODUCING GASTRIC CANCER

We experienced four clinical cases of alpha-fetoprotein (AFP) producing gastric carcinoma. Those four cases were difinitely diagnosed as AFP producing gastric carcinoma, because the serum levels of AFP decreased significantly after surgical removal of the tumors and the localizations of AFP in tumor tissues were demonstrated in all resected specimens by immunohistochemical study (peroxidase-anti-peroxidase; PAP method). It is said that AFP producing gastric carcinomas are characterized by advanced gastric cancer and histologically undifferentiated or poorly differentiated adenocarcinoma.These four cases also had advanced gastric carcinoma, which were histopathologically papillary, medullary, poorly differentiated and well differentiated adenocarcinomas respectively. AFP producing gastric carcinomas are frequently accompanied with liver metastasis and the prognosis is miserable. Of these 4 cases, 2 already had liver metastasis at surgery, and one developed liver metastasis in an early period after surgery. Their prognoses were poor such as one year survival rate of 25% and the 2 year rate of 0%. From the above, the importance of some strategies or efforts for preventing liver metastasis is emphasized even after curative resection could be attained.